FIELD OF THE INVENTION

The present invention relates generally to the field of antibody detection, and in particular relates to methods involving the detection of autoantibodies relating to Sjogren’s syndrome in a sample comprising patient bodily fluid.

BACKGROUND OF THE INVENTION

Sjogren's syndrome (SjS) is a systemic autoimmune disorder characterized by a lymphocytic infiltration of the salivary and lachrymal glands and, as a consequence of that, oral and ocular dryness. In addition, 50% of the patients develop extraglandular manifestations such as neurological, pulmonary or articular involvement. The prevalence of Sjogren’s syndrome, which is affecting mostly middle-aged women, has been estimated to be in the range between 1 :200 and 1 :1 ,000. Sjogren’s syndrome can be challenging to recognise or diagnose because symptoms may mimic those of menopause, drug side effects, or medical conditions such as lupus, rheumatoid arthritis, fibromyalgia, chronic fatigue syndrome, and multiple sclerosis and thus makes receiving a prompt diagnosis difficult. Since all symptoms are not always present at the same time and because Sjogren’s is so complex, physicians and dentists sometimes treat each symptom individually and do not recognize that a systemic disease is present, (i.e. an eye care provider may be treating dry eye and not realize the patient has other symptoms, or a primary care physician may be treating joint pain but not realise a patient has dryness symptoms too.) Even when diagnostic procedures have been started, Sjogren's syndrome still is difficult to diagnose.

At present, there is no single test that will confirm the diagnosis of Sjogren's and thus physicians must conduct a series of tests and ask about symptoms the patient is experiencing. Blood tests are used to measure the presence of the antibodies anti-Ro (SSA) and anti-La (SSB). Extra-glandular manifestations, which include vasculitis, peripheral neuropathy, renal tubular acidosis, pulmonary involvement, lymphoproliferative disease and/or immunological abnormalities, are present in a subset of patients and found most commonly among those with high levels of SSA and SSB autoantibodies. Approximately 40% of Sjogren’s patients are double positive for SSA and SSB autoantibody reactivity, 28% have single autoantibody reactivity and 28% are tested negative for SSA or SSB autoantibodies (Motors et al., J Intern Med. 2019. 286(4):458-468). Eyes tests are used to measure tear production (Schirmer test), and the surface of the eyes for dry spots (Rose Bengal and Lissamine Green dyes). A salivary gland biopsy (usually in the lower lip) can confirm lymphocytic infiltration of the minor salivary glands and a salivary flow can measure the amount of saliva produced over a certain period of time.

Whilst SSA and SSB antibodies are the only well-established diagnostic marker, the remaining 28% of the patients negative to these antibodies currently can only be identified using invasive procedures (gland biopsies) and a small subset of the patients by ultrasounds of the large salivary glands. These techniques are not established at most sites in the world, and therefore easily measurable biomarkers are required in order to identify SjS patients without SSA/SSB antibodies. Antibodies to salivary gland protein 1 (SP1), carbonic anhydrase 6 (CA6) and parotid secretory protein (PSP) were discovered in an animal model of Sjogren’s syndrome, but clinical studies have shown that these antibodies are only identified in a minority of patients (Shen et al., Clin Immunol. 2012. 145(3):251 -255). Approximately 10% of the patients with Sjogren’s syndrome develop pulmonary problems (Sogkas et aL, Front Med (Lausanne). 2020. 7:332) and up to 30% develop neurological involvement (Seeliger et aL, Front Immunol. 2019. 10:1600). In order to prevent irreversible damage, it would be helpful to identify predictors of these complications, so that patients identified as “at-risk“ individuals could be monitored more closely for the development of pulmonary or neurological involvement. Novel autoantibodies associated with pulmonary or neurological involvement would be suitable to identify these subsets of Sjogren’s syndrome in an early, pre-clinical phase.

Sjogren's syndrome is not a uniform disease, and so far, no effective anti-inflammatory therapies have been identified in randomized trials, although a number of controlled trials have been conducted. Recently, four clusters were identified based on transcriptome analysis from peripheral blood cells in 227 patients with Sjogren's syndrome (Soret P et al, Nat Common 2021 ; 12:3523). The four clusters were characterized by a dominant type I IFN signal, an IFNgamma signal, a lymphocytic signature, and in one cluster no inflammatory signature. That suggests, that the pathomechanisms leading to Sjogren's syndrome differ, and that current anti-inflammatory therapies directed against specific cytokines or cell subsets may be efficacious only in subsets of the patients, in which the particular cytokine or cell subset has a dominant pathophysiological role. Novel biomarkers are needed which are associated with the Sjogren’s syndrome patient subgroups and which could be used by pharmaceutical companies to stratify therapeutic trials in order to predict the response to immunomodulatory therapies. Clustering of patients in clinical trials helps to identify response groups, patient outliers augmenting the possible success of trials in order to identify subgroups of misdiagnosed patients or subgroups with lack of efficacy for the application of certain drugs.

Antibodies, and in particular autoantibodies, can serve as biological markers of disease or disease susceptibility. Autoantibodies are naturally occurring antibodies directed to an antigen which an individual's immune system recognises as foreign even though that antigen actually originated in the individual. They may be present in the circulation as circulating free autoantibodies or in the form of circulating immune complexes consisting of autoantibodies bound to their target protein. Differences between a wild-type protein expressed by "normal" cells and an altered form of the protein produced by a diseased cell or during a disease process may, in some instances, lead to the altered protein being recognised by an individual's immune system as "non-self" and thus eliciting an immune response in that individual. This may be a humoral (/.e. B cell-mediated) immune response leading to the production of autoantibodies immunologically specific for the altered protein.

Assays which measure the immune response of the individual to the presence of a marker protein in terms of autoantibody production provide an alternative to the direct measurement or detection of a marker protein in bodily fluids. Such assays essentially constitute indirect detection of the presence of marker protein. The nature of the immune response means it is likely that autoantibodies can be elicited by a very small amount of a circulating marker protein and indirect methods which rely on detecting the immune response to a marker will consequently be more sensitive than methods for the direct measurement of a marker in bodily fluids. Assay methods based on the detection of autoantibodies may therefore be of particular value early in the disease process and possibly also in relation to screening of asymptomatic patients, for example in screening to identify individuals "at risk" of developing disease amongst a population of asymptomatic individuals. In addition, methods based on the detection of autoantibodies may be of particular value early in the disease process and may also be used to identify individuals who have developed a disease amongst a population of symptomatic individuals.

Whilst the autoantibodies SSA and SSB have been identified as important markers in the diagnosing of Sjogren’s syndrome, there is still a requirement for a diagnostic set of markers to fully diagnose Sjogren’s disease, particularly since not all Sjogren’s patients test positive for SSA and SSB autoantibodies. SUMMARY OF INVENTION

The present application describes new marker antigens that can be used to detect autoantibodies associated with Sjogren’s syndrome. Surprisingly it has been found that a group of marker antigens can be used to help in the characterisation of Sjogren’s syndrome. Through the detection of autoantibodies directed to these novel marker antigens, the inventors have devised effective and non-invasive screening methods for Sjogren’s syndrome, and a corresponding kit.

The inventors of the present application have screened a group of antigens and developed a panel of antigen markers suitable for relatively accurate prediction of Sjogren’s syndrome. These markers can be used in conjunction with the known markers for Sjogren’s syndrome (SSA and SSB) to improve the diagnosis of Sjogren’s syndrome.

According to a first aspect, the present invention provides a method of detecting Sjogren’s syndrome in a mammalian subject, the method comprising:

(a) determining in a test sample comprising a bodily fluid obtained from the mammalian subject the level(s) of autoantibodies specifically binding to one or more of the antigens selected from the following:

DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , TMPO, CCL4, OAS3, NUMA1 , CBLC and SPEG, and

(b) comparing the level(s) of autoantibodies determined in (a) with a predetermined cut-off value for autoantibodies specifically binding to the same one or more antigens, wherein if the level of autoantibodies determined in the test sample is higher than the predetermined cut-off value, the presence of Sjogren’s syndrome is determined.

In certain embodiments, when the mammalian subject has no detectible SSA or SSB antibodies, the method comprises:

(a) determining in a test sample comprising a bodily fluid obtained from the mammalian subject the level(s) of autoantibodies specifically binding to one or more of the antigens selected from DNMBP, CLCN2, and TONSL, and

(b) comparing the level(s) of autoantibodies determined in (a) with a predetermined cut-off value for autoantibodies specifically binding to the same one or more antigens, wherein if the level of autoantibodies determined in the test sample is higher than the predetermined cut-off value, the presence of Sjogren’s syndrome is determined. According to a second aspect, the present invention provides a method of determining whether a mammalian subject has Sjogren’s syndrome with peripheral neuropathy, the method comprising:

(a) determining in a test sample comprising a bodily fluid obtained from the mammalian subject the level(s) of autoantibodies that are specifically binding to one or more of the antigens selected from the following:

CD24, TMEM98, PMF1 , HES1 and SPEG, and

(b) comparing the level(s) of autoantibodies determined in (a) with a predetermined cut-off value for autoantibodies specifically binding to the same one or more antigens, wherein if the level of autoantibodies determined in the test sample is higher than the predetermined cut-off value, the presence of Sjogren’s syndrome with peripheral neuropathy is determined.

According to a third aspect, the present invention provides for a method of determining whether a mammalian subject has Sjogren’s syndrome with pulmonary complications, the method comprising:

(a) determining in a test sample comprising a bodily fluid obtained from the mammalian subject the level(s) of autoantibodies that are specifically binding to one or more of the antigens selected from the following:

EXOSC10, TONSL, CENPH, POLR3B, and FGF21 , and

(b) comparing the level(s) of autoantibodies determined in (a) with a predetermined cut-off value for autoantibodies specifically binding to the same one or more antigens, wherein if the level of autoantibodies determined in the test sample is higher than the predetermined cut-off value, the presence of Sjogren’s syndrome with pulmonary complications is determined.

According to a fourth aspect, the present invention provides for the use of a panel of four or more antigens in a method of detecting Sjogren’s syndrome in a mammalian subject by detecting autoantibodies specifically binding to four or more of the antigens selected from DNMBP, SOX13, PPL, SNRPA, HSP90AA1 , CDH10, TRIM21 , and DHX29, which method comprises the steps of:

(a) determining in a test sample comprising a bodily fluid obtained from the mammalian subject the levels of autoantibodies specifically binding to four or more of the antigens selected from DNMBP, SOX13, PPL, SNRPA, HSP90AA1 , CDH10, TRIM21 , and DHX29; and

(b) comparing the levels of autoantibodies determined in (a) with a predetermined cut-off value for autoantibodies specifically binding to the same four or more antigens, wherein if the level of autoantibodies determined in the test sample is higher than the predetermined cut-off value, the presence of Sjogren’s syndrome is determined.

According to a fifth aspect, the present invention provides for the use of a panel of four or more antigens in a method of detecting Sjogren’s syndrome in a mammalian subject that has no detectible SSA or SSB antibodies by detecting autoantibodies specifically binding to four or more of the antigens selected from CD47, GAL, IL36RN, KRT73 and TNFRSF10B, which method comprises the steps of:

(a) determining in a test sample comprising a bodily fluid obtained from the mammalian subject the levels of autoantibodies specifically binding to four or more of the antigens selected from CD47, GAL, IL36RN, KRT73 and TNFRSF10B; and

(b) comparing the levels of autoantibodies determined in (a) with a predetermined cut-off value for autoantibodies specifically binding to the same four or more antigens, wherein if the level of autoantibodies determined in the test sample is higher than the predetermined cut-off value, the presence of Sjogren’s syndrome is determined.

According to a sixth aspect, the present invention provides a kit for the detection of autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, the kit comprising:

(a) a panel of one or more antigens selected from DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , TMPO, CCL4, OAS3, NUMA1 , CBLC, EXOSC10, TONSL, CENPH, POLR3B, FGF21 and SPEG; and

(b) a reagent capable of detecting complexes of the antigens bound to autoantibodies present in the test sample.

In certain embodiments, the kit further comprises:

(c) means for contacting the antigens with the test sample comprising a bodily fluid from the mammalian subject.

In certain embodiments, the means for contacting the antigens with the test sample comprising a bodily fluid from the mammalian subject comprises the antigens immobilised on a chip, slide, plate, wells of a microtitre plate, bead, membrane or nanoparticle.

In certain embodiments, the kit is for the detection of Sjogren’s syndrome. According to a seventh aspect, the present invention provides a kit for the detection of autoantibodies in a test sample comprising a bodily fluid from a mammalian subject comprising:

(a) a panel of the antigens DNMBP, SOX13, PPL, SNRPA, HSP90AA1 , CDH10, TRIM21 , and DHX29; and

(b) a reagent capable of detecting complexes of the antigens bound to autoantibodies present in the test sample.

According to an eighth aspect, the present invention provides a kit for the detection of autoantibodies in a test sample comprising a bodily fluid from a mammalian subject comprising:

(a) a panel of the antigens CD47, GAL, IL36RN, KRT73 and TNFRSF10B; and

(b) a reagent capable of detecting complexes of the antigens bound to autoantibodies present in the test sample.

According toa ninth aspect, the present invention provides an in vitro method of determining an autoantibody profile of an individual suffering from Sjogren’s syndrome by detecting one or more autoantibodies in a test sample comprising a bodily fluid from the mammalian subject, wherein the autoantibodies are immunologically specific for the antigens DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 and SPEG, which method comprises the steps of:

(a) contacting the test sample with a panel of one or more antigens selected from DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 and SPEG; and

(b) determining the presence or absence of complexes of the antigens bound to autoantibodies present in the test sample, wherein the method is repeated to build up a profile of autoantibody production.

In certain embodiments of the ninth aspect, the method comprises:

(a) contacting the test sample with a panel of three or more antigens selected from DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 and SPEG. According to a tenth aspect, the present invention provides for a method of diagnosing and treating Sjogren’s syndrome in a mammalian subject by detecting one or more autoantibodies in a test sample comprising a bodily fluid from the mammalian subject, wherein the autoantibodies are immunologically specific for the antigens DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 and SPEG, which method comprises the steps of:

(a) contacting the test sample with a panel of one or more antigens selected from DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 and SPEG;

(b) determining the presence or absence of complexes of the antigens bound to autoantibodies present in the test sample;

(c) diagnosing the subject with Sjogren’s syndrome when complexes containing at least one of the antigens DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 and SPEG bound to autoantibodies present in the test sample are detected; and

(d) administering a Sjogren’s syndrome treatment to the diagnosed subject.

According to an eleventh aspect, the present invention provides for a method of predicting response to a Sjogren’s syndrome treatment, the method comprising detecting one or more autoantibodies in a test sample comprising a bodily fluid obtained from a mammalian subject, wherein the autoantibodies are immunologically specific for the antigens DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 , CBLC and SPEG, which method comprises the steps of:

(a) contacting the test sample with a panel of one or more antigens selected from DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 , CBLC and SPEG;

(b) determining the presence or absence of complexes of the antigens bound to autoantibodies present in the test sample;

(c) detecting the amount of specific binding between the antigens and autoantibodies present in the test sample; and (d) comparing the amount of specific binding between the antigens and the autoantibodies with a previously established relationship between the amount of binding and the likely outcome of treatment, wherein a change in the amount of specific binding, when compared to controls, predicts that the subject will or will not respond to the Sjogren’s syndrome treatment.

In all aspects of the invention the antigen may be a naturally occurring protein or polypeptide, a recombinant protein or polypeptide, a synthetic protein or polypeptide, a synthetic peptide, a peptide mimetic, a polysaccharide or a nucleic acid.

In all aspects of the invention the bodily fluid may be selected from the group comprising plasma, serum, whole blood, urine, sweat, lymph, faeces, cerebrospinal fluid, ascites fluid, pleural effusion, seminal fluid, sputum, nipple aspirate, post-operative seroma, saliva, amniotic fluid, tears and wound drainage fluid.

In all aspects of the invention the method is preferably carried out in vitro on a test sample comprising a bodily fluid obtained or prepared from the mammalian subject.

In all aspects of the invention the mammalian subject is preferably a human.

DETAILED DESCRIPTION

A. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by the ordinary person skilled in the art to which the invention pertains. Without limiting any term, further clarifications of some of the terms used herein are provided below.

As used herein, the term autoantibody refers to a naturally occurring antibody directed to an antigen which an individual’s immune system recognises as foreign even though that antigen actually originated in the individual. In general, autoantibodies include antibodies directed against altered forms of naturally occurring proteins produced by a diseased cell or during a disease process. The altered form of the protein originates in the individual but may be viewed by the individual’s immune system as "non-self" and thus elicit an immune response in that individual in the form of autoantibodies immunologically specific to the altered protein. Such altered forms of a protein can include, for example, mutants having altered amino acid sequence, optionally accompanied by changes in secondary, tertiary or quaternary structure, truncated forms, splice variants, altered glycoforms etc. In other embodiments, the autoantibody may be directed to a protein which is overexpressed in a disease state, or as a result of gene amplification or abnormal transcriptional regulation. Overexpression of a protein which is not normally encountered by cells of the immune system in significant amounts can trigger an immune response leading to autoantibody production. In further embodiments the autoantibody may be directed to a foetal form of a protein which becomes expressed in a disease state. If a foetal protein which is normally expressed only in early stages of development, before the immune system is functional, becomes expressed in a disease state, the foetal form expressed in a disease state in the fully developed human may be recognised by the immune system as "foreign", triggering an immune response leading to autoantibody production. In still further embodiments the autoantibody may be directed against a protein which is expressed at a different location in a disease state. For example, the protein may be expressed at an internal location in healthy individuals but is expressed at a surface exposed location in a disease state such that it is exposed to the circulation and therefore the immune system in the disease state but not in the healthy individual. Herein the protein to which the autoantibody is directed will be referred to as a “a marker protein” or “marker antigen”.

As used herein, the term autoantibody biomarker refers to an autoantibody, the levels of which are associated with a particular phenotype, response or outcome. Autoantibody biomarkers in accordance with the present invention are associated with Sjogren’s syndrome and/or the response of Sjogren’s syndrome patients to treatment. As described herein, the levels of autoantibody biomarkers can be detected in samples obtained from subjects and the levels can be compared with pre-determined cut-off values. This assessment of autoantibody biomarkers can be used to detect/diagnose Sjogren’s syndrome as well as inform decisions relating to treatment of Sjogren’s syndrome patients.

As used herein, the term antigen or marker antigen refers to an immunospecific reagent which complexes with autoantibodies present in the test sample. An antigen is a substance comprising at least one antigenic determinant or epitope capable of interacting specifically with the target autoantibody it is desired to detect, or any capture agent interacting specifically with the variable region or complementary determining regions of said autoantibody. The antigen will typically be a naturally occurring or synthetic biological macromolecule such as, for example, a protein or peptide, a polysaccharide or a nucleic acid and can include antibodies or fragments thereof such as anti-idiotype antibodies. As used herein, the term specifically bind refers to the higher affinity of a binding molecule for a target molecule compared to the binding molecule's affinity for non-target molecules. A binding molecule that specifically binds a target molecule does not substantially recognize or bind non-target molecules, e.g., an antibody "specifically binds" and/or "specifically recognizes" another molecule, meaning that this interaction is dependent on the presence of the binding specificity of the molecule structure, e.g., an antigenic epitope.

As used herein, the term distinct antigens encompasses antigens derived from different proteins or polypeptides (such as antigens derived from unrelated proteins encoded by different genes).

As used herein, the term antigen variants refers to allelic or other variants of a single antigen, such as a single protein antigen as defined above. Antigen variants will generally be derived from a single gene, and different antigen variants may be expressed in different members of the population or in different disease states. Antigen variants may differ by amino acid sequence or by a post translational modification such as glycosylation, phosphorylation or acetylation. In addition, the term “antigen variant” encompasses antigen mutations such as amino acid substitutions, additions or deletions. Generally an antigen variant will contain less than five (e.g. less than four, less than three, less than two, or one) mutations relative to the wild-type antigen.

As used herein, the term bodily fluid when referring to the material to be tested for the presence of autoantibodies using the method of the invention, includes inter alia plasma, serum, whole blood, urine, sweat, lymph, faeces, cerebrospinal fluid, ascites fluid, pleural effusion, seminal fluid, sputum, nipple aspirate, post-operative seroma, saliva, amniotic fluid, tears or wound drainage fluid. As aforesaid, the methods of the invention are preferably carried out in vitro on a test sample comprising bodily fluid removed from the test subject. The type of bodily fluid used may vary depending upon the identity of the autoantibody to be tested and the clinical situation in which the assay is used. In general, it is preferred to perform the assays on samples of serum or plasma. The test sample may include further components in addition to the bodily fluid such as for example diluents, preservatives, stabilising agents, buffers etc. Because the assay method is performed on a sample of bodily fluids it is essentially non-invasive. This means that the assay can be repeated as often as necessary, for example, to build up a profile of the patient’s immune response throughout the course of the disease. As used herein, the terms mammalian subject and subject will be used interchangeably to refer to a subject who is mammalian, preferably human. The subject may have Sjogren’s syndrome. The subject may be suspected of having Sjogren’s syndrome. The subject may have tested positive for Sjogren’s syndrome using clinical observation, blood tests to measure the presence of the antibodies anti-Ro (SSA) and anti-La (SSB), eye tests to measure tear production (Schirmer test) and the surface of the eyes for dry spots, or a salivary gland biopsy (usually in the lower lip) to confirm lymphocytic infiltration of the minor salivary glands and a salivary flow to measure the amount of saliva produced over a certain period of time.

B. Method of detecting an autoantibody

The invention provides, in general, an immunoassay method for the detection of autoantibodies immunologically specific for marker proteins associated with Sjogren’s syndrome. The immunoassay method may be used to detect or diagnose Sjogren’s syndrome.

According to the first aspect, the present invention provides a method of detecting Sjogren’s syndrome in a mammalian subject, the method comprising:

(a) determining in a test sample comprising a bodily fluid obtained from the mammalian subject the level(s) of autoantibodies specifically binding to one or more of the antigens selected from the following:

DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , TMPO, CCL4, OAS3, NUMA1 , CBLC and SPEG, and

(b) comparing the level(s) of autoantibodies determined in (a) with a predetermined cut-off value for autoantibodies specifically binding to the same one or more antigens, wherein if the level of autoantibodies determined in the test sample is higher than the predetermined cut-off value, the presence of Sjogren’s syndrome is determined.

In certain embodiments, when the mammalian subject has no detectible SSA or SSB antibodies, the method comprises:

(a) determining in a test sample comprising a bodily fluid obtained from the mammalian subject the level(s) of autoantibodies specifically binding to one or more of the antigens selected from the DNMBP, CLCN2, and TONSL, and

(b) comparing the level(s) of autoantibodies determined in (a) with a predetermined cut-off value for autoantibodies specifically binding to the same one or more antigens, wherein if the level of autoantibodies determined in the test sample is higher than the predetermined cut-off value, the presence of Sjogren’s syndrome is determined.

In certain embodiments, the level(s) of autoantibodies is determined by detecting the amount of specific binding between the marker antigens and autoantibodies present in the test sample, and the presence of Sjogren’s syndrome is based upon a comparison between the amount of specific binding observed and a pre-determined cut-off.

Within this embodiment the amount of specific binding between the marker antigens and autoantibodies present in the test sample may be the relative amount of binding or the absolute amount of binding.

Generally the autoantibody is considered to be present if the amount of specific binding between the marker antigen and autoantibodies present in the test sample is above a predetermined cut-off. The pre-determined cut-off value may be different for different autoantibodies. The pre-determined cut-off may be determined by performing a control assay on known negative samples (e.g. normal individuals) in case-controlled studies. The “normal” individuals will preferably be age-matched controls not having any diagnosis of Sjogren’s syndrome based on clinical and/or biochemical criteria. Preferably the normal individuals do not have any diagnosis of any Sjogren’s syndrome. Here the amount of specific binding between the marker antigen and autoantibodies present in test samples from normal patients may be detected and averaged to provide a pre-determined cut-off. The control cohort of normal individuals from which the pre-determined cut-off value is calculated for any given antigen may be any reasonably-sized cohort, for example at least 50 individuals, at least 100 individuals, at least 200 individuals, at least 500 individuals. In certain embodiments the pre-determined cut-off may be determined by selecting the cut-off value giving the largest Youden’s value which keeps specificity greater than 90%.

For embodiments wherein the autoantibody level is assessed as “higher” than the predetermined cut-off value, a threshold may be applied. For example, a threshold may be applied such that the autoantibodies in the test sample must be at least 1 .5 fold higher, at least 2 fold higher, or at least 2.5 fold higher than the pre-determined cut-off value for the presence of Sjogren’s syndrome to be determined. A threshold may be applied such that the autoantibodies in the test sample must be at least 10%, at least 20%, at least 50% higher than the pre-determined cut-off value for the presence of Sjogren’s syndrome to be determined. Once the level of autoantibodies in the test sample has been compared with the predetermined cut-off value for autoantibodies specifically binding to the same target antigen, an assessment is made as to whether the level of autoantibodies in the patient sample is higher than the predetermined cut-off value. As reported herein, this comparison allows a decision to be made as to whether or not the presence of Sjogren’s syndrome is determined.

For embodiments wherein the method involves determining the levels of autoantibodies specifically binding to multiple antigens, the presence of Sjogren’s syndrome is determined if the autoantibody levels for at least one of the antigens are higher than the pre-determined cut-off value for autoantibodies specifically binding to that antigen. For embodiments wherein the method involves determining the levels of autoantibodies specifically binding to multiple antigens, the presence of Sjogren’s syndrome is determined if the autoantibody levels for at least two, at least three, at least four, at least five of the antigens are higher than the pre-determined cut-off values for autoantibodies specifically binding to the corresponding antigens. In some embodiments wherein the method involves determining the levels of autoantibodies binding to multiple antigens, the presence of Sjogren’s syndrome is determined if the levels of autoantibodies specifically binding to each antigen tested are higher than the pre-determined cut-off values for autoantibodies specifically binding to the corresponding antigens.

The subject may be suspected of having Sjogren’s syndrome due to the presence of a known risk factor for Sjogren’s syndrome. Any methods of determining these risk factors are contemplated and the subject may or may not be undergoing or have undergone treatment relevant to the risk factor. For the purposes of the invention, subjects which are undergoing treatment for Sjogren’s syndrome or which have previously undergone treatment for Sjogren’s syndrome may still be considered “suspected of having Sjogren’s syndrome”. Herein the treatment for Sjogren’s syndrome may have been performed at any time and the subject may or may not have subsequently been tested for the presence of Sjogren’s syndrome.

C. Panels of marker antigens

The present invention provides methods involving the detection of one or more autoantibodies in a test sample comprising a bodily fluid obtained from a mammalian subject, wherein the autoantibodies are immunologically specific for the marker antigens DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 , CBLC and SPEG.

In certain embodiments of the invention the methods may detect one or more autoantibodies, two or more autoantibodies, three or more autoantibodies, four or more autoantibodies, or five or more autoantibodies. For example, the methods may detect three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more autoantibodies.

It is generally accepted that the sensitivity of an assay will be increased by testing for the presence of multiple autoantibodies. Therefore, in some embodiments the methods of the invention contemplate the use of a panel comprising multiple marker antigens, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more marker antigens.

In certain embodiments of the invention, the additional detection of autoantibodies immunologically specific for the marker antigens SSA, SSB and R06O may be employed.

In certain embodiments of the invention where the mammalian subject has no detectible SSA or SSB antibodies, the method provides for the detection of one or more autoantibodies in a test sample comprising a bodily fluid obtained from a mammalian subject, wherein the autoantibodies are immunologically specific for one or more, or all, of the marker antigens DNMBP, CLCN2, and TONSL.

For embodiments involving use of panels comprising multiple marker antigens, the methods may require immune complexes containing two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more of the antigens to be present for a positive assay result.

These methods may be hereinafter referred to as “panel assays”. Such assays are generally more sensitive than the detection of autoantibodies to a single marker antigen and give a much lower frequency of false negative results (see WO99/58978, W02004/044590 and W02006/126008, the contents of which are incorporated herein by reference).

The panel of marker antigens may be tailored having regard to the particular ethnic background of the subject. In certain embodiments, the panel may comprise two or more marker antigens which are distinct antigens. The invention also contemplates methods utilising a panel which comprises two or more antigen variants of one or more distinct antigens.

Also provided herein is a method of determining whether a mammalian subject has Sjogren’s syndrome with peripheral neuropathy, the method comprising:

(a) determining in a test sample comprising a bodily fluid obtained from the mammalian subject the level(s) of autoantibodies specifically binding to one or more of the antigens selected from the following:

CD24, TMEM98, PMF1 , HES1 and SPEG, and

(b) comparing the level(s) of autoantibodies determined in (a) with a predetermined cut-off value for autoantibodies specifically binding to the same one or more antigens, wherein if the level of autoantibodies determined in the test sample is higher than the predetermined cut-off value, the presence of Sjogren’s syndrome with peripheral neuropathy is determined.

Also provided herein is a method of determining whether a mammalian subject has Sjogren’s syndrome with pulmonary complications, the method comprising:

(a) determining in a test sample comprising a bodily fluid obtained from the mammalian subject the level(s) of autoantibodies specifically binding to one or more of the antigens selected from the following:

EXOSC10, TONSL, CENPH, POLR3B, and FGF21 , and

(b) comparing the level(s) of autoantibodies determined in (a) with a predetermined cut-off value for autoantibodies specifically binding to the same one or more antigens, wherein if the level of autoantibodies determined in the test sample is higher than the predetermined cut-off value, the presence of Sjogren’s syndrome with pulmonary complications is determined.

In certain embodiments, two, three, four, five or more autoantibodies are detected, and the method comprises the step of

(a) contacting the test sample with a panel of two or more, three or more, four or more, five or more, six or more or seven or more marker antigens, wherein at least two, at least three, at least four, or at least five of the marker antigens are selected from the group consisting of CD24, TMEM98, PMF1 , HES1 and SPEG, wherein the presence of complexes containing at least two, at least three, at least four, or five of the marker antigens selected from the group consisting of CD24, TMEM98, PMF1 , HES1 and SPEG is indicative of the presence of Sjogren’s syndrome with peripheral neuropathy. D. Assay formats

The actual steps of detecting autoantibodies in a sample of bodily fluids may be performed in accordance with immunological assay techniques known perse in the art.

The general features of immunoassays, for example ELISA, radioimmunoassays and the like, are well known to those skilled in the art (see Immunoassay, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996). Immunoassays for the detection of antibodies having a particular immunological specificity generally require the use of a reagent (antigen) that exhibits specific immunological reactivity with the antibody under test. Depending on the format of the assay this antigen may be immobilised on a solid support. A sample to be tested for the presence of the antibody is brought into contact with the antigen and if antibodies of the required immunological specificity are present in the sample they will immunologically react with the antigen to form antibody-antigen complexes which may then be detected or quantitatively measured.

The methods of the invention may be carried out in any suitable format which enables contact between a test sample suspected of containing the autoantibody and the antigen. Conveniently, contact between the test sample and the antigen may take place in separate reaction chambers such as the wells of a microtitre plate, allowing different antigens or different amounts of antigen to be assayed in parallel, if required. In embodiments in which varying amounts of the antigen are required (see antigen titration method below), these can be coated onto the wells of the microtitre plate by preparing serial dilutions from a stock of antigen across the wells of the microtitre plate. The stock of antigen may be of known or unknown concentration. Aliquots of the test sample may then be added to the wells of the plate, with the volume and dilution of the test sample kept constant in each well. The absolute amounts of antigen added to the wells of the microtitre plate may vary depending on such factors as the nature of the target autoantibody, the nature of the test sample, dilution of the test sample etc. as will be appreciated by those skilled in the art. Generally, the amounts of antigen and the dilution of the test sample will be selected so as to produce a range of signal strengths which fall within the acceptable detection range of the read-out chosen for detection of antigen / autoantibody binding in the method. Conveniently the tested amounts of antigen may vary in the range of from 1 .6 nM to 160 mM.

In a further embodiment of the invention the antigen may be immobilised at a discrete location or reaction site on a solid support. In embodiments where different amounts of the antigen are required (see antigen titration method below), these may each be immobilised at discrete locations or reaction sites on a solid support. The entire support may then be brought into contact with the test sample and binding of autoantibody to antigen detected or measured separately at each of the discrete locations or reaction sites. Suitable solid supports include microarrays. Where different amounts of antigen are required, microarrays can be prepared by immobilising different amounts of a particular antigen at discrete, resolvable reaction sites on the array. In other embodiments the actual amount of immobilised antigen molecules may be kept substantially constant but the size of the sites or spots on the array varied in order to alter the amount of binding epitope available, providing a titration series of sites or spots with different amounts of available binding epitope. In such embodiments the two-dimensional surface concentration of the binding epitope(s) on the antigen is important in preparing the titration series, rather than the absolute amount of antigen. Techniques for the preparation and interrogation of protein/peptide microarrays are generally known in the art.

Microarrays may be used to perform multiple assays for autoantibodies of different specificity on a single sample in parallel. This can be done using arrays comprising multiple antigens or sets of antigens.

Certain antigens may comprise or be derived from proteins or polypeptides isolated from natural sources, including but not limited to proteins or polypeptides isolated from patient tissues or bodily fluids (e.g. plasma, serum, whole blood, urine, sweat, lymph, faeces, cerebrospinal fluid, ascites fluid, pleural effusion, seminal fluid, sputum, nipple aspirate, postoperative seroma and wound drainage fluid). In such embodiments the antigen may comprise substantially all of the naturally occurring protein, i.e. protein substantially in the form in which it is isolated from the natural source, or it may comprise a fragment of the naturally occurring protein. To be effective as an antigen in the method of the invention any such fragment must retain immunological reactivity with the autoantibodies for which it will be used to test. Suitable fragments might, for example, be prepared by chemical or enzymatic cleavage of the isolated protein.

In certain embodiments, and depending on the precise nature of the assay in which it will be used, the antigen may comprise a naturally occurring protein, or fragment thereof, linked to one or more further molecules which impart some desirable characteristic not naturally present in the protein. For example, the protein or fragment may be conjugated to a revealing label, such as for example a fluorescent label, coloured label, luminescent label, radiolabel or heavy metal such as colloidal gold. In other embodiments the protein or fragment may be expressed as a recombinantly produced fusion protein. By way of example, fusion proteins may include a tag peptide at the N- or C- terminus to assist in purification of the recombinantly expressed antigen.

Depending on the format of the assay in which it is to be used the antigen may be immobilised on a solid support such as, for example, a chip, slide, wells of a microtitre plate, bead, membrane or nanoparticle. Immobilisation may be effected via non-covalent adsorption, covalent attachment or via tags.

Any suitable attachment means may be used provided this does not adversely affect the ability of the antigen to immunologically react with the target autoantibody to a significant extent.

The invention is not limited to solid phase assays, but also encompasses assays which, in whole or in part, are carried out in liquid phase, for example solution phase bead assays or competition assays.

In one embodiment, antigens may be labelled with a ligand that would facilitate immobilisation, such as biotin. The antigen can then be diluted to a suitable titration range and allowed to react with autoantibodies in patient samples in solution. The resulting immune complexes can then be immobilised on to a solid support via a ligand-receptor interaction (e.g. biotin-streptavidin) and the remainder of the assay performed as described below.

To facilitate the production of biotinylated antigens for use in the assay methods of the invention, cDNAs encoding a full length antigen, a truncated version thereof or an antigenic fragment thereof may be expressed as a fusion protein labelled with a protein or polypeptide tag to which the biotin co-factor may be attached, for example via an enzymatic reaction.

Vectors for the production of recombinant biotinylated antigens are commercially available from a number of sources. Alternatively, biotinylated antigens may be produced by covalent linkage of biotin to the antigen molecule following expression and purification.

As aforesaid, the immunoassay used to detect autoantibodies according to the invention may be based on standard techniques known in the art. In a most preferred embodiment the immunoassay may be an ELISA. ELISAs are generally well known in the art. In a typical indirect ELISA an antigen having specificity for the autoantibodies under test is immobilised on a solid surface (e.g. the wells of a standard microtiter assay plate, or the surface of a microbead or a microarray) and a sample comprising bodily fluid to be tested for the presence of autoantibodies is brought into contact with the immobilised antigen. Any autoantibodies of the desired specificity present in the sample will bind to the immobilised antigen. The bound antigen / autoantibody complexes may then be detected using any suitable method. In one preferred embodiment a labelled secondary anti-human immunoglobulin antibody, which specifically recognises an epitope common to one or more classes of human immunoglobulins, is used to detect the antigen / autoantibody complexes. Typically, the secondary antibody will be anti-IgG, anti-lgA, anti-lgE or anti-IgM. The secondary antibody is usually labelled with a detectable marker, typically an enzyme marker such as, for example, peroxidase or alkaline phosphatase, allowing quantitative detection by the addition of a substrate for the enzyme which generates a detectable product, for example a coloured, chemiluminescent or fluorescent product. Other types of detectable labels known in the art may be used with equivalent effect.

A given microarray may include exclusively sets of distinct antigens derived from different proteins or polypeptides, or exclusively sets of distinct antigens derived from different peptide epitopes of a single protein or polypeptide, or a mixture of the two in any proportion. It should be noted that each individual set of antigens of different amounts in any embodiment of the invention will generally comprise just one antigen and not mixtures thereof.

A set of antigen variants refers to a single antigen variant to be tested at different amounts in the method of the invention.

E. Key applications of the method

The immunoassay methods according to the invention may be employed in a variety of different clinical situations. In accordance with the invention, the methods are useful for the detection of Sjogren’s syndrome. In particular, the methods may be used in the detection or diagnosis of Sjogren’s syndrome or in screening a population of asymptomatic human subjects in order to diagnose the presence of Sjogren’s syndrome.

Diagnosing and treating Sjogren’s syndrome

In certain embodiments, there is provided a method of diagnosing and treating Sjogren’s syndrome in a mammalian subject by detecting one or more autoantibodies in a test sample comprising a bodily fluid from the mammalian subject, wherein the autoantibodies are immunologically specific for the antigens DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 and SPEG, which method comprises the steps of:

(a) contacting the test sample with a panel of one or more antigens selected from DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 , CBLC and SPEG;

(b) determining the presence or absence of complexes of the antigens bound to autoantibodies present in the test sample;

(c) diagnosing the subject with Sjogren’s syndrome when complexes containing at least one of the antigens DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 , CBLC and SPEG bound to autoantibodies present in the test sample are detected; and

(d) administering a Sjogren’s syndrome treatment to the diagnosed subject.

Within this aspect, the autoantibody may be considered to be present if the amount of specific binding between the marker antigen and autoantibodies present in the test sample is above a pre-determined cut-off, as explained above.

It should be noted that the invention is in no way limited to any specific treatment for Sjogren’s syndrome.

Within the bounds of the invention, the Sjogren’s syndrome treatment may be administered at any time following the diagnosis of Sjogren’s syndrome. For example, the Sjogren’s syndrome treatment may be administered one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, ten hours, eleven hours, twelve hours, twenty four hours, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, one year or more after the diagnosis of Sjogren’s syndrome. Multiple administrations of Sjogren’s syndrome treatment with any spacing between rounds of treatment are also contemplated.

Administration of the Sjogren’s syndrome treatment at a geographical location different from the geographical location at which the Sjogren’s syndrome diagnosis was performed is contemplated. Further, the Sjogren’s syndrome treatment may be administered by a person different from the person performing the diagnosis, irrespective of whether the diagnosis and treatment are performed at the same or different geographical locations.

Within this embodiment of the invention all limitations discussed above in relation to the various methods of the invention are contemplated in relation to the method of diagnosing and treating Sjogren’s syndrome.

Predicting response to a Sjogren’s syndrome treatment

In one aspect, the autoantibody detection method of the invention may be used for treatment stratification, i.e. to determine whether a particular subject or group of subjects is more or less likely to respond to a particular Sjogren’s syndrome treatment. The methods may be used in predicting the response of a Sjogren’s syndrome patient to a treatment, in the selection of a Sjogren’s syndrome therapy, in the selection of a Sjogren’s syndrome therapy for use in a particular patient, in predicting response to therapy, in predicting survival responsive to treatment, or in predicting the risk of immune-related adverse events (irAEs) in patients undergoing immunotherapy.

The invention therefore provides a method of predicting response to a Sjogren’s syndrome treatment, the method comprising detecting one or more autoantibodies in a test sample comprising a bodily fluid obtained from a mammalian subject, wherein the autoantibodies are immunologically specific for the antigens DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 , CBLC and SPEG, which method comprises the steps of:

(a) contacting the test sample with a panel of one or more antigens selected from DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 , CBLC and SPEG;

(b) determining the presence or absence of complexes of the antigens bound to autoantibodies present in the test sample;

(c) detecting the amount of specific binding between the antigens and autoantibodies present in the test sample; and

(d) comparing the amount of specific binding between the antigens and the autoantibodies with a previously established relationship between the amount of binding and the likely outcome of treatment, wherein a change in the amount of specific binding, when compared to controls, predicts that the patient will or will not respond to the Sjogren’s syndrome treatment

Herein, the control may be a sample of bodily fluid derived from a subject known to have Sjogren’s syndrome and known not to respond to the Sjogren’s syndrome treatment being tested i.e. to be a non-responding control. In other embodiments, the control may be a sample of bodily fluid derived from a subject known to have Sjogren’s syndrome and known to have suffered an irAE following treatment.

It should be noted that the invention is in no way limited to any specific Sjogren’s syndrome treatment.

Within this embodiment of the invention all limitations discussed above in relation to the various methods of the invention are contemplated in relation to the method of predicting response to a Sjogren’s syndrome treatment.

Determining an antibody profile

The aspects of the invention described above will usually be performed once. However in vitro immunoassays are non-invasive and can be repeated as often as is thought necessary to build up a profile of autoantibody production in a subject, either prior to the onset of Sjogren’s syndrome, as in the screening of “at risk” individuals, or throughout the course of the disease. The methods therefore may be used in determining an antibody profile in a subject having or suspected of having Sjogren’s syndrome.

In certain embodiments, there is provided an in vitro method of determining an autoantibody profile of an individual suffering from Sjogren’s syndrome by detecting one or more autoantibodies in a test sample comprising a bodily fluid from the mammalian subject, wherein the autoantibodies are immunologically specific for the antigens DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 , CBLC and SPEG, which method comprises the steps of:

(a) contacting the test sample with a panel of one or more antigens selected from DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 , CBLC and SPEG; and (b) determining the presence or absence of complexes of the antigens bound to autoantibodies present in the test sample, wherein the method is repeated to build up a profile of autoantibody production.

In certain embodiments, the method comprises:

(a) contacting the test sample with a panel of three or more antigens selected from DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 , CBLC and SPEG.

Within this embodiment of the invention all limitations discussed above in relation to the various methods of the invention are contemplated in relation to the in vitro method of determining an antibody profile.

Use of a panel of marker antigens to detect Sjogren’s syndrome

The present invention provides use of a panel of one or more marker antigens for the detection of Sjogren’s syndrome in a mammalian subject by detecting autoantibodies immunologically specific for DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 , CBLC and SPEG in a test sample comprising a bodily fluid from the mammalian subject.

In another aspect, the present invention provides use of a panel of four or more antigens in a method of detecting Sjogren’s syndrome in a mammalian subject by detecting autoantibodies specifically binding to four or more of the antigens selected from DNMBP, SOX13, PPL, SNRPA, HSP90AA1 , CDH10, TRIM21 , and DHX29, which method comprises the steps of

(a) determining in a test sample comprising a bodily fluid obtained from the mammalian subject the levels of autoantibodies specifically binding to four or more of the antigens selected from DNMBP, SOX13, PPL, SNRPA, HSP90AA1 , CDH10, TRIM21 , and DHX29; and

(b) comparing the levels of autoantibodies determined in (a) with a predetermined cut-off value for autoantibodies specifically binding to the same four or more antigens, wherein if the level of autoantibodies determined in the test sample is higher than the predetermined cut-off value, the presence of Sjogren’s syndrome is determined. In another aspect, the present invention provides use of a panel of four or more antigens in a method of detecting Sjogren’s syndrome in a mammalian subject that has no detectible SSA or SSB antibodies by detecting specifically binding to four or more of the antigens selected from CD47, GAL, IL36RN, KRT73 and TNFRSF10B, which method comprises the steps of:

(a) determining in a test sample comprising a bodily fluid obtained from the mammalian subject the levels of autoantibodies specifically binding to four or more of the antigens selected from CD47, GAL, IL36RN, KRT73 and TNFRSF10B; and

(b) comparing the levels of autoantibodies determined in (a) with a predetermined cut-off value for autoantibodies specifically binding to the same four or more antigens, wherein if the level of autoantibodies determined in the test sample is higher than the predetermined cut-off value, the presence of Sjogren’s syndrome is determined.

Within this embodiment of the invention all limitations discussed above in relation to the various methods of the invention are contemplated in relation to this use.

Other applications of the method

The methods may be used for the identification of individuals at risk of developing Sjogren’s syndrome in a population of asymptomatic individuals.

The assay method may be repeated on a number of occasions to provide continued monitoring for recurrence of disease. The methods may be used in the detection of recurrent disease in a patient previously diagnosed as having Sjogren’s syndrome who has undergone Sjogren’s syndrome treatment.

The methods may be used in monitoring the progress of Sjogren’s syndrome in a patient, or in monitoring the response of a Sjogren’s syndrome patient to a Sjogren’s syndrome treatment.

The immunoassay methods may complement existing methods of screening, diagnosis and surveillance. For example, the methods of the invention may be used in combination with existing methods to confirm a diagnosis of Sjogren’s syndrome. In certain embodiments, the methods of the invention are performed in combination with blood tests to measure the presence of the antibodies anti-Ro (SSA) and anti-La (SSB), eye tests to measure tear production (Schirmer test) and the surface of the eyes for dry spots, a salivary gland biopsy (usually in the lower lip) to confirm lymphocytic infiltration of the minor salivary glands and a salivary flow to measure the amount of saliva produced over a certain period of time.

F. Kits

The present invention also encompasses a kit for the detection of autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, the kit comprising:

(a) a panel of one or more antigens selected from DNMBP, CLCN2, KDM6B, RPLP2, FKBP15, PARP8, CDH10, HSP90AA1 , SNRPA, TONSL, THAP3, PPL, NONO, DHX29, SOX13, CD24, TMEM98, PMF1 , HES1 , EXOSC10, TONSL, CENPH, POLR3B, FGF21 , TMPO, CCL4, OAS3, NUMA1 , CBLC and SPEG; and

(b) a reagent capable of detecting complexes of the antigens bound to autoantibodies present in the test sample.

In another aspect, the present invention also encompasses a kit for the detection of autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, the kit comprising:

(a) a panel of the antigens DNMBP, SOX13, PPL, SNRPA, HSP90AA1 , CDH10, TRIM21 , and DHX29; and

(b) a reagent capable of detecting complexes of the antigens bound to autoantibodies present in the test sample.

In another aspect, the present invention also encompasses a kit for the detection of autoantibodies in a test sample comprising a bodily fluid from a mammalian subject, the kit comprising:

(a) a panel of the antigens CD47, GAL, IL36RN, KRT73 and TNFRSF10B and

(b) a reagent capable of detecting complexes of the antigens bound to autoantibodies present in the test sample.

In certain embodiments, the kit further comprises

(c) means for contacting the marker antigens with the test sample comprising a bodily fluid from the mammalian subject.

Examples of means for contacting the marker antigen with the test sample comprising a bodily fluid from the mammalian subject include the immobilisation of the marker antigen on a chip, slide, wells of a microtitre plate, bead, membrane or nanoparticle. Within the kits of the invention, the marker antigen is a naturally occurring protein or polypeptide, a recombinant protein or polypeptide, a synthetic protein or polypeptide, a synthetic peptide, a peptide mimetic, a polysaccharide or a nucleic acid.

Within the kits of the invention, the bodily fluid may be selected from the group consisting of plasma, serum, whole blood, urine, sweat, lymph, faeces, cerebrospinal fluid, ascites fluid, pleural effusion, seminal fluid, sputum, nipple aspirate, post-operative seroma, saliva, amniotic fluid, tears and wound drainage fluid.

The kits of the invention are suitable for performing any one of the methods of the invention described above. In particular, the kits of the invention are suitable for the detection of Sjogren’s syndrome. Accordingly, in certain embodiments the kits are for the detection of Sjogren’s syndrome.

The invention will now be further understood with reference to the following non-limiting examples.

EXAMPLES

The use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.

Example 1 : Production of recombinant autoantigens

Recombinant antigens were produced in Escherichia coli. Five cDNA libraries originating from different human tissues (fetal brain, colon, lung, liver, CD4-induced and non-induced T cells) were used for the recombinant production of human antigens. All of these cDNA libraries were oligo(dT)-primed, containing the coding region for an N-terminally located hexa-histidine-tag and were under transcriptional control of the lactose inducible promoter (from E. coli). Sequence integrity of the cDNA libraries was confirmed by 5’ DNA sequencing. Additionally, expression clones representing the full-length sequence derived from the human ORFeome collection were included. Individual antigens were designed in silico, synthesized chemically (Life Technologies, Carlsbad, USA) and cloned into the expression vector pQE30-NST fused to the coding region for the N-terminal-located His6- tag. Recombinant gene expression was performed in E. coli SCSI cells carrying plasmid pSE111 for improved expression of human genes. Cells were cultivated in 200 ml autoinduction medium (Overnight Express auto-induction medium, Merck, Darmstadt, Germany) overnight and harvested by centrifugation. Bacterial pellets were lysed by resuspension in 15 ml lysis buffer (6 M guanidinium-HCI, 0.1 M NaH2PO4, 0.01 M Tris-HCI, pH 8.0).

Soluble proteins were affinity-purified after binding to Protino® Ni-IDA 1000 Funnel Column (Macherey-Nagel, Duren, Germany). Columns were washed with 8 ml washing buffer (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCI, pH 6.3). Proteins were eluted in 3 ml elution buffer (6 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCI, 0.5 % (w/v) trehalose pH 4.5). Each protein preparation was transferred into 2D-barcoded tubes, lyophilized and stored at -20°C.

Example 2: Selection of antigens

A bead-based array was designed to screen for autoantibodies binding to proteins playing a role in autoimmune signalling pathways and in the pathogenesis of SjS. Furthermore, self- reactive antigens of normal humans and typical autoimmune antigens were included. In total, 1629 potential antigens were selected for screening. Multiplex bead-based antigen arrays were designed to include the well-known Sjogren’s syndrome-associated antigens SSA (TRIM21 , Ro60) and SSB/La as well as 45 antigens associated with various rheumatic or connective tissue diseases to assess whether there is overlapping autoantibody reactivity between Sjogren’s syndrome and other autoimmune diseases. The majority of antigens were selected by consulting relevant Gene Ontology (GO) terms, tissue expression (Uniprot tissue annotation database), and KEGG (Kyoto Encyclopaedia of Genes and Genomes database) pathway annotations using the Database Visualization and Integrated Discovery software (DAVID Gene Functional Classification Tool, version 6.7). Proteins were selected from the following categories, numbers of proteins per category are given in brackets: Salivary gland expression (286), brain (701), eye (110), lung (323), saliva (17), non-hodgkin lymphoma (65), type I interferon signalling pathway (19), inflammatory response (95), response to virus (34), apoptotic process (167), response to estrogen (22), innate immune response (87), RNA-binding (193), DNA-binding (143) and ion channel (16).

Example 3: Coupling of antigens to beads

For the production of bead-based arrays (BBA), the proteins were coupled to magnetic carboxylated color-coded beads (MagPlex™ microspheres, Luminex Corporation, Austin, TX, USA). The manufacturer’s protocol for coupling proteins to MagPlex™ microspheres was adapted to use liquid handling systems. A semi-automated coupling procedure of one BBA encompassed 384 single, separate coupling reactions, which were carried out in four 96-well plates. For each single coupling reaction, up to 12.5 pg antigen and 8.8 x 10 ⁵ MagPlex™ beads of one colour region (ID) were used. All liquid handling steps were carried out by either an eight-channel pipetting system (Starlet, Hamilton Robotics, Bonaduz, Switzerland) or a 96-channel pipetting system (Evo Freedom 150, Tecan, Mannderdorf, Switzerland). For semi-automated coupling, antigens were dissolved in H2O, and aliquots of 60 pl were transferred from 2D barcode tubes to 96-well plates. MagPlex™ microspheres were homogeneously resuspended and each bead ID was transferred in one well of a 96- well plate. The 96-well plates containing the microspheres were placed on a magnetic separator (LifeSep™, Dexter Magnetic Technologies Inc., Elk Grove Village, USA) to sediment the beads for washing steps and on a microtiter plate shaker (MTS2/4, IKA) to facilitate permanent mixing for incubation steps.

For coupling, the microspheres were washed three times with activation buffer (100 mM NaH2PO4, pH 6.2) and resuspended in 120 pl activation buffer. To obtain reactive sulfo- NHS-ester intermediates, 15 pl 1-ethly-3-(3-dimethlyaminopropyl) carbodiimide (50 mg/ml) and 15 pl N-hydroxy-succinimide (50 mg/ml) were applied to microspheres. After 20 minutes incubation (900 rpm, room temperature (RT)) the microspheres were washed three times with coupling buffer (50 mM MES, pH 5.0) and resuspended in 65 pl coupling buffer. Immediately, 60 pl antigen solution was added to reactive microspheres and coupling took place over 120 minutes under permanent mixing (900 rpm, RT). After three wash cycles using washing buffer (PBS, 0.1 % Tween20) coupled beads were resuspended in blocking buffer (PBS, 1 % BSA, 0.05 % ProClin300), incubated for 20 minutes (900 rpm, RT) and then transferred to be maintained at 4-8 °C for 12-72 h.

Finally, a multiplex BBA was produced by pooling 384 antigen-coupled beads.

Example 4: Incubation of serum samples with antigen-coupled beads

Serum samples were transferred to 2D barcode tubes and a 1 :100 serum dilution was prepared with assay buffer (PBS, 0.5 % BSA, 10 % E. coli lysate, 50 % Low-Cross buffer (Candor Technologies, Nurnberg, Germany)) in 96-well plates. The serum dilutions were first incubated for 20 minutes to neutralize any human IgG eventually directed against E. coli proteins. The BBA was sonicated for 5 minutes and the bead mix was distributed in 96-well plates. After three wash cycles with washing buffer (PBS, 0.05 % Tween20) serum dilutions (50 pl) were added to the bead mix and incubated for 20 h (900 rpm, 4-8°C). Supernatants were removed from the beads by three wash cycles, and secondary R-phycoerythrin- labelled antibody (5 pg/ml, goat anti-human, Dianova, Hamburg, Germany) was added for a final incubation of 45 minutes (900 rpm, RT). The beads were washed three times with washing buffer (PBS, 0.1 % Tween20) and resuspended in 100 pl sheath fluid (Luminex Corporation). Subsequently, beads were analysed in a FlexMap3D device for fluorescent signal readout (DD gate 7.500-15.000; sample size: 80 pl; 1000 events per bead ID; timeout 60 sec). The binding events were displayed as median fluorescence intensity (MFI). Measurements were disregarded when low numbers of bead events (<30 beads) were counted per bead ID.

Example 5: Collection of serum samples from patients

In total 221 samples were collected from patients diagnosed with Sjogren’s syndrome (sourced from the Rheumatology and Immunology, Medical School Hannover, Hannover Germany), which were analysed in different screens as two independent cohorts of 50 and 171 samples, respectively. Of those 19/50 and 42/171 samples were from patients diagnosed with Sjogren’s syndrome with peripheral neuropathy (PNP) and 31/50 and 129/171 from patients diagnosed with Sjogren’s syndrome without peripheral neuropathy. The same samples were assessed for the presence of the known antibody markers SSA and SSB, and it was found that 32/50 and 112/171 samples were positive for SSA, 18/50 and 58/171 were negative for SSA, 25/50 and 51/171 were positive for SSB, 25/50 and 119/171 were negative for SSB and 1/171 sample was not assessed. The majority of the samples were from females with a mean age of 55.

In addition, samples from healthy subjects (n = 31 and 89) with no evidence of Sjogren’s syndrome were used as controls. These were matched with regards to gender and age.

Example 6: Statistical analysis

Different statistical tests were applied including SAM (Significance Analysis of Microarrays; Tusher et aL, Proc. Natl. Acad. Sci. U. S. A. 2001 98: 5116) to identify autoantibodies elevated in the SjS patients or subgroups of SjS patients. Groups were compared using a variance stabilised, permutation based “t-test” called "significance analysis of microarrays" (SAM) in “R” (https://cran.r-project.org/web/packages/samr/index.html.).

All available autoantibodies were tested. Results were sorted according to d-scores (SAM’s modified T-scores), with higher scores ranking better. To obtain a top list of markers the following filtering rules were applied: P-values filtering: p<0.05, |D| >1.2 for identifying autoantibodies elevated in Sjogren’s syndrome patients. Afterwards random forest machine learning algorithm with forward feature selection of prefiltered markers was applied to evaluate the predictive performance of marker combinations (Tusher, V. G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. U. S. A. 98, 5116-5121 (2001)).

Example 7: Characterisation of the autoantibody response in Sjogren’s syndrome patients

The statistical analyses provide a limited number of antibodies that were significantly found in the samples from all Sjogren’s syndrome patients compared to the healthy controls. Table 1 lists those antigens that bind to the antibodies in the samples. The indicated p-value and D-scores are obtained from the best analysis of one of the two SjS cohorts and include patients with lung involvement and polyneuropathy.

Table 1 : Autoantibody response in Sjogren’s syndrome patients Interestingly the antigens can be characterised as having certain structural characteristics, for example the antigens KDM6B, HSP90AA1 , SSB, FKBP15, NUMA1 , NONO, SOX13, TONSL, DNMBP, DHX29, SNRPA, TMPO all have a bias to having more basic and acidic residues that the average, and the antigens KDM6B, FKBP15, NUMA1 , SOX13, TONSL, CLCN2, PARP8, THAP3, DNMBP, OAS3, DHX29, TMPO all have a bias to having more polar residues than the average.

Example 8: Characterisation of the autoantibody response of patients with Sjogren’s syndrome with peripheral neuropathy compared to Sjogren’s syndrome patients without peripheral neuropathy

The statistical analyses of the cohort consisting of 50 patients provide a limited number of antibodies that were significantly found in the samples from Sjogren’s syndrome patients with peripheral neuropathy compared to Sjogren’s syndrome patients without peripheral neuropathy compared to the healthy controls. Table 2 lists those antigens that bind to the antibodies in the samples.

Table 2: Autoantibody response in Sjogren’s syndrome patients with peripheral neuropathy

Interestingly the antigens can be characterised as having certain structural characteristics, for example the antigens SPEG, HES1 , CD24 all have a bias to having more polar residues than the average.

Interestingly the known antigens SSA/R06O and SSB showed higher reactivity in patients without PNP compared to those with PNP. The known antigen SSA/TRIM21 was not significantly different between patients with PNP compared to those without PNP. Example 9: Characterisation of the autoantibody response of patients with Sjogren’s syndrome that are negative to the antibodies SSA and SSB

The statistical analyses provide a limited number of antibodies that were significantly found in the samples from Sjogren’s syndrome patients that were negative to the antibodies SSA and SSB. Table 3 lists those antigens that bind to the antibodies in the samples. The indicated p-value and D-scores are obtained from the best analysis of one of the two Sjogren’s syndrome cohorts.

Table 3: Autoantibody response in Sjogren’s syndrome patients that are negative to the antibodies SSA and SSB

Interestingly, one of the identified antigens is voltage-gated chloride channel (CLCN2 or CIC- 2), which plays an important role in salivary secretion and showed increased expression in a rabbit model of SjS (Nandoskar et aL, Cornea. 2012 3:273-279).]

Example 10: Panels of antigens for improved diagnosis of Sjogren’s syndrome

Whilst there is a high performance of SSA (TRIM21 ), SSB and Ro60 (TROVE2) as a panel of markers as a predictor of Sjogren’s syndrome, the addition of other markers improves the sensitivity at a given specificity. Table 4 shows the results of a combination of markers. The indicated sensitivity and specificity relate to the cohort consisting of 50 patients and 31 healthy controls.

Table 4: Panels of markers for predicting Sjogren’s syndrome

Example 11 : Panels of markers for diagnosis of Sjogren’s syndrome in SSA negative patients

In analysing samples from Sjogren’s patients that do not have antibodies to the marker SSA (n = 58), it was found that there were a set of markers (CD47, GAL, IL36RN, KRT73 and TNFRSF10B) that provided a panel that identified 44.8% of SSA-negative patients, while only 10% of healthy subjects were falsely classified. Example 12: Characterisation of the autoantibody response of patients with Sjogren’s syndrome with pulmonary complications compared to Sjogren’s syndrome patients without

The statistical analyses of the cohort consisting of 39 patients characterised with Sjogren’s syndrome with pulmonary complications provide a limited number of antibodies that were significantly found in the samples from these patients compared to Sjogren’s syndrome patients (n = 132) without such complications. Table 5 lists those antigens that bind to the antibodies in the samples.

Table 5: Autoantibody response in Sjogren’s syndrome patients with pulmonary complications

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those taken from other aspects of the invention (including in isolation) as appropriate.

Various publications and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.