WO2016141441A1 - Treatment and detection of inflammatory disease - Google Patents

Abstract

Methods for determining a predisposition to and presence of an inflammatory disease, disorder or condition in a subject, which include evaluating the expression level of PAG1 in a biological sample from the subject, are provided. Also provided is a method for preventing or treating an inflammatory disease, disorder or condition in a subject, including administration of an agent that inhibits or prevents expression and/or activity of of PAG1 to said subject. A method for identifying, designing and/or engineering an agent for the inhibition of PAG1, as well as the agent identified, designed and/or engineered therefrom, is further provided.

Description

TITLE

TREATMENT AND DETECTION OF INFLAMMATORY DISEASE

TECHNICAL FIELD

THIS INVENTION relates to the diagnosis and/or treatment of inflammatory diseases, and more particularly allergic or autoimmune diseases, using an agent that binds PAG1 expressed by cells, such as immune cells.

BACKGROUND

The incidence of allergic disease is rapidly increasing. To date, genome-wide association studies (GWAS) have identified 41 genetic associations with allergic diseases, including asthma, hayfever and eczema (Table 1). The identification of allergy risk variants is expected to provide new insights into the molecular pathways involved in disease pathophysiology and, in this way, facilitate the development of new disease treatments. However, these expectations have been hard to meet and this represents a major bottleneck in the field. There are two main reasons for this. First, for most loci, there is no functional evidence linking the risk variant with changes in the expression or protein sequence of any nearby genes; the likely target gene(s) is therefore unknown. This is the case for 27 of the 41 allergy risk loci discovered to date; of note, for 10 of these 27 loci, experimental mouse models of allergic disease implicate nearby genes in disease pathophysiology. Second, often there is little or no information available to determine if and how disruption of the expression or sequence of the target gene impacts cellular function and, ultimately, disease pathophysiology. Addressing these knowledge gaps is critical to help translate GWAS findings into clinically useful information. To this end and notwithstanding the existence of a number of existing therapies for allergic diseases, there remains a need for new compounds and/or molecular targets for treating allergic diseases, especially given the fact that such diseases are often poorly managed and/or do not respond to current treatments.

SUMMARY

The present invention is directed to methods for detecting, diagnosing, preventing, and/or treating diseases, disorders or conditions associated with inflammation. In a broad form, the method of detecting an inflammatory disease, disorder or condition in a subject involves detection of the presence or increased expression of Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (hereinafter PAGl or Pagl), also known as Csk-binding protein (Cbp), in one or more cells or tissues of said subject, such as with an antibody or antibody fragment. Additionally, a method of treating an inflammatory disease, disorder or condition in a subject may involve administering to said subject a therapeutic agent that inhibits the expression and/or activity of PAGl in one or more cells or tissues of the subject.

In a first aspect, the invention provides a method of detecting an inflammatory disease, disorder or condition in a subject, said method including the step of determining an expression level of a PAGl protein or nucleic acid in a biological sample obtainable from the subject to thereby detect the inflammatory disease, disorder or condition.

Suitably, the inflammatory disease, disorder or condition is detected if said expression level of PAGl protein or nucleic acid is at an increased level or upregulated in the biological sample.

In one embodiment, the expression level of PAGl protein or nucleic acid in the biological sample is determined by binding an antibody, an antibody fragment and/or a small molecule to PAGl protein.

In a second aspect, the invention provides a method of determining a predisposition of a subject to an inflammatory disease, disorder or condition, the method including the step of determining an expression level of a PAGl protein or nucleic acid in a biological sample from the subject to thereby evaluate the predisposition to the inflammatory disease, disorder or condition of the subject.

Suitably, an increased or upregulated expression level of a PAGl protein or nucleic acid in the biological sample is indicative of said predisposition to the inflammatory disease, disorder or condition in the subject.

In one embodiment, the expression level of a PAGl protein or nucleic acid in the biological sample is determined by binding an antibody, an antibody fragment and/or a small molecule to a PAGl protein.

In a third aspect, the invention provides a method of preventing or treating an inflammatory disease, disorder or condition in a subject, the method including the step of administering a therapeutically effective amount of an agent that prevents and/or inhibits expression and/or activity of PAGl to thereby treat the inflammatory disease, disorder or condition in the subject.

Suitably, the agent prevents and/or inhibits the expression and/or activity of PAGl in one or more immune cells of the subject. Preferably, the one or more immune cells are or comprise B-cells and/or T-cells.

In an embodiment, the agent is or comprises an antibody or antibody fragment which binds a PAGl protein. Preferably, the antibody is a monoclonal antibody or fragment thereof.

In an embodiment, the agent is or comprises a small molecule which binds a PAGl protein.

Suitably, for the method of the first, second and third aspects, the inflammatory disease, disorder or condition is selected from the group consisting of allergic rhinitis, asthma, eczema and rheumatoid arthritis.

In a fourth aspect, the invention provides a method of screening, designing and/or engineering of an agent for the inhibition of PAGl, said method including the steps of:

(i) contacting a PAGl protein, or variant or fragment thereof, with a test

agent; and

(ii) determining whether the test agent at least partly reduces, eliminates,

suppresses or inhibits expression and/or an activity of PAGl. In one embodiment, the activity of the PAGl is associated with B-cell and/or T- cell activation.

In another embodiment, the activity of the PAGl is an ability to bind one or more molecules or atoms, such as a tyrosine kinase.

Suitably, the agent possesses or displays little or no significant off-target and/or nonspecific effects.

Preferably, the agent is an antibody or a small molecule.

In a fifth aspect, the invention provides an agent identified, designed and/or engineered by the method of the third aspect for use in the detection and/or treatment of an inflammatory disease, disorder or condition.

Suitably, the agent may be used in the methods of the first, second and third aspects.

Suitably, the subject referred to herein is a mammal. Preferably, the subject is a human. As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further elements, components, integers or steps but may include one or more unstated further elements, components, integers or steps.

It will be appreciated that the indefinite articles "a" and "an" are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example, "a" cell includes one cell, one or more cells and a plurality of cells. BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Allergy risk SNPs are associated with PAG1 expression and overlap putative regulatory elements (PREs). (A) Location of the core region of association (blue rectangle) and genes located within 1 Mb of the GWAS sentinel SNP, rs7009110. (B) Results from multivariate association analysis (N=373) between exon expression levels and variants in the core region of association. The color of each variant reflects linkage disequilibrium with rs7009110 (purple square). (C) Location of four PREs, based on histone marks and DNase I hypersensitive sites in an LCL analysed by the ENCODE project.

Figure 2. Chromatin interactions at the 8q21 risk region with PAG1. (A) 3C interaction profiles indicating frequent interactions between PRE2 and PRE3 and the PAG1 promoter region. 3C libraries were generated with EcoRI, with the anchor point set at the PAG1 promoter. A physical map of the region interrogated by 3C is shown below, with the blue bars representing the position of the PREs and the black lines representing the EcoRI fragments interrogated. Triangles show position of variants in linkage disequilibrium (LD) with rs7009110, with color gradient representing the strength of association with PAG1 gene expression levels (cf. Figure IB), from most associated in red to least associated in grey. Graph shows 3C profiles generated from four different LCLs assayed in duplicate. Error bars denote SD. (B) Allele-specific chromatin looping between PRE2 and the PAG1 promoter. 3C followed by direct sequencing of the PCR product was performed in two heterozygous LCLs using rsl 1783496 as the surrogate marker. The rsl l783496:C and rs7009110:C alleles are in phase (r² = 0.75). Chromatograms represent one of two independent 3C libraries generated and sequenced. Figure 3. PRE3 containing the rs2370615 risk allele acts as an enhancer on the PAG1 promoter. PRE2 or PRE3 was cloned upstream of a PAG1 promoter-driven luciferase reporter with and without the candidate causal S Ps. 1 ug of each plasmid was electroporated into lxl 0⁶ cells using Amaxa Nucleofectorll with the SF buffer and the program EH- 100. Luciferase activity was measured after 24h using the Dual-Glo luciferase assay system on a Beckman-Coulter DTX-800 plate reader. Graphs represent three independent experiments assayed in duplicate. Error bars denote 95% CI and P- values were determined with a two-way ANOVA followed by Dunnett's multiple comparisons test (* PO.01, ** <0.001).

Figure 4. Effect of rs7009110 genotype on variation in PAGl expression. The rs7009110:T allele increases the risk of allergic disease. Plotted are the read counts (N=373) for exon 2 of PAGl (exon with largest weight on the multivariate analysis, cf. Table 3), previously- normalized by library depth, with technical variation removed by PEER normalization and after quantile normalization. The association between rs7009110 (coded additively: 0, 1 or 2 copies of the T minor allele) and variation in gene expression levels was significant ( =0.0018, cf. Table 4).

Figure 5. Results from multivariate association analysis (N=373) between gene expression levels and variants in the core region of association. Color of each variant reflects linkage disequilibrium with rs7009110 (purple square). Results are shown for five of the eight genes located within 1 Mb of rs7009110; results for PAGl are shown in Figure 1. The remaining two genes, ZNF704 and STMN2, were only expressed in 56% and 12% of samples, respectively, and so were not analysed.

Figure 6. Results from independent biological replicates for the chromatin interaction analysis between the 8q21 risk region and PAGl. For details, see legend to Figure 2A.

Figure 7. Independent 3C biological replicates showing allele-specific chromatin looping between PRE2 and the PAGl promoter. 3C followed by direct sequencing of the PCR product was performed in two heterozygous LCLs using rsl 1783496 as the surrogate marker. Chromatograms represent one of two independent 3C libraries generated and sequenced.

Figure 8. Location of the PRE2 and PRE3 fragments used in the luciferase assay experiments. Also shown are the location of SNPs in linkage disequilibrium with rs7009110, peak 3C interaction fragments, histone modifications and DNase I hypersensitive sites from an ENCODE LCL, transcription Factor ChlP-seq from ENCODE and RelA binding based on ChlP-seq data in LCLs. The latter track was downloaded from: ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1329nnn/GSM1329660/suppl/GSM1329660_ RelA.bw.

Figure 9. Effect of PAGl KO on the proportion of mucus producing airway epithelial cells (AECs). (A) HDM challenge increases the proportion of AECs that secrete mucus in WT mice. This effect was significantly reduced in PAGl KO mice. Bars represent mean (n=6) ± SEM. (B) Representative lung section images, showing that mucus producing AECs (stained red) are common in HDM-treated WT but not in PAGl KO mice.

Figure 9. Cytokine levels measured in BALF of WT and PAGl KO mice, exposed to HDM or control vehicle. Bars represent mean (n=6) ± SEM. *P<0.05, **P<0.005 HDM versus Vehicle. §P<0.05, §§P<0.005 HDM KO versus HDM WT.

DETAILED DESCRIPTION

The present invention is predicated, at least in part, on the surprising discovery that the risk-associated allele of rs2370615 predisposes to allergic disease by increasing PAGl expression which may promote immune cell activation, including B-cell activation, and thereby have a pro-inflammatory effect. Accordingly, PAGl expression, including nucleic acid and protein expression thereof, may function as a diagnostic or predisposition marker for inflammatory diseases, disorders or conditions. Furthermore, the data provided herein suggest that inhibition of PAGl expression or function may have therapeutic potential for such diseases.

In one aspect, the invention provides a method of detecting an inflammatory disease, disorder or condition in a subject, said method including the step of determining an expression level of PAGl in a biological sample from the subject to thereby detect the inflammatory disease, disorder or condition.

Suitably, the inflammatory disease, disorder or condition is detected if said expression level of PAGl is at an increased level or upregulated in the biological sample.

As used herein, "PAGl " refers to a protein or nucleic acid encoded by a mammalian PAGl gene. Also contemplated are fagments and/or variants of PAGl nucleic acids and proteins. Non-limiting examples of a nucleotide sequence of human PAGl and/or its encoded protein, include HGNC:30043, ENSG00000076641, HPRD:05772, NCBI Gene ID 55824, NP_060910, NP_001007550, XP_496979, Q9NWQ8 (Uniprot), NM_018440, NM_001007549, XM_496979, EAW87086 (GenBank), XP_011515863, XP_011515864, XP_011515865, XP_011515866, XP_011515867, EAW87085 (GenBank) and XP 011515868. The nucleotide or protein sequence provided by these accessions is incorporated by reference herein.

As generally used herein, "inflammation " and "inflammatory " refer to the well known localised response to various types of injury or infection, which is characterised by redness, heat, swelling, and pain, and often also including dysfunction or reduced mobility. Inflammation represents an early defence mechanism to contain an infection and prevent its spread from the initial focus. Major events in inflammation include dilation of capillaries to increase blood flow, changes in the microvasculature structure, leading to escape of plasma and proteins and leukocytes from the circulation, and leukocyte emigration from the capillaries and accumulation at the site of injury or infection.

Inflammation is often associated with, or secondary to, a disease, disorder and/or condition in a subject, including an immunological disease, disorder and/or condition (such as an autoimmune disease, disorder and/or condition) and an allergic disease, disorder and/or condition. Examples of inflammatory diseases, disorders and/or conditions include, without limitation, Addison's disease, allergic rhinitis, ankylosing spondylitis, asthma, celiac disease, chronic bronchitis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic obstructive pulmonary disease (COPD), chronic recurrent multifocal ostomyelitis (CRMO), Crohn's disease, ulcerative colitis, demyelinating neuropathies, eczema, emphysema, glomerulonephritis, food allergy, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), insulin-dependent diabetes (typel), juvenile arthritis, Kawasaki syndrome, multiple sclerosis, myasthenia gravis, postmyocardial infarction syndrome, primary biliary cirrhosis, psoriasis, idiopathic pulmonary fibrosis, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus (SLE), thrombocytopenic purpura (TTP), ulcerative colitis, vasculitis, vitiligo, and Wegener's granulomatosis.

In particular embodiments, the inflammatory disease, disorder or condition is or comprises an allergic disease, disorder or condition. As used herein, "allergy" and "allergic", in the context of a disease, disorder or condition refer to an individual that is susceptible to, or has an increased likelihood or probability of, following contact with that particular allergen, inducing an allergen-specific immune response. This includes situations where the individual is not yet exhibiting clinical symptoms of sensitivity or allergy as well as where the individual is displaying symptoms of sensitivity or allergy. By "immune response " is meant the response of a subject's immune system comprising recognizing and responding to an immunogen, such as an allergen, which may neutralize and/or remove said immunogen from the subject. Immunogens may be on the surface of cells, viruses, fungi, or bacteria or may be nonliving substances such as toxins, chemicals, drugs, and foreign particles. An allergen is a type of immunogen that produces an abnormal or aberrant immune response in which the subject's immune system recognises and responds to a perceived harmful immunogen {e.g., the allergen) that would otherwise be largely harmless to the body.

A subject's immune response to an allergen may comprise the production of allergen-specific antibodies, such as IgE, by cells, such as B-cells, of the subject's immune system. As would be acknowledged by those skilled in the art, allergy or allergic conditions at least partly involve circulating IgE that binds to high-affinity IgE receptors on immune effector cells {e.g., mast cells) located throughout the body triggering mast cell degranulation and an immediate allergic response. Such responses may comprise the release of histamine, leukotrienes, cytokines or other immunologically relevant mediators from allergy effector cells, such as basophils, mast cells or eosinophils. The allergic response in human beings may also be, at least partly, mediated by T lymphocytes, which may proliferate and/or secrete cytokines, such as IL-4, IL-5, and IL-13, in response to activation by allergen-derived peptides.

Allergic conditions commonly include signs and symptoms that can be: (i) cutaneous (e.g. urticaria); (ii) respiratory (e.g. acute bronchospasm, rhinoconjunctivitis); (iii) cardiovascular (e.g. tachycardia, hypotension); (iv) gastrointestinal (e.g. vomiting, diarrhoea); and/or (v) systemic (e.g. anaphylactic shock) in nature.

In particular embodiments, the inflammatory disease, disorder or condition is or comprises an autoimmune disease, disorder or condition. It would be readily appreciated that autoimmune diseases arise, at least in part, from an inappropriate immune response of the body against substances and tissues normally present in the body. This may be restricted to certain organs (e.g. in autoimmune thyroiditis) or involve a particular tissue in different places (e.g. Goodpasture's disease which may affect the basement membrane in both the lung and the kidney). By way of example, organ-specific autoimmune diseases may include type I diabetes mellitus, Crohn's disease, ulcerative colitis, myasthenia gravis, vitiligo, Graves' disease, Hashimoto's disease, Addison's disease, autoimmune gastritis and autoimmune hepatitis. Non-organ specific autoimmune diseases may include, for example, rheumatoid disease, systemic lupus erythematosus, progressive systemic sclerosis and variants, polymyositis and dermatomyositis. Additional autoimmune diseases include pernicious anemia including some of autoimmune gastritis, primary biliary cirrhosis, autoimmune thrombocytopenia, Sjogren's syndrome, multiple sclerosis and psoriasis.

As will be understood by one of ordinary skill in the art, allergic diseases, such as asthma, eczema and allergic rhinitis, and autoimmune diseases, such as rheumatoid arthritis, have an inflammatory component, and thus are particularly amenable to detection and treatment using the disclosed methods.

Accordingly, in one embodiment, the inflammatory disease, disorder or condition is selected from the group consisting of allergic rhinitis, asthma, eczema and rheumatoid arthritis.

The term "determining" includes any form of measurement, and includes determining if an element, for example a PAGl RNA or protein, is present or not, and/or if its expression is enhanced or reduced. As used herein, the terms "determining", "measuring", "evaluating", "assessing" and "assaying" are used interchangeably and include quantitative and qualitative determinations. Determining may be relative or absolute. "Determining the presence of" includes determining the amount of something present (e.g., an RNA and/or protein biomarker), and/or determining whether it is present or absent.

By "enhanced", "up regulated" or "increased" as used herein to describe the expression level of PAGl (e.g., RNA and/or protein), refers to the increase in and/or amount or level of PAGl, including variants, in a biological sample when compared to a control sample or further biological sample from a subject. The expression level of PAGl may be measured or expressed in relative or absolute terms. In some embodiments, the expression of PAGl is increased if its level of expression, such as its protein and/or mRNA expression, is more than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400% or at least about 500% greater than the level of expression of PAGl in a control sample or further biological sample from a subject.

The terms, "reduced" and "down regulated", as used herein to describe the expression level of PAGl (e.g., RNA and/or protein), refer to a reduction in and/or amount or level of PAGl, including variants in a biological sample when compared to a control sample or further biological sample from a subject. The expression level of PAGl may be measured or expressed in relative or absolute terms. In some embodiments, the expression of PAG1 is reduced if its level of expression, such as its protein and/or mRNA expression, is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, or even less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or 0.0001% of the level of expression of PAG1 in a control sample or further biological sample from a subject.

The term "control sample" typically refers to a biological sample from a (healthy) non-diseased individual not having an inflammatory disease, disorder or condition. In one embodiment, the control sample may be from a subject known to be free of an inflammatory disease, disorder or condition. Alternatively, the control sample may be from a subject in remission from an inflammatory disease, disorder or condition. The control sample may be a pooled, average or an individual sample. An internal control is a marker from the same biological sample being tested.

Accordingly, in certain embodiments, a diagnostic PAG1 expression level is correlated to an inflammatory disease, disorder or condition by merely its presence or absence. In other embodiments, a threshold level of a diagnostic PAG1 expression level can be established, and the level of PAG1 in a subject's biological sample can simply be compared to the threshold level.

A threshold level of expression is generally a quantified level of expression of a particular gene, such as PAG1, or set of genes, including gene products thereof. Typically, an expression level of a gene or set of genes in a sample that exceeds or falls below the threshold level of expression is predictive of a particular disease state or outcome. The nature and numerical value (if any) of the threshold level of expression will vary based on the method chosen to determine the expression the one or more genes or proteins used in determining, for example, a diagnosis of or predisposition to an inflammatory disease, disorder or condition, in the mammal. In light of this disclosure, any person of skill in the art would be capable of determining the threshold level of gene/protein (e.g., PAG1) expression in a mammal sample that may be used in determining, for example, a diagnosis of or predisposition to an inflammatory disease, disorder or condition, using any method of measuring gene or protein expression known in the art, such as those described herein. In one embodiment, the threshold level is a mean and/or median expression level (median or absolute) of PAG1 in a reference population that, for example, do not have a particular inflammatory disease, disorder or condition. Additionally, the concept of a threshold level of expression should not be limited to a single value or result. In this regard, a threshold level of expression may encompass multiple threshold expression levels that could signify, for example, a high, medium, or low probability of, for example, developing an inflammatory disease, disorder or condition.

In another aspect, the invention provides a method of determining a subject's predisposition to an inflammatory disease, disorder or condition, the method including the step of determining an expression level of PAGl in a biological sample from the subject to thereby evaluate the predisposition to the inflammatory disease, disorder or condition of the subject.

For the present aspect that the expression level of PAGl may be determined by any method known in the art, including those hereinbefore described.

Preferably, the method is to be performed on those subjects that do not already demonstrate symptoms manifested by the inflammatory disease, disorder or condition.

Suitably, an increased or upregulated expression level of PAGl in the biological sample is indicative of said predisposition to the inflammatory disease, disorder or condition in the subject. In this regard, a subject having an increased or upregulated expression level of PAGl typically has a higher likelihood or increased risk or a statistically significant higher frequency of occurrence for developing or having an inflammatory disease, disorder or condition than one not having such an expression level of PAGl.

In certain embodiments, a PAGl expression level is indicative of a predisposition to an inflammatory disease, disorder or condition by merely its presence or absence. In other embodiments, a threshold level of a PAGl expression level can be established, and the level of PAGl in a subject's biological sample can simply be compared to the threshold level in order to evaluate said predisposition.

As would be understood by the skilled person, by determining a predisposition can provide for predicting a clinical outcome {i.e., the development of an inflammatory disease, disorder or condition) with or without medical treatment in the subject, and selecting an appropriate course of treatment, if any, such as a prophylactic or preventative treatment. This may be at least partly based on determining expression levels of PAGl by the methods of the invention, which may be in combination with determining the expression levels of additional protein and/or other nucleic acid biomarkers.

In one embodiment, the predisposition is used, at least in part, to develop a treatment strategy and/or determine suitability of a treatment for the subject.

It will be appreciated that the methods of the invention include methods of determining the expression level of PAGl alone or in combination with protein and/or nucleic acid biomarkers which have been identified as being diagnostic for an inflammatory disease, disorder or condition. Suitably, when the expression level of PAGl and the additional one or more protein and/or nucleic acid biomarkers are determined, they can be derived from the same or different samples. For example, the expression level of PAGl can be determined in a blood derived sample and the expression level of a nucleic acid biomarker can be determined in a tissue sample.

As used herein, an expression level of PAGl may be an absolute or relative amount of an expressed PAGl gene or gene product thereof inclusive of nucleic acids such as RNA, mRNA and cDNA and protein, inclusive of fragments or variants thereof.

The term "nucleic acid" as used herein designates single- or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and/or thiouridine, although without limitation thereto.

Also included are, "variant" nucleic acids that include nucleic acids that comprise nucleotide sequences of naturally occurring (e.g., allelic) variants and orthologs {e.g., from a different species). Preferably, nucleic acid variants share at least 70% or 75%, preferably at least 80% or 85% or more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a nucleotide sequence oiPAGl.

Also included are nucleic acid fragments. A "fragment" is a segment, domain, portion or region of a nucleic acid, which respectively constitutes less than 100%) of the nucleotide sequence. A non-limilting example is an amplification product or a primer or probe. In particular embodiments, a nucleic acid fragment may comprise, for example, at least 10, 15, 20, 25, 30 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 contiguous nucleotides of said nucleic acid.

By "protein" is meant an amino acid polymer. The amino acids may be natural or non- natural amino acids, D- or L- amino acids as are well understood in the art. As would be appreciated by the skilled person, the term "protein" also includes within its scope phosphorylated forms of a protein {i.e., phosphoproteins). In this regard, the protein of PAGl possesses a number of sites capable of phosphorylation, including, but not limited to, tyrosine residues 106, 163, 181, 227, 299, 317, 341, 359, 387 and 417. A "peptide" is a protein having no more than fifty (50) amino acids.

A "polypeptide^'" is a protein having more than fifty (50) amino acids.

Also provided are protein "variants" such as natrually occurring (eg allelic variants) and orthologs. Preferably, protein variants share at least 70% or 75%, preferably at least 80% or 85% or more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%) or 99% sequence identity with an amino acid sequence of PAG1.

Also provided are protein fragments, inclusive of peptide fragments that comprise less than 100% of an entire amino acid sequence of PAG1. In particular embodiments, a protein fragment may comprise, for example, at least 10, 15, 20, 25, 30 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375 and 400 contiguous amino acids of said protein.

Determining, assessing or measuring protein levels may be performed by any technique known in the art that is capable of detecting cell- or tissue-expressed proteins whether on the cell surface or intracellularly expressed, or proteins that are isolated, extracted or otherwise obtained from the cell of tissue source. These techniques include antibody-based detection that uses one or more antibodies which bind the protein, electrophoresis, isoelectric focussing, protein sequencing, chromatographic techniques and mass spectroscopy and combinations of these, although without limitation thereto. Antibody-based detection may include flow cytometry using fluorescently-labelled antibodies that bind the protein, ELISA, immunoblotting, immunoprecipitation, in situ hybridization, immunohistochemistry and immuncytochemistry, although without limitation thereto. Suitable techniques may be adapted for high throughput and/or rapid analysis such as using protein arrays such as a TissueMicroArray™ (TMA), MSD MultiArrays™ and multiwell ELISA, although without limitation thereto.

Determining, assessing or measuring nucleic acids such as RNA, mRNA and cDNA may be performed by any technique known in the art. These may be techniques that include nucleic acid sequence amplification, nucleic acid hybridization, nucleotide sequencing, mass spectroscopy and combinations of any these.

Nucleic acid amplification techniques typically include repeated cycles of annealing one or more primers to a "template" nucleotide sequence under appropriate conditions and using a polymerase to synthesize a nucleotide sequence complementary to the target, thereby "amplifying" the target nucleotide sequence. Nucleic acid amplification techniques are well known to the skilled addressee, and include but are not limited to polymerase chain reaction (PCR); strand displacement amplification (SDA); rolling circle replication (RCR); nucleic acid sequence-based amplification (NASBA), Q-β replicase amplification; helicase-dependent amplification (HAD); loop-mediated isothermal amplification (LAMP); nicking enzyme amplification reaction (NEAR) and recombinase polymerase amplification (RPA), although without limitation thereto. As generally used herein, an "amplification product" refers to a nucleic acid product generated by a nucleic acid amplification technique.

PCR includes quantitative and semi-quantitative PCR, real-time PCR, allele- specific PCR, methylation-specific PCR, asymmetric PCR, nested PCR, multiplex PCR, touch-down PCR and other variations and modifications to "basic" PCR amplification.

Nucleic acid amplification techniques may be performed using DNA or RNA extracted, isolated or otherwise obtained from a cell or tissue source. In other embodiments, nucleic acid amplification may be performed directly on appropriately treated cell or tissue samples.

Nucleic acid hybridization typically includes hybridizing a nucleotide sequence (typically in the form of a probe) to a target nucleotide sequence under appropriate conditions, whereby the hybridized probe-target nucleotide sequence is subsequently detected. Non-limiting examples include Northern blotting, slot-blotting, in situ hybridization and fluorescence resonance energy transfer (FRET) detection, although without limitation thereto. Nucleic acid hybridization may be performed using DNA or RNA extracted, isolated, amplified or otherwise obtained from a cell or tissue source or directly on appropriately treated cell or tissue samples.

It will also be appreciated that a combination of nucleic acid amplification and nucleic acid hybridization may be utilized.

In certain embodiments of the methods described herein, a gene expression level may be assessed indirectly by the measurement of a non-coding RNA, such as miRNA, that regulate gene expression. MicroRNAs (miRNAs or miRs) are post-transcriptional regulators that bind to complementary sequences in the 3' untranslated regions (3' UTRs) of target mRNA transcripts, usually resulting in gene silencing. miRNAs are short RNA molecules, on average only 22 nucleotides long. The human genome may encode over 1000 miRNAs, which may target about 60% of mammalian genes and are abundant in many human cell types. Each miRNA may alter the expression of hundreds of individual mRNAs. In particular, miRNAs may have multiple roles in negative regulation {e.g., transcript degradation and sequestering, translational suppression) and/or positive regulation (e.g., transcriptional and translational activation). Additionally, aberrant miRNA expression has been implicated in various types of cancer.

In one embodiment, the expression level of PAGl in the biological sample is determined by binding an antibody or antibody fragment thereto.

As used herein an "antibody" is or comprises an immunoglobulin. The term

"immunoglobulin" includes any antigen-binding protein product of a mammalian immunoglobulin gene complex, including immunoglobulin isotypes IgA, IgD, IgM, IgG and IgE and antigen-binding fragments thereof. Included in the term "immunoglobulin" are immunoglobulins that are chimeric or humanised or otherwise comprise altered or variant amino acid residues, sequences and/or glycosylation, whether naturally occurring or produced by human intervention (e.g. by recombinant DNA technology).

Antibody fragments include Fab and Fab'2 fragments, demibodies, diabodies and single chain antibody fragments (e.g. scVs), although without limitation thereto. Typically, an antibody comprises respective light chain and heavy chain variable regions that each comprise CDR 1, 2 and 3 amino acid sequences. A preferred antibody fragment comprises at least one light chain variable region CDR and/or at least one heavy chain variable region CDR.

Antibodies and antibody fragments may be polycolonal or preferably monoclonal. Monoclonal antibodies may be produced using the standard method as for example, described in an article by Kohler & Milstein, 1975, Nature 256, 495, or by more recent modifications thereof as for example described in Chapter 2 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with a PAGl protein or fragment thereof. It will also be appreciated that antibodies may be produced as recombinant synthetic antibodies or antibody fragments by expressing a nucleic acid encoding the antibody or antibody fragment in an appropriate host cell. Recombinant synthetic antibody or antibody fragment heavy and light chains may be co-expressed from different expression vectors in the same host cell or expressed as a single chain antibody in a host cell. Non-limiting examples of recombinant antibody expression and selection techniques are provided in Chapter 17 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY and Zuberbuhler et al, 2009, Protein Engineering, Design & Selection 22 169.

Antibodies and antibody fragments may be modified so as to be administrable to one species having being produced in, or originating from, another species without eliciting a deleterious immune response to the "foreign" antibody. In the context of humans, this is "humanization" of the antibody produced in, or originating from, another species. Such methods are well known in the art and generally involve recombinant "grafting" of non-human antibody complementarity determining regions (CDRs) onto a human antibody scaffold or backbone.

To facilitate detection the antibody or antibody fragment may be directly labelled as hereinafter described or a labelled secondary antibody may be used. The labelled secondary antibody may be as hereinafter described.

Suitably, the method includes detecting the PAG1 expressed by one or more cells or tissues present in, or obtained from, a biological sample. In certain embodiments, the biological sample may be a pathology sample that comprises one or more fluids, cells, tissues, organs or organ samples obtained from a subject. Non-limiting examples include blood, plasma, serum, lymphocytes, urine, faeces, amniotic fluid, cervical samples, cerebrospinal fluid, tissue biopsies, bone marrow, bronchoalveolar lavage fluid, sputum and skin.

In a particular embodiment, the agent binds PAG1 in or on one or more immune cells, including lymphocytes, of the subject. Suitably, the one or more immune cells are or comprise B-cells and/or T-cells. As would be well understood, B-cells, B lymphocytes or plasma cells are the terminal effectors of the B lymphoid lineage of white blood cells and are typically responsible for the production of antibodies {i.e. the humoral arm of adaptive immunity), such as IgE. Further, T-cells or T lymphocytes play a central role in the cell- mediated arm of adaptive immunity and may be distinguished from other lymphocytes by the presence of a T-cell receptor (TCR) on their cell surface.

In particular embodiments, the PAG1 expression level in cells or tissues of the subject is determined by binding an antibody or antibody fragment thereto. In this regard, the antibody or antibody fragment may be a monoclonal antibody, such as that hereinbefore described.

In certain embodiments, the expression level of PAG1 in the subject is determined by binding a small molecule thereto.

Suitably, determining the expression level of PAG1 includes the step of forming a detectable complex between an antibody, antibody fragment or small molecule and PAG1. The complex so formed may be detected by any technique, assay or means known in the art including immunoblotting, immunohistochemistry, immunocytochemistry, immunoprecipitation, ELISA, flow cytometry, magnetic bead separation, and biosensor- based detection systems such as surface plasmon resonance, although without limitation thereto.

To facilitate detection the antibody may be directly labelled or a labelled secondary antibody may be used. Additionally, the small molecule may be directly labelled.

The label may be selected from a group including a chromogen, a catalyst, biotin, digoxigenin, an enzyme, a fluorophore, a chemiluminescent molecule, a radioisotope, a drug or other chemotherapeutic agent, a magnetic bead and/or a direct visual label.

In the case of a direct visual label, use may be made of a colloidal metallic or non- metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.

The fluorophore may be, for example, fluorescein isothiocyanate (FITC), Alexa dyes, tetramethylrhodamine isothiocyanate (TRITL), allophycocyanin (APC), Texas Red, Cy5, Cy3, or R-Phycoerythrin (RPE) as are well known in the art.

The enzyme may be horseradish peroxidase (HRP), alkaline phosphatase (AP), β- galactosidase or glucose oxidase, although without limitation thereto.

In some embodiments, detection methods may be performed in "high throughput" diagnostic tests or procedures such as performed by commercial pathology laboratories or in hospitals.

It would be further appreciated, that such detection methods of PAG1 expression may have potential utility in characterising disease progression and/or severity of a given patient. Additionally, such methods may be used for selecting patients for anti-PAGi treatment, such as by a so-called "companion diagnostic".

In another aspect, the invention provides a method of preventing or treating an inflammatory disease, disorder or condition in a subject, the method including the step of administering a therapeutically effective amount of an agent that inhibits the expression and/or activity of PAG1 to thereby treat the inflammatory disease, disorder or condition in the subject.

It would be appreciated that inhibiting the expression and/or activity of PAG1 may relate to an expressed PAG1 gene or gene product thereof inclusive of nucleic acids such as RNA, mRNA and cDNA and protein, or fragment thereof.

As used herein, "treating", "treat" or "treatment" refers to a therapeutic intervention, course of action or protocol that at least ameliorates a symptom of an inflammatory disease, disorder or condition after said disease, disorder or condition and/or its symptoms have at least started to develop. As used herein, "preventing", "prevent" or "prevention" refers to therapeutic intervention, course of action or protocol initiated prior to the onset of an inflammatory disease, disorder or condition and/or a symptom thereof so as to prevent, inhibit or delay or development or progression of said disease, disorder or condition or the symptom.

By "administration " or "administering" is meant the introduction of a composition (e.g., a composition comprising an agent that binds PAG1 protein or mRNA) into a subject by a chosen route.

The term "therapeutically effective amount" describes a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this can be the amount of a composition comprising one or more agents that binds PAG1 necessary to reduce, alleviate and/or prevent an inflammatory disease, disorder or condition. In some embodiments, a "therapeutically effective amount" is sufficient to reduce or eliminate a symptom of such a disease, disorder or condition. In other embodiments, a "therapeutically effective amount" is an amount sufficient to achieve a desired biological effect, for example an amount that is effective to decrease the immune response associated with an inflammatory disease, disorder or condition.

Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The effective amount of an agent useful for reducing, alleviating and/or preventing an inflammatory disease, disorder or condition will be dependent on the subject being treated, the type and severity of any associated disease, disorder and/or condition, and the manner of administration of the therapeutic composition.

In a particular embodiment, the agent prevents and/or inhibits the expression and/or activity of PAG1 in one or more immune cells of the subject. Suitably, the one or more immune cells are or comprise B-cells and/or T-cells.

In one embodiment, the agent prevents and/or inhibits the activation and/or maturation of and/or promotes death of the one or more immune cells, such as B-cells and T-cells.

In an embodiment, the agent is an antibody or antibody fragment which binds a PAG1 protein or fragment thereof. Preferably, the antibody is a monoclonal antibody or fragment thereof.

In particular embodiments, the agent is a small molecule which binds PAG1. In light of the foregoing, therapeutic application of mRNA-based gene silencing technologies is also contemplated. Useful references describing such technology include RNAi: Design and Application (Methods in Molecular Biology, vol. 442, Humana Press N.Y. USA, 2008) and RNAi: A Guide to Gene Silencing (Cold Spring Harbor Laboratory Press N.Y. USA, 2003).

Suitably, the antibody or fragment thereof or the small molecule is administered to a mammal as a pharmaceutical composition comprising a pharmaceutically-acceptable carrier, diluent or excipient.

By " pharmaceutically -acceptable carrier, diluent or excipient" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, liposomes and other lipid-based carriers, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water.

A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991), which is incorporated herein by reference.

Any safe route of administration may be employed for providing a patient with the composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intranasal, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Intra-muscular and subcutaneous injection is typical, for example, for administration of immunotherapeutic compositions, proteinaceous vaccines and nucleic acid vaccines.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.

Compositions suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of one or more therapeutic agents of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective. The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial response in a patient over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.

In a further aspect, the invention provides a method of screening, designing and/or engineering an agent for use in the prevention and/or treatment of an inflammatory disease, disorder or condition, said method including the steps

(i) contacting a PAG1 protein, or a fragment thereof, with a test agent; and

(ii) determining whether the test agent at least partly reduces, eliminates,

suppresses or inhibits expression and/or an activity of PAG1.

Suitably, the inflammatory disease, disorder or condition is of a type hereinbefore described, albeit without limitation thereto. Preferably, the inflammatory disease, disorder or condition has an overexpressed PAG1 gene product, such as mRNA and/or protein.

Suitably, the agent possesses or displays little or no significant off-target and/or nonspecific effects.

Preferably, the agent is an antibody or a small organic molecule.

In embodiments relating to antibody inhibitors, the antibody may be polyclonal or monoclonal, native or recombinant, as hereinbefore described. Such antibodies may be prepared by any method known in the art, including those described herein. Generally, antibodies of the invention bind to or conjugate with an isolated protein, fragment, variant, or derivative of the protein product of PAG1.

Typically, the inhibitory activity of candidate inhibitor antibodies may be assessed by in vitro and/or in vivo assays that detect or measure the expression levels and/or activity of the protein products of PAG1 in the presence of the antibody.

In embodiments relating to small organic molecule inhibitors, this may involve screening of large compound libraries, numbering hundreds of thousands to millions of candidate inhibitors (chemical compounds including synthetic, small organic molecules or natural products, for example) which may be screened or tested for biological activity at any one of hundreds of molecular targets in order to find potential new drugs, or lead compounds. Screening methods may include, but are not limited to, computer-based ("in silico") screening and high throughput screening based on in vitro assays.

Typically, the active compounds, or "hits", from this initial screening process are then tested sequentially through a series of other in vitro and/or in vivo tests to further characterize the active compounds. A progressively smaller number of the "successful" compounds at each stage are selected for subsequent testing, eventually leading to one or more drug candidates being selected to proceed to being tested in human clinical trials.

At the clinical level, screening a test agent may include obtaining samples from test subjects before and after the subjects have been exposed to a test compound. The levels in the samples of the protein product of the overexpressed genes may then be measured and analysed to determine whether the levels and/or activity of the protein products change after exposure to a test agent. By way of example, protein product levels in the samples may be determined by mass spectrometry, western blot, ELISA and/or by any other appropriate means known to one of skill in the art. Additionally, the activity of the protein products, such as their enzymatic activity, may be determined by any method known in the art. This may include, for example, enzymatic assays, such as spectrophotometric, fluorometric, calorimetric, chemiluminescent, light scattering, microscale thermophoresis, radiometric and chromatographic assays.

It would be appreciated that subjects who have been treated with test agents may be routinely examined for any physiological effects which may result from the treatment. In particular, the test agents will be evaluated for their ability to prevent the development of an inflammatory disease, disorder or condition in a subject. Alternatively, if the test agents are administered to subjects who have previously been diagnosed with an inflammatory disease, disorder or condition, they will be screened for their ability to alleviate symptoms, at least in part, or stop the progression of the inflammatory disease, disorder or condition as well as induce disease remission.

In one embodiment, the activity of PAG1 is associated with lymphocyte, preferably B-cell or T-cell, activation and/or maturation.

As would be understood by the skilled artisan, PAG1 is a raft-associated transmembrane adaptor protein, which may not be directly associated with any receptor, but still may be directly or indirectly involved in the regulation of receptor signalling, including tyrosine kinase signalling. Accordingly, in one embodiment, the activity of

PAG1 is an ability to modulate receptor signalling, and more preferably tyrosine kinase signalling, and even more preferably Src family kinase signalling. The Src family kinases represent a family of non-receptor tyrosine kinases that includes nine members: Src, Yes,

Fyn, Fgr, Lck, Hck, Blk, Lyn and Frk.

In another embodiment, the activity of Pagl is an ability to bind one or more molecules or atoms, such as Lck, Fyn, Lyn, Csk, She, Vav, GAP, phosphatidylinositol 3- kinase (PI3K), ZAP-70, Syk, Grb2, SLP-76, SHP-1, SHP-2 and rasGAP, but without limitation thereto.

In a related aspect, the invention provides an agent identified by the method of the third aspect for use in the treatment and/or detection of an inflammatory disease, disorder or condition.

Suitably, the agent may be used in methods of the first and second aspects as hereinbefore described.

The methods described herein may be applicable to any mammal in which PAG1 expression may be indicative of an inflammatory disease, disorder or condition. In particular embodiments, the term "mamma/" includes but is not limited to humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, goats, sheep, horses) and companion animals (such as cats and dogs). Preferably, the subject is a human.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

So that the present invention may be more readily understood and put into practical effect, the skilled person is referred to the following non-limiting examples.

EXAMPLE 1 In a recent Genome Wide Association Study (GWAS), the present inventors compared 6,685 individuals with both asthma and hayfever against 14,091 asthma- and hayfever-free controls and identified two new risk loci for allergic disease: 8q21 and 16p 13¹. Variants in both were associated with the individual risks of asthma and hayfever, but the association was stronger with the combined asthma with hayfever phenotype. In the 16pl3 locus, evidence from gene expression studies suggests that DEXI is the most likely target gene of this association²'³; functional studies of this gene are now warranted to understand how variation in its expression might affect disease risk. Currently, the gene whose expression is regulated by allergy risk variants in the 8q21 locus is unknown. Therefore, the aim of this study was to use both population genetics and functional approaches to identify the target gene(s) and likely causal variant(s) underlying the 8q21 association with allergy risk.

Eight genes and one miRNA are located within 1 Mb of the sentinel single nucleotide polymorphism (SNP) rs7009110, which has a 36% risk allele frequency and was associated with a 1.14 per-allele odds of disease¹; the nearest gene being ZBTB10 (Figure 1A). rs7009110 was the variant with the strongest association with disease risk after imputation of unmeasured variants from the 1000 Genomes project¹. Forty-three gene expression quantitative trait loci (eQTL) have been reported in this 2 Mb region across six relevant tissue or cell types, but rs7009110 is not in linkage disequilibrium (LD) with any of these (Table 2). However, rs7009110 is in modest LD with two nearby risk variants associated with eczema (rs7000782, r²=0.51)⁴ and rheumatoid arthritis (rs998731, r²=0.20)⁵; in Europeans, the risk alleles for these three variants (rs7009110:T, rs7000782:A and rs998731 :T) are on the same haplotype. The associations at this locus with asthma, hayfever, eczema and rheumatoid arthritis suggest that the underlying disrupted molecular mechanism affects a component of the immune system that is shared between allergic and auto-immune diseases. The most plausible candidate target genes are TPD52 [MIM604068], which is involved in B- cell maturation⁶; PAG1 [MIM605767], involved in T- and B-cell activation^7"10; and ZBTB10, a putative repressor of the Spl transcription factor (TF)¹¹, which regulates multiple immune-related genes^{12 13}.

We first considered the possibility that the lack of a published association between rs7009110 and the expression of nearby genes could represent a false-negative finding, arising because: (i) a true association did not exceed the false discovery rate threshold in the original eQTL studies and so was not reported; and/or (ii) most published eQTL studies surveyed used expression microarrays, which have incomplete coverage of gene expression patterns. To test this possibility, we analysed RNA-seq data obtained by the Geuvadis Project¹⁴ for lymphoblastoid cell line (LCL) samples of 373 individuals of European descent from the 1000 Genomes Project¹⁵. LCLs are derived from peripheral blood B-cells and therefore represent a practical and effective in vitro model to study gene expression patterns relevant to immune-related conditions.

Genotype and RNA-seq data were downloaded from EBI ArrayExpress (accessions E-GEUV-1 and E-GEUV-2). As in Lappalainen¹⁴, we selected the exon (not the gene) as the quantification unit and restricted the analysis to exons expressed in >90% of all the individuals. Read counts normalized by library depth and with technical variation removed by PEER normalization¹⁴ were available for all exons of six of the eight genes located within a 1 Mb region around rs7009110. Read counts were quantile normalized and adjusted for ancestry informative covariates and genotype imputation status, as in Lappalainen¹⁴. For each of these six genes, the association between rs7009110 allelic dosage and variation in the expression of all exons was tested simultaneously using a multivariate test of association that improves power over the alternative strategy of testing each exon individually¹⁶. The remaining two genes, ZNF704 and STMN2 [MIM600621], were only expressed in 56% and 12% of samples, respectively. Given their low relative abundance and because there was no association between dichotomised read counts (expressed vs not expressed) and rs7009110 genotype (not shown), both genes were excluded from further analysis. Likewise, the expression of miR5708 was not associated with rs7009110 (not shown) and so this miRNA was not considered further.

Multivariate association was tested between rs7009110 and the expression levels of five exons in each of HEY1 [MIM602953], MRPS28 [MIM611990] and FABP5 [MIM605168]; seven in ZBTB10 nine in PAG1 and 10 in TPD52. In this analysis, rs7009110 was significantly associated with the expression of PAG1 ( =0.0017) but with no other gene (Table 3). The weights attributed to individual PAG1 exons in the multivariate analysis were consistent with an effect of rs7009110 on the expression of exons 1, 2 and 3; this observation was confirmed with individual univariate analyses of these three exons (Table 4). The rs7009110:T allele that increases the risk of allergic disease was associated with an increased expression of PAG1 (Figure 4), explaining 2.6% of the observed variation.

To help fine-map the eQTL results for PAG1, we extended the multivariate association analysis to an additional 167 common variants (MAF>=5%, call rate >95%, Hardy- Weinberg equilibrium test P-value > 10 ) located in the 69.4 kb core region of association with allergic disease, delimited by the left-most (rs7822328, chr8:81246659) and right-most (rs4739746, chr8:81316034) variants in LD (r²>0.6) with rs7009110. This is the region most likely to include the underlying causal variant(s); most variants were in LD with rs7009110 (70% with r²>0.6). Multiple S Ps in high LD (r²>0.8) with rs7009110 were associated with the expression of PAG1 (Figure IB). In general, the strength of the association increased with the physical proximity to rs7009110, but did not exceed that seen with this SNP. However, no SNPs in LD (r²>0.6) with rs7009110 showed an association with the expression of the other five genes tested ( >0.05, Figure 5).

Although the association between 8q21 allergy risk variants and PAG1 expression in LCLs was relatively modest, it pointed to the possibility that a regulatory element in this region might control the expression of PAG1. Thus, we next queried genome-wide maps of epigenetic profiles to search for putative regulatory elements (PREs) in the 8q21 core region of association. Analysis of histone modifications associated with regulatory activity (e.g. H3K4mel/2) and DNase I hypersensitive sites assayed by the ENCODE Project¹⁷ in an LCL (GM12878) identified four PREs in this region, named PRE1 to PRE4 (Figure 1C). Consistent with these results, Seumois et al¹⁸ detected a significant gain in H3K4me2, which marks both active and poised enhancers, in PREl and PRE2 when comparing primary human CD4+ memory T cells against naive CD4+ T cells. Similarly, Hnisz et al¹⁹ predicted PRE3 to be an enhancer in multiple human immune cell types. Of the 118 SNPs that are in LD (r²>0.6) with rs7009110, 35 overlap one of the four PREs identified (Table 8); rs7009110 overlaps PRE3. We therefore hypothesized that (i) one (or more) of these four PREs in this region regulates the expression of PAG1 and (ii) rs7009110 or a correlated variant disrupts the function of the PRE.

To test the first hypothesis, we used Chromosome Conformation Capture (3C) as described previously²⁰ to quantify the frequency of long-range chromatin interactions that take place between the core region of association and the promoter of PAG1, 710 kb apart. Briefly, 3C libraries were created by cross-linking the chromatin from LCLs of individuals homozygote for the rs7009110:T risk allele (n=2) or the C protective allele (n=2); DNA was then digested with EcoRI which flanks 17 contiguous fragments that cover the core region of association, as well as the PAG1 promoter (Table 6); DNA was religated and decrosslinked; and quantitative PCR (qPCR) with primers for the bait (PAG1 promoter) and interactors (17 fragments) was then performed to detect the presence of ligation products, which represent gene loops. BAC clones covering the regions of interest were used to normalize for PCR efficiency.

The frequency of chromatin interactions between the PAGl promoter and 15 of the 17 fragments that covered the core region of association was low and consistent with being due to chance (Figure 2 A). However, for two fragments - numbered F9 and Fl 1— the interaction frequency was significantly above the background level. The size of the interaction products amplified was confirmed by gel electrophoresis and their sequence verified by Sanger sequencing; similar results were also obtained in two additional independent replicates of each sample (Figure 6). Fragment F9 is 1.1 kb long and overlaps the centromeric end of PRE2, including the peak region of H3K4me2 gain observed by Seumois et al¹⁸ in memory CD4+ T cells. Interactions with fragment F9 were observed in LCLs homozygote for the rs7009110:C protective allele but not (or less frequently) for the T predisposing allele. Consistent with this, allele-specific 3C from two heterozygous LCLs indicate that the protective C-allele is more strongly associated with looping of PRE2 to the PAGl promoter (Figures 2B and 7). These results suggest that an allergy risk allele disrupts the establishment of long-range chromatin interactions between the centromeric end of PRE2 and the PAGl promoter. The interaction observed with fragment Fl l - which is 10.6 kb long and overlaps both the telomeric end of PRE2 and the centromeric end of PRE3 - was detected in all four samples, irrespective of rs7009110 genotype. This suggests that allergy risk variants do not influence the establishment of chromatin looping with this fragment.

Having established that DNA fragments overlapping PRE2 and PRE3 physically interact with the promoter of PAGl, next we tested the hypothesis that the regulatory ability of these two PREs is modulated by allergy risk variants. To assess this, a PAGl promoter-driven luciferase reporter construct was generated by inserting a 1,660 bp fragment containing the PAGl promoter into pGL3-basic, as described previously²¹. A fragment overlapping PRE2 (1,893 bp) or PRE3 (1,560 bp) and containing the major (ie. allergy protective) allele for SNPs in LD (r2>0.6) with rs7009110 was inserted downstream of luciferase. The coordinates of these fragments were selected such that they coincided with the two interaction peaks observed in the 3C experiments (fragments F9 and Fl l) and, within these regions, with the highest density of histone modifications and DNase I hypersensitive sites from ENCODE (Figure 8). To assess the effect of individual SNPs on PRE activity, the minor (ie. allergy predisposing) allele for SNPs in LD with rs7009110 was individually incorporated into PRE2 (rsl 1783496, rsl 1786685, rsl 1786685 or rsl3275449) and PRE3 (rs4739737, rsl0957979 or rs2370615) via standard DNA cloning. All constructs were sequenced to confirm variant incorporation (AGRF, Australia). Primers used to generate all constructs are listed in Table 7. LCLs were electroporated with the reporter plasmids and luciferase activity was measured 24 hr post-transfection. To correct for any differences in transfection efficiency or cell lysate preparation, Firefly luciferase activity was normalized to Renilla luciferase. The activity of each test construct was calculated relative to PAGl promoter construct.

Our 3C studies provided evidence for allele-specific chromatin looping between PRE2 and PA Gl. Despite this, PRE2 did not enhance or silence the PA Gl promoter in reporter assays (Figure 3 A). It is nonetheless possible that such an effect is only present upon cell activation, in different cell types or in vivo. For PRE3, we found that the construct containing the major (allergy protective) allele at the three S Ps (rs4739737, rsl0957979 and rs2370615) in LD with rs7009110 also did not increase PAGl promoter activity (Figure 3B). A similar lack of regulatory activity was observed for PRE3 when the fragments cloned contained the minor (allergy predisposing) allele for rs4739737 or rsl0957979. However, the PRE3 construct containing the minor allele for rs2370615 increased PAGl promoter activity by 1.8-fold when compared to the promoter-only construct ( =0.0051), and by 2.3-fold when compared to the construct with the promoter plus the enhancer containing the major alleles P=0.0005). These results demonstrate that PRE3 acts as a transcriptional enhancer in the presence of the rs2370615:C allergy predisposing allele. Consistent with this effect, rs2370615:C was associated with increased expression of PAGl in the eQTL analyses described above ( =0.0018); this variant is in complete LD (r²=l) with rs7009110. Collectively, these results confirm that PAGl is a target gene of 8q21 allergy risk variants and suggest that rs2370615 represents the underlying putative functional variant.

Based on ENCODE ChlP-seq data for LCLs¹⁷, two transcription factors (TFs) bind to the region containing rs2370615: the RelA (p65) subunit of the nuclear factor kB (NF-kb) TF, which is critical for innate and adaptive immune responses²², and the POU domain class 2 transcription factor 2 (POU2F2, Oct-2), which regulates B-cell-specific genes²³. These TFs have been shown to co-occur in LCLs²⁴ and to synergistically regulate enhancer activity in B-cells²⁵. Furthermore, TNF-a-induced recruitment of RelA to enhancers involved in long-range looping interactions is associated with transcriptional induction of target genes²⁶. These observations suggest that binding of RelA and Oct-2 to PRE3 may be required for long-range activation of PAGl transcription. However, despite RelA binding to PRE3 being highest precisely over rs2370615 (Figure 8), this T/C polymorphism is not predicted to disrupt the binding motifs reported for either RelA or Oct-2²⁴'²⁷. Based on these observations, it is possible that the rs2370615:C allele disrupts the binding of another transcription factor that recruits RelA to PRE3 and so promotes long-range activation of PAG1 transcription. Notably, rs2370615 is predicted to disrupt the binding motif (TTGTTTAC) for five Forkhead (Fox) TFs²⁸, namely Foxo3a, an NF- kB antagonist that inhibits lymphocyte activation and proliferation²⁹. Further studies that dissect the molecular mechanisms underlying the regulation of PAG1 expression by rs2370615 are warranted.

Importantly, our results suggest that increased PAG1 transcription is associated with increased risk of allergic disease. To our knowledge, there are no published studies investigating the contribution of PAG1 to the pathophysiology of allergic diseases. PAG1 encodes the phosphoprotein associated with glycosphingolipid microdomains (or Csk- binding protein, Cbp), a transmembrane adaptor protein localized to lipid rafts that has highest expression in the immune system, notably in T- and B-cells^30"32. One of the main cellular functions of PAG1 is the regulation of Csk activity³⁰, with direct effects on immunoreceptor signalling³³. The role of PAG1 in the development of immune responses has been studied extensively using in vitro and in vivo experimental systems, with conflicting results (reviewed in reference 33). Briefly, in vitro studies suggest that PAG1 overexpression inhibits T-cell, B-cell and mast cell activation^7"10' ⁴ and so, by extension, would be expected to have an anti-inflammatory effect. In contrast, in vivo studies have shown that T- and B-cell development and function are normal in PAG-deficient mice³⁵' 6. Our finding that allergy predisposing alleles increase the transcription of PAG1 in human B-cell lines is not consistent with results from these experimental studies; instead, it suggests that PAG1 overexpression might promote B-cell activation and so have a proinflammatory effect. Given the potential role of the NF-kB pathway in PAG1 transcription, and the observation that this pathway is activated by allergen and/or pathogen engagement of Toll like receptors³⁷, characterization of the immune response to allergen or viral challenge in PAG1 -deficient mice might help resolve these conflicting observations.

In conclusion, we showed that an allergy risk allele on chromosome 8q21 increases PAG1 transcription by activating a poised enhancer located 732 kb away from the gene promoter. The significance of these results is two-fold. First, they highlight the fact that the target gene(s) underlying an observed genetic association can be a considerable distance away from the GWAS sentinel S P. Second, our results suggest that inhibition of PAGl expression or function may have therapeutic potential to treat allergic diseases.

EXAMPLE 2

Mouse experiments were performed to support the data in Example 1 that inhibition of PAGl expression can be used to prevent or treat asthma.

Methods

C57BL/6 wild-type (WT, n=6) and PAGl KO mice on the same background (n=6) were lightly anesthetized with isoflurane and challenged intranasally with 100 μg of house dust mite extract (HDM, Dermatophagoides pteronyssinus; Greer Laboratories, Lenoir, NC, USA) on day 0. Subsequently, mice were challenged with 5 μg of HDM at day 14, 15, 16 and 17 and sacrificed 3 hours later for endpoint assessment. In this model of experimental allergic asthma, WT mice challenged with HDM develop all the hallmark features of asthma, including airway hyperresponsiveness, mucous cell hyperplasia and granulocytic airway inflammation (Ullah et al, J Allergy Clin Immunol 2015; 136: 1065- 73). As controls, two additional groups of mice (WT and PAGl KO, n=6 each) received control vehicle instead of HDM. The following asthma endpoints were then measured as previously described in Ullah et al.¹: cytokine levels and numbers of white-blood cells in bronchoalveolar lavage fluid (BALF); and the number of mucus-producing airway epithelial cells. The Mann-Whitney U test was used to determine if differences in endpoints between two groups were statistically significant.

Results and Discussion

As previously described (Ullah et al.)¹, HDM challenge increased the proportion of mucus-producing airway epithelial cells (AEC) in WT mice ( =0.02, Figure 9). This effect was significantly reduced in PAGl KO mice ( =0.03). These results indicate that inhibition of PAGl expression might help reduce mucus production in the airways.

The number of white-blood cells recovered in BALF, based on total counts or for major cell subtypes, was not significantly different between WT and PAGl KO mice, either exposed to vehicle or allergen (not shown). However, in mice challenged with allergen, there was a trend for a reduction in the number of BALF eosinophils in PAGl KO when compared to WT mice (6xl0³ vs 19xl0³ cells, 70% reduction, =0.24). These results suggest that inhibition of PAGl expression has the potential to reduce allergen- induced eosinophilia.

We also measured three pro-inflammatory cytokines in BALF: IL-5, IL-17A and IL-33. When compared to WT mice, PAGl KO mice had significantly lower levels of IL- 5 and IL-17A after HDM challenge (Figure 10). A similar trend was observed for IL-33. These results show that PAGl inhibition can attenuate the pro-inflammatory responses triggered by allergen challenge.

Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

All scientific and patent literature and genetic information at database accession numbers referred to herein are incorporated by reference in their entirety.

Table 1. Published risk loci for allergic disease (asthma, eczema, allergies and hayfever)

32 Table 2: SNPs located within 1 Mb of rs70091 10 and associated with the expression of nearby genes in published genome-wide association studies of gene expression. Index SNPs are in low linkage disequilibrium (r²<0.1) with each other; from each study queried, we selected the SNP correlated with the Index SNP (r²>0.1) that had the most significant association with gene expression.

2 FAM164A rs2705496 22 Monocytes rs2705496 9.60E-04 0.00

3 FTHL11 rs3812459 22 Monocytes rs3812459 4.38E-06 0.01

4 rsl026830 22 Monocytes rsl026830 1.53E-05 0.00

5 HEY1 rs282850 23 Blood rs282850 5.81E-56 0.01

6 rsl2544114 23 Blood rsl2544114 7.27E-43 0.00

7 rsl7534643 23 Blood rsl7534643 4.76E-17 0.00

8 rs390691 23 Blood rs390691 1.38E-15 0.00

9 rs6996298 23 Blood rs6996298 2.00E-15 0.01

10 rs2956251 23 Blood rs2956251 8.00E-14 0.01

11 rs2029824 23 Blood rs2029824 5.44E-06 0.01

12 rs2920944 23 Blood rs2920944 5.75E-06 0.00

13 rsl2543376 23 Blood rsl2543376 6.89E-06 0.00

14 rs2467778 23 Blood rs2467778 1.59E-04 0.01

15 rs2278667 23 Blood rs2278667 4.51E-04 0.00

16 rs7845273 23 Blood rs7845273 7.10E-04 0.00

17 rs2979707 23 Blood rs2979707 1.09E-03 0.00

18 rsl3281919 23 Blood rsl3281919 1.19E-03 0.01

19 rsl 157639 23 Blood rsl 157639 1.22E-03 0.00

20 IL7 rs 1863593 22 Monocytes rsl863593 4.87E-04 0.00

21 IMPA1 rsl 7582647 21 PBMCs rsl 7582647 7.38E-06 0.01

22 LOC646374 rs3812460 22 Monocytes rs3812460 7.97E-04 0.00

23 rsl0110615 22 Monocytes rsl0110615 2.63E-03 0.00

24 MRPS28 rs903583 22 Monocytes rs903583 1.53E-04 0.01

25 PAG1 rsl3279056 24 LCLs rs4500045 5.20E-11 0.00

25 22 B cells rsl2677218 3.37E-04 0.00

25 22 Monocytes rsl6908663 2.85E-17 0.01

25 1 LCLs rsl2677218 5.35E-08 0.00

25 25 Lung rsl 3266020 7.59E-08 0.00

25 23 Blood rsl3279056 1.5 E-97 0.00

25 21 PBMCs rs6473263 3.54E-38 0.01

26 rsl 0097731 24 LCLs rsl0504730 5.00E-08 0.00 22 B cells rsl0504730 2.54E-04 0.00

1 LCLs rsl0504730 1.97E-08 0.00

23 Blood rsl0097731 1.64E-88 0.00 rs4739791 22 Monocytes rsl896216 2.51E-03 0.00

23 Blood rs4739791 2.54E-31 0.00

21 PBMCs rsl0957999 2.65E-17 0.00 rs4237093 23 Blood rs4237093 7.38E-25 0.00 rs920983 22 Monocytes rsl445558 5.20E-04 0.00

23 Blood rs920983 3.52E-11 0.00 rsl 1988289 23 Blood rsl 7494473 8.95E-04 0.00

21 PBMCs rsl 1988289 7.1 1E-07 0.00 rs9650270 22 Monocytes rs9650270 1.39E-06 0.00

23 Blood rs7009399 4.39E-06 0.00 rs7831986 21 PBMCs rs7831986 2.51E-06 0.01 rs2705499 23 Blood rs2705499 2.06E-05 0.00 rs7017984 23 Blood rs7017984 5.09E-04 0.00

TPD52 rs l863436 25 Lung rsl0755965 9.02E-07 0.01

23 Blood rsl863436 6.31E-52 0.00 rs6473202 23 Blood rs6473202 3.94E-11 0.01 r_s4440674 23 Blood _rs4440674 8.81E-06 0.01 rs4739729 23 Blood rs4739729 1.58E-05 0.13

ZBTB10 rs9298338 25 Lung rs9298338 O.OOE+00 0.00

1 LCLs rs201499880 1.90E-11 0.01 rs3863246 25 Lung rs3863246 9.76E-11 0.00

1 LCLs rs368280 5.39E-09 0.01 rsl l781854 1 LCLs rsl 1781854 9.86E-09 0.02

ZFAND1 rs3812459 22 Monocytes rs3812459 1.71E-04 0.01 rsl026830 22 Monocytes rsl026830 4.19E-04 0.00 rs6473199 22 Monocytes rs6473199 9.32E-04 0.01

Table 3. Results from multivariate association analysis between rs70091 10 and exon expression levels measured in LCLs from 373 individuals of European descent studied by the Geuvadis Project. For each gene, the association between rs70091 10 (coded additively: 0, 1 and 2 copies of the T minor allele) and all exons of that gene were tested using the multivariate test described in Ferreira et al.

MRPS2

5 0.5247 0.29 -0.06 0.45 -0.02 0.68 - - - -

8

TPD52 10 0.8366 -0.25 -0.25 -0.49 -0.59 0.25 -0.15 -0.11 0.01 0.10 -0.01

ZBTBl

7 0.3411 -0.34 0.52 0.63 0.61 0.34 0.53 0.66 - - 0

PAGl 9 0.0017 0.51 0.61 0.46 0.33 0.14 0.24 0.01 0.25 0.16

FABP5 5 0.3932 0.19 0.58 0.25 0.62 0.67

Table 4. Results from univariate association analysis between rs70091 10 and the individual expression levels of exons 1, 2 and 3 from PAGl . For each exon, the association between rs70091 10 (coded additively: 0, 1 and 2 copies of the T minor allele) and expression levels were tested using linear regression

Exon iiiii End Beta SE /Maine

3 81942231 81942324 0.165 0.070 0.0189

2 81982347 81982405 0.217 0.069 0.0018

1 82023826 82024303 0.189 0.072 0.0086

20

Table 5. List of variants in linkage disequilibrium (r²>0.6) with rs7009110 and located in one of the four putative regulatory elements (PREs) identified in the core region of association.

/·^" with

\ ria nl Position, bp PRE

i s7009I I0

rs76079837 81259105 0.842

rsl0957975 81259446 0.826 1

rsl3275219 81259826 0.786 1

rs4739735 81259877 0.842 1

rsl 1776367 81260051 0.837 1

rs3913968 81261038 0.847 1

rs3913969 81261064 0.847 1

rs62517191 81262527 0.847 1

rs62517192 81262544 0.783 1

rs62517193 81262839 0.808 1

rs7824993 81262896 0.721 1

rs5892724 81263715 0.847 1

rs7462675 81263962 0.797 1

indel:2D 8 812640 81264051 0.765 1

rs201311214 81264052 0.759 1

rsl 1783496 81275652 0.751 2

rsl 1786685 81275835 0.753 2

rsl 1786704 81275860 0.856 2

rsl3275449 81276113 0.792 2

rs4739736 81277369 0.784 2

rsl2543811 81278885 0.792 2

rsl3272328 81280496 0.781 2

rsl3270496 81280666 0.868 2

rs7826521 81280778 0.858 2

rs6473225 81281007 0.868 2

rs952559 81288634 0.889 3

rs952558 81288734 0.956 3

rs952557 81288925 0.889 3

rs4739737 81289625 0.889 3

rsl0957979 81289787 0.889 3

rs2370615 81290387 1.000 3

rs4739738 81291645 0.919 3

rs7009110 81291879 - 3

rs28656344 81304496 0.687 4

rs4739739 81304576 0.656 4 Table 6. Primers and coordinates of fragments tested in 3C experiments.

Table 7. Primers used to generate luciferase assay constructs.

( loiiiim I'riiiK' i ^• Svillll'IICl' ( 5' m ^■) Primer 1- r lillK'llt

Primer Coordinates Size ( l)D )

PAG 1 promoter C CACCATTAGTCAACTCATGTCCA 82.025.770- 1660 fwd G G 82.025.795

PAG 1 promoter C AGGGAATCACGGCTCAATTAG 82.024.136- rev G 82.024.159

PRE2 fwd G AGGTACCACCGGTGCAAATTCC 81.275.368- 1893

T CTGTGGTAGGATGTCTGG 81.275.395

PRE2 rev G ATCTAGAC CTGC AGGGTAGGGT 81.277.234- C CAGTGGGAGAGACACTTGC 81.277.260

PRE3 fwd G AGAAGCTTACCGGTCCTTTGATC r 81.289.408- 1560

G ACATGATGTCACC 81.289.433

PRE3 rev G ATCTAGAC CTGC AGGC ACGTGG 81.290.942- C TGC CTTCTTATGAAA AGC 81.290.967

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Claims

1. A method of detecting an inflammatory disease, disorder or condition in a subject, said method including the step of determining an expression level of a PAGl protein, nucleic acid or fragment or variant thereof in a biological sample obtained from the subject to thereby detect the inflammatory disease, disorder or condition in the subject.

2. The method of Claim 1, wherein the inflammatory disease, disorder or condition is detected if said expression level of PAGl protein, nucleic acid fragment or variant thereof is at an increased level or upregulated in the biological sample.

3. A method of determining a predisposition of a subject to an inflammatory disease, disorder or condition, the method including the step of determining an expression level of a PAGl protein, nucleic acid, fragment or variant thereof in a biological sample from the subject to thereby evaluate the predisposition to the inflammatory disease, disorder or condition of the subject.

4. The method of Claim 3, wherein an increased or upregulated expression level of PAGl protein or nucleic acid in the biological sample is indicative of said predisposition to the inflammatory disease, disorder or condition in the subject.

5. The method of any one of the preceding claims, wherein the expression level of PAGl protein in the biological sample is determined by binding an antibody, an antibody fragment and/or a small molecule to the PAGl protein.

6. A method of preventing or treating an inflammatory disease, disorder or condition in a subject, the method including the step of administering a therapeutically effective amount of an agent that inhibits or prevents expression and/or activity of a PAGl protein or nucleic acid to thereby treat the inflammatory disease, disorder or condition in the subject.

7. The method of Claim 6, wherein the agent prevents and/or inhibits expression and/or activity of the PAGl protein or nucleic acid in one or more immune cells of the subject.

8. The method of Claim 7, wherein the one or more immune cells are or comprise B-cells and/or T-cells.

9. The method of any one of Claims 6 to 8, wherein the agent is or comprises an antibody, an antibody fragment and/or a small molecule that binds a PAGl protein.

10. The method of any preceding claim, wherein the subject is a mammal.

11. The method of Claim 10, wherein the subject is a human.

12. The method of any one of the preceding claims wherein the inflammatory disease, disorder or condition is selected from the group consisting of allergic rhinitis, asthma, eczema and rheumatoid arthritis.

13. A method of identifying, designing and/or engineering an agent for the inhibition of PAG1, said method including the steps of:

(i) contacting a PAG1 protein, variant or fragment thereof, with a test agent; and

(ii) determining whether the test agent at least partly reduces, eliminates, suppresses or inhibits expression and/or an activity of PAG1.

14. The method of Claim 13, wherein the activity of PAG1 is associated with B-cell and/or T-cell activation.

15. The method of Claim 14, wherein the activity of PAG1 is an ability to bind one or more tyrosine kinases.

16. The method of any one of Claims 13 to 15, wherein the agent is an antibody, an antibody fragment or a small molecule.

17. An agent, identified, designed and/or engineered by the method of any one of Claims 13 to 16, for use in the treatment and/or detection of an inflammatory disease, disorder or condition.

18. The agent of Claim 17 for use in the method of any one of Claims 1 to 12.