FIELD OF THE INVENTION The present invention relates to new methods of diagnosing several autoimmune diseases. More particularly the present invention relates to methods and compositions for detecting anti-heterogeneous nuclear ribonucleoprotein autoantibodies for diagnosing these autoimmune diseases. More in particular, the present invention relates to methods and compositions for detecting anti- heterogeneous nuclear ribonucleoproteins-EI , -F, -H1, -A1 , and -B1, including any combinations of these, for diagnosing autoimmune diseases, more in particular for diagnosing Sjogren's syndrome, mixed connective tissue disease (MCTD) and dermatomyositis (DM).

BACKGROUND Autoantibodies to intracellular antigens are a common feature of systemic autoimmune disorders such as systemic lupus erythematosus (SLE), systemic sclerosis (Ssc), polymyositis (PM), mixed connective tissue disease (MCTD), dermatomyositis (DM), primary Sjogren's syndrome (SSp) or rheumatic arthritis (RA). These autoantibodies are typically directed to sets of proteins associated with discrete supramolecular entities built up of either DNA or RNA and proteins, such as the nucleosome, the centromere, the spliceosome, the ribosome or other RNPs. These autoantibodies are starting to be regarded as important tools to obtain deeper insight into the pathogenesis of autoimmune rheumatic diseases. (Caporali R et al., Autoimmunity 2005; 38:25-32.)

HnRNPs comprise a group of approximately 30 different proteins. They mainly play a role in processing of precursor mRNA, but are also involved in telomeric DNA synthesis and cellular apoptotic processes. HnRNPs are composed of at least 1 RNA recognition motif. HnRNP proteins appear to be an important target of autoimmune responses. Several different hnRNP proteins have been described as autoantigens in autoimmune connective tissue diseases.

Autoantibodies to hnRNP A1 were first detected by Jensen et al. in 63 out of 160 SLE patients (37%) and in 17 out of 71 RA patients (24 %), but only in 1 out of 24 polymyositis patients and in 7 out of 98 healthy controls. Anti-hnRNP A2 antibodies target the 36 kDa hnRNP A2 as well as its alternatively spliced variants hnRNP B1 (37 kDa) en B2 (38 kDa). These 3 autoantigens may be designated as the "RA33 complex", that shows reactivity with autoantibodies in 35% of RA patients, 38% of MCTD patients, 23% of SLE patients and not in patients with other rheumatic diseases. In German Shepherd dogs with lupus-like syndrome, Soulard ef al. identified antibodies reacting with hnRNP G. The same research group characterized a new antibody specificity in a panel of sera from dogs developing SLE. The target antigen was found to be a C protein of the hnRNP complex. Valai et al. found in 350 sera with different autoimmune rheumatic diseases hnRNP K as autoantigen in 44% of SLE patients, in 18% of patients with MCTD and in 14% of RA patients. Caporali et al. found reactivit against hnRNP I in patients with systemic sclerosis, but not in patients with SLE, RA of MCTD. HnRNP D was identified by Skriner et al. as a new autoantigen in patients with SLE and related disorders. Antibodies to hnRNP R were found in 1998 by Hassfeld et al. in 1 patient with vage auto-immuun complaints. In 2007 Siapka ef al. Found high antibody titers against hnRNP L in patients with systemic sclerosis.

Recently, hnRNP H1 was described as a newly recognized target of autoantibodies. These antibodies were mainly found in patients with Sjogren's syndrome, but were also present in patients with other autoimmune-mediated diseases (WO2009055880).

Sjogren's syndrome is an autoimmune exocrinopathy, characterized by dryness of the mouth and eyes resulting from a chronic loss of secretory function of the salivary and lacrimal glands. Extraglandular systemic manifestations are common and include abnormalities of skin, arthralgia, myalgia, thyroiditis, and pulmonary, renal, gastrointestinal, hematological, cardiac, and neurological abnormalities. There is an increased propensity to develop B cell lymphoma. The etiology of the disease remains unclear. The prevalence of the disease is estimated to be 0.5% and there is a female preponderance. Pathological findings involve focal lymphocytic infiltration of affected tissues. Laboratory findings comprise hypergammaglobulinemia, rheumatoid factor and auto-antibodies against salivary ductal cells and SSA, SSB, and hnRNP H1. Depending on the technique used, anti-SSA antibodies are found in 45-90% of the patients with Sjogren's syndrome. Anti-SSB antibodies are found slightly less commonly. Anti-SSA antibodies are not specific for Sjogren syndrome as they are found in other systemic diseases such as systemic lupus erythematosus, subacute cutaneous lupus erythematosus and neonatal lupus.

Sjogren's syndrome can occur in two forms: primary Sjogren's syndrome (not associated with other autoimmune diseases) and secondary Sjogren's syndrome (associated with other autoimmune diseases). Because of the non-specific symptoms (dry eyes and mouth), establishment of the diagnosis of Sjogren's syndrome may be difficult. There is an unmet need for additional accurate and valid diagnostic markers. A number of new autoantigens have recently been suggested: oc-fodrin, muscarinic M3 acetylcholine receptor, SS-56, which is structurally related to the 52 kDa SSA antigen, and hnRNP H1. These markers, however, have not shown adequate specificity and sensitivity to serve as valuable clinical markers, or are not available for routine diagnostics (Kovacs L et al., Rheumatology (Oxford). 2005;44:1021-5, WO2009055880).

The fact that there are patients with autoimmune diseases, particularly with sicca symptoms, who have antinuclear antibodies in high titer but in whom no antibodies to extractabie nuclear antigens (e.g. SSA or SSB) can be identified is indicative for the need to identify novel and preferentially early diagnostic markers. Moreover, the fact that particular antibodies (such as hnRNP H1) are found in patients with a particular autoimmune disease (such as Sjogren's syndrome), and where said antibodies are also present in patients with other autoimmune-mediated diseases, is indicative for the need to identify additional and preferentialy early diagnostic markers and combinations of these diagnostic markers for diagnosing particular autoimmune diseases.

SUMMARY OF THE INVENTION

The present invention shows several hnRNPs as a new targets of autoantibodies in patients with autoimmune diseases. These hnRNPs are selected from the group consisting of hnRNP-A1 , hnRNP-B1 , hnRNP-C1 , hnRNP-E1 , hnRNP-F, hnRNP-H1 , hnRNP-l, hnRNP-K, and hnRNP-P2. More in particular the hnRNPs as targets of antibodies in Sjogren's syndrome are one or more hnRNPs selected from the group consisting of hnRNP-A1 , hnRNP-B1 , hnRNP-CI , hnRNP-E1 , hnRNP-F, hnRNP-l, hnRNP-K, and hnRNP- P2. The one or more hnRNPs from said group can be combined with hnRNP-H1 as combined markers for Sjogren's syndrome.

More in particular, the present invention identifies hnRNP-F; hnRNP-E1 ; and the combination of hnRNP-F and hnRNP-E1 as very valuable diagnostic markers and marker-combinations in Sjogren's syndrome. The present invention also identifies the following combination of antibodies as very valuable diagnostic combination markers in Sjogren's syndrome: hnRNP-F and hhRNP-H1 ; hnRNP-E1 and hnRNP-H1; and the combination of hnRNP-F, -E1 , and -H1 ; and the combination of hnRNP-B1 , -F, and -E1 ; and the combination of hnRNP-B1 , -F, -E1 , and -H1. In addition to the 4 diagnostic antibody markers (hnRNP- E1 , -F, -H1 and -B1) including any of their combinations as very valuable diagnostic markers and marker- combinations in Sjogren's syndrome, the present invention also identifies additional antibody markers (for example hnRNP-A1) which in combination with any of the above mentioned 4 diagnostic antibody markers and their combinations (hnRNP-E1 , -F, -H1 and -B1) that are useful diagnostic antibody marker combinations in Sjogren's syndrome (examples are the combination of hnRNP-A1 , -B1 , and -F; the combination of hnRNP-A1 , -B1 , -F, and -E1 ; the combination of hnRNP-A1 , -B1 , -F, and -H1; and the combination of hnRNP-A1 , -B1 , -F, -E1 , and -HI).

Included in the above described markers are markers and markercombinations that are also valuable diagnostic markers and marker-combinations for other autoimmune diseases, more in particular the hnRNP-F; hnRNP-E1 ; and the combination of hnRNP-F and hnRNP-E1 markers (including the combinations of these markers with the markers hnRNP-H1 and/or hnRNP-B1 , example given hnRNP-E1 and hnRNP-H1; hnRNP-F and hnRNP-H1 ; hnRNP-F, -E1 , and -HI ; hnRNP-E1 , -F, and -B1 ; and hnRNP-E1 , -F, -H1 and -B1,) are useful markers for mixed connective tissue disease, systemic lupus erythematosus, and dermatomyositis.

The present invention provides for a method for diagnosing Sjogren's syndrome. More particularly the present invention provides methods and compositions for detecting the above mentioned anti-hnRNP autoantibodies for diagnosing Sjogren's syndrome. The method of this invention is applicable to a large group of patients, including patients having Sjogren's syndrome, patients being suspected as having Sjogren's syndrome or at risk of developing Sjogren's syndrome, or patients suffering from other autoimmune diseases.

One aspect of the present invention relates to a method of diagnosing an autoimmune disease, more particularly Sjogren's syndrome, characterized in that the presence of autoantibodies against the above mentioned hnRNPs and the above mentioned combinations is determined in an isolated biological sample derived from a subject. The present invention relates to methods of diagnosing autoimmune diseases, more in particular Sjogren's syndrome, by measuring the presence of autoantibodies against the above mentioned hnRNPs and the above mentioned combinations in an isolated biological sample derived from a subject, and wherein the presence of the above mentioned hnRNPs or the above mentioned combinations is indicative for the diagnosis of said autoimmunedisease for said subject, or wherein the presence of the above mentioned hnRNPs or the above mentioned combinations is indicative for the predisposition of said subject to develop said autoimmunedisease.

The present invention further relates to a method for detecting the above mentioned hnRNP antibodies and the above mentioned combinations in an isolated biological sample, the method comprising the steps of (a) obtaining an isolated biological sample from a patient; and (b) analyzing said isolated biological sample for the presence of antibodies against said hnRNPs.

In particular embodiments of the invention, said method is characterized in that the corresponding hnRNP proteins, peptides, variants or fragments thereof are used for determining the presence of the above mentioned hnRNP antibodies and the above mentioned combinations. In particular embodiments of the invention, said method is characterized in that a hnRNP antigen, more specific an immobilized hnRNP antigen is used for determining the presence of the corresponding hnRNP antibodies.

In particular embodiments of the invention, said analysis or determination of the presence of autoantibodies against the hnRNPs of this invention (including the above mentioned hnRNP antibodies and the above mentioned combinations) is performed in an immunoassay. In particular embodiments of the invention, said immunoassay is selected from the group consisting of ELISA, FEIA, western blot, dot blot, bead-based assay, antigen array and Radio Immuno Assay.

In a particular embodiment of the invention, the methods of the invention are used for predicting responsiveness to a medicament. Another aspect of the present invention relates to the use of hnRNP proteins of this invention (including the above mentioned hnRNPs and the above mentioned combinations), peptides, variants or fragments thereof to detect the presence of autoantibodies against said hnRNPs in an isolated biological sample derived from a subject. In particular embodiments of the invention, the present invention further relates to the use of said hnRNP proteins, peptides, variants or fragments thereof to detect the presence of autoantibodies against said hnRNPs in an isolated biological sample derived from a patient for diagnosing an autoimmune disease, more particularly Sjogren's syndrome. In a particular embodiment of the invention, said hnRNP proteins, peptides, variants or fragments thereof are used for carrying out the methods of the invention. In particular embodiments of the invention, the isolated biological sample is a fluid selected from the group consisting of blood, serum, plasma, saliva, tears, mucus and ascites fluid; and preferentially said biological sample is serum. In another embodiment of the invention, said isolated biological sample is derived from blood, plasma, serum, saliva, tears, lymph, urine, cerebrospinal fluid, any biopsy material or tissue sample, including bone marrow, lymph nodes, nervous tissue, skin, hair, fetal material including amniocentesis material, uterine tissue, faeces or semen. In yet another embodiment of the invention, said isolated biological sample is an antibody-containing isolated biological sample.

Another aspect of the present invention relates to a test kit for detecting the presence of antibodies against the hnRNPs of this invention (including the above mentioned hnRNPs and the above mentioned combinations) in an isolated biological sample. In a particular embodiment, the test kit additionally comprises a solid phase onto which said antigen is or can be bound. In a more particular embodiment of the foregoing, the test kit additionally comprises a labeling group which is bound to said antigen or can be bound thereto. In another embodiment of the foregoing, the test kit additionally comprises at least one other antibody class-specific test reagent. The test kit may additionally comprise other conventional reagents such as buffers, substrates and wetting solutions. The present invention further relates to the use of said test kit for carrying out any of the methods of the invention.

Another aspect of the present invention relates to a method of treating an autoimmune disease, more particularly Sjogren's syndrome, comprising administering to a patient an inhibitor that specifically binds to the hnRNP autoantibodies of said patient, in an amount effective to treat the autoimmune disease, more particularly Sjogren's syndrome. In a particular embodiment, said inhibitor is selected from the group consisting of hnRNP-E1 and hnRNP-F proteins, peptides, variants, fragments, epitopes, or combinations thereof and an antibody, in particular an antibody that recognizes and inhibits said autoantibodies.

The present invention further relates to the use of a compound which specifically binds to at least one group of hnRNP autoantibodies or the combination of certain autoantibodies (as described herein above) for the production of a medicine. In a particular embodiment said medicine is used for the treatment of an auto-immunedisease, more particularly Sjogren's syndrome.

In particular embodiments of the foregoing the subject or the patient is a human being. Said human being can be a patient having Sjogren's syndrome, a patient being suspected as having Sjogren's syndrome or at risk of developing Sjogren's syndrome, or a patient suffering from other autoimmune diseases.

Yet another aspect of the present invention relates to the identification of the combinations of hnRNP-F- and hnRNP-H1 ; and the combination of hnRNP-F, hnRNP-E1 and hnRNP-H1 ; and the combination of hnRNP-A1 and hnRNP-B1 ; and the combination of hnRNP-B1 and hnRNP-K antibodies as valuable diagnostic marker-combinations in dermatomyositis.

Yet another aspect of the present invention relates to the identification of the combination of hnRNP-A1- and hnRNP-B1 -antibodies as valuable diagnostic marker-combinations in scleroderma.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 Amino Acid (AA) sequence alignments of hnRNP-H1 from mouse, rat and humans. The three sequences (numbers from Swiss-Prot Database) Q8VHV7_RAT (SEQ ID NO:1) , HNRH1 HUMAN

(SEQ ID NO:2) and HNRH1_MOUSE (SEQ ID NO:3) show 99 % homology with exclusion of the missing 77 amino acids from hnRNP-H1Q8VHV7 from rat (SEQ ID NO:1). RNA recognition motifs (RRMs) are indicated by arrows above the AA sequence.

Figure 2 Homolgy between hnRNP-H1 and hnRNP-F. The complete amino acid sequences were used to calculate the percentage homology. Some regions with particularly high homology between the proteins are shown. Figure 3 Prevalence of anti-hnRNP-H1 antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-H1-fragment 1. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. The cutoff (black horizontal line) was set at the 97,5 % specificity in CVS (the disease control group), also indicated as series9.

Figure 4 Prevalence of anti-hnRNP-H1 antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-H1-fragment 2. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. The cutoff (black horizontal line) was set at the 97,5 % specificity in CVS (the disease control group), also indicated as series9. Figure 5 Prevalence of anti-hnRNP-H1 antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-H1-fragment

3. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. The cutoff (black horizontal line) was set at the 97,5 % specificity in CVS (the disease control group), also indicated as series9.

Figure 6 Prevalence of anti-hnRNP-H1 antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-H1-fragment

4. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. The cutoff (black horizontal line) was set at the 97,5 % specificity in CVS (the disease control group), also indicated as series9.

Figure 7 Prevalence of anti-hnRNP-A1 antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-A1. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. The cutoff (black horizontal line) was set at the 97,5 % specificity in CVS (the disease control group), also indicated as series9.

Figure 8 Prevalence of anti-hnRNP-B1 antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-B1. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. The cutoff (black horizontal line) was set at the 97,5 % specificity in CVS (the disease control group), also indicated as series9. Figure 9 Prevalence of anti-hnRNP-C1 antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-C1. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. The cutoff (black horizontal line) was set at the 97,5 % specificity in CVS (the disease control group), also indicated as series9.

Figure 10 Prevalence of anti-hnRNP-E1 antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-E1. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. The cutoff (black horizontal line) was set at the 97,5 % specificity in CVS (the disease control group), also indicated as series9.

Figure 11 Prevalence of anti-hnRNP-F antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP^F. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CFS: chronic fatigue syndrome. The cutoff (black horizontal line) was set at the 97,5 % specificity in CFS (the disease control group), also indicated as series9.

Figure 12 Prevalence of anti-hnRNP-Gi antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-Gi. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CFS: chronic fatigue syndrome. The cutoff (black horizontal line) was set at the 97,5 % specificity in CVS (the disease control group), also indicated as series9. Figure 13 Prevalence of anti-hnRNP-H1 antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-H1. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. The cutoff (black horizontal line) was set at the 97,5 % specificity in CVS (the disease control group), also indicated as series9.

Figure 14 Prevalence of anti-hnRNP-l antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-l. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. The cutoff (black horizontal line) was set at the 97,5 % specificity in CVS (the disease control group), also indicated as series9.

Figure 15 Prevalence of anti-hnRNP-K antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-K. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. The cutoff (black horizontal line) was set at the 97,5 % specificity in CVS (the disease control group), also indicated as series9.

Figure 16 Prevalence of anti-hnRNP-P2 antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-P2. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. The cutoff (black horizontal line) was set at the 97,5 % specificity in CVS (the disease control group), also indicated as series9.

Figure 17 Left panel: 10 hnRNP proteins after 1-dimensional gelelectrophoresis and staining with Coomassie Brilliant Blue. Middle and right panel: 10 hnRNP proteins subjected to SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. After incubation of the membrane with patient serum, protein-antibody interactions were visualized by use of 3,3'-Diaminobenzidinetetrahydrochloride. Middle panel: serum from a healthy control with negative to light reactivity to different hnRNP's. Right panel: serum from a patient with primary Sjogren's syndrome with strong reactivity to different hnRNP's. Figure 18 Serum samples obtained from patients with chronic fatigue syndrome (CFS) (n= 106), Sjogren's syndrome (SSp) (n=56 ), systemic sclerosis (n=58), and various autoimmune connective tissue diseases (CTD) [(dermatomyositis (n=29), mixed connective tissue disease (n=19), polymyositis (n=18), systemic lupus erythematosus (n=71), rheumatoid arthritis (n=47)] were tested for reactivity to a recombinant human hnRNP A1 , B1 , C1 , and E1 by Western blot analysis. The figure shows the peak x area value as measured by densitometry for several patient groups. The figure shows box plots, from the first to the third quartile, with the addition of a notched section for the confidence interval around the median. The whiskers extend to the furthest observations within +1.5 interquartile ranges (IQR) of the 1st or 3rd quartile. Observations outside 1.5 IQRs are marked as (+), and those outside 3.0 IQRs are marked as C).

Figure 19 Serum samples obtained from controls (n= 106 ), Sjogren's syndrome (SSp) (n=56), systemic sclerosis (n=58), and various autoimmune connective tissue diseases (CTD) [(dermatomyositis (n=29), mixed connective tissue disease (n=19), polymyositis (n=18), systemic lupus erythematosus (n=71 ), rheumatoid arthritis (n=47)] were tested for reactivity to a recombinant human hnRNP F, H1 , 1, and K by Western blot analysis. The figure shows the peak x area value as measured by densitometry for several patient groups. The figure shows box plots, from the first to the third quartile, with the addition of a notched section for the confidence interval around the median. The whiskers extend to the furthest observations within ±1.5 interquartile ranges (IQR) of the 1st or 3rd quartile. Observations outside 1.5 IQRs are marked as (+), and those outside 3.0 IQRs are marked as (*). Figure 20 Serum samples obtained from controls (n= 89 ), blood donors (n=82), and diagnostic serum samples from patients with various connective tissue diseases [systemic sclerosis (SSc) (n=65), polymyositis (PM) (n=11 ), dermatomyositis (DM) (n=22), rheumatoid arthritis (RA) (n=23), systemic lupus erythematosus (SLE) (n=62), mixed connective tissue disease (MCTD) (n=11), and Sjogren's syndrome (SS) (n=34)] were tested for reactivity to recombinant human hnRNP B1 and hnRNP E1 by ELISA. The figure shows box plots, from the first to the third quartile, with the addition of a notched section for the confidence interval around the median. The whiskers extend to the furthest observations within ±1.5 interquartile ranges (IQR) of the 1st or 3rd quartile. Observations outside 1.5 IQRs are marked as (+), and those outside 3.0 IQRs are marked as (*). Figure 21 Serum samples obtained from controls (n= 89 ), blood donors (n=82), and diagnostic serum samples from patients with various connective tissue diseases [systemic sclerosis (SSc) (n=65), polymyositis (PM) (n=11), dermatomyositis (DM) (n=22), rheumatoid arthritis (RA) (n=23), systemic lupus erythematosus (SLE) (n=62), mixed connective tissue disease (MCTD) (n=11 ), and Sjogren's syndrome (SS) (n=34)] were tested for reactivity to recombinant human hnRNP F and hnRNP H1 by ELISA. The figure shows box plots, from the first to the third quartile, with the addition of a notched section for the confidence interval around the median. The whiskers extend to the furthest observations within ±1.5 interquartile ranges (IQR) of the 1st or 3rd quartile. Observations outside 1.5 IQRs are marked as (+), and those outside 3.0 IQRs are marked as (*). DETAILED DESCRIPTION OF THE INVENTION

Detailed description of embodiments of the invention

In a preferred embodiment, the invention provides a method of detecting anti-hnRNP-E1 and/or anti- hnRNP-F antibodies in a patient suffering from or susceptible to an autoimmune disease such as Sjogren's syndrome. In another preferred embodiment said method additionally detects anti-hnRNP-H1 antibodies in said patient. This combined presence of anti-hnRNP-H1 , anti-hnRNP-E1 , and anti-hnRNP-F antibodies; and combinations of these antibodies with anti-hnRNP-B1 antibodies (example given anti- hnRNP-H1 and -B1 ; anti-hnRNP-E1 and -B1 ; anti-hnRNP-H1 , -B1 and -E1 ; and anti-hnRNP-H1 , -F, -B1 and-E1) are highly specific for SSp and to a lesser extent for MCTD, SLE and DM. Therefore, other embodiments provide methods of detecting combinations of antibodies in patients suffering from or susceptible to an autoimmune disease such as Sjogren's syndrome, systemic lupus erythematosus, mixed connective tissue disease, scleroderma, dermatomyositis, polymyositis, and rheumatoid arthritis; some of the most relevant combinations associated with a certain autoimmune disease are depicted in grey in Table 4 in Example 2. Preferably, the method of detecting anti-hnRNP antibodies of the invention (such as anti-hnRNP-F antibodies) includes the step of analyzing a biological sample for the presence of an these antibodies that specifically binds to the specific hnRNP of this invention (such as hnRNP-F). Any suitable biological sample might be used in the method. For example, a biological sample that would normally be expected to contain immunoglobulins might be used. Typically, the sample would take the form of a bodily fluid, such as blood (and fractions thereof, such as serum or plasma), saliva, tears, mucus, and the like. Because immunoassays are known to generally work well with blood or blood fractions, these are preferred. Biological samples can be collected from a subject by any suitable method. For example, a blood sample can be collected using conventional phlebotomy procedures; a saliva sample can be collected by spitting or merely by placing a stick in the mouth and is the preferred patient sample. Blood samples can be used for verification of detection of autoantibodies.

A subject from which a biological sample can be obtained for analysis according to the invention is an animal such as a mammal, e.g. a dog, cat, horse, cow, pig, sheep, goat, primate, rat, or mouse. A preferred subject is a human being, particularly a patient suspected of having or at risk for developing an autoimmune disorder such as Sjogren's syndrome (e.g. an individual suffering from dry eye and/or dry mouth), or a patient with a connective tissue disease (e.g. an individual diagnosed with SLE, rheumatoid arthritis, or scleroderma).

In a preferred embodiment, analysis of a biological sample for the presence of antibodies that specifically binds to the specific hnRNPs of this invention (such as hnRNP-F and hnRNP-H1), agents to which these anti-hnRNP antibodies specifically bind are used. Such agents might include a native (i.e. naturally occurring) hnRNP (such as hnRNP-F and hnRNP-H1) or fragments (such as fragment 3 and 4 of hnRNP- H1 , as described in Example2), mutants or variants thereof; or a non-hnRNP test reagent such as an anti- idiotypic antibody. A number of different native hnRNPs have been characterized (e.g. amino acid sequenced), for hnRNP-H1 these include those from human, mouse and rat. Generally, those hnRNPs from the same species as the biological sample are preferred for particular variations of the method of the invention. Nonetheless, due to cross-reactivity, non-species matched assays may also be used. For example, rat hnRNP-H1 can be used to detect human anti-hnRNP-H1 antibodies.

To analyze a biological sample for the presence of an anti-hnRNP antibody of this invention (such as anti- hnRNP-F), anti-idiotypic anti-hnRNP (such as anti-hnRNP-F) or hnRNP (such as hnRNP-F) proteins, peptides, variants or fragments thereof, a test reagent that specifically binds to these proteins, a number of different methods might be used. In general, the sample, or a purified portion thereof, is contacted with the agent under conditions that allow agent-antibody binding. The presence of the formed agent-antibody complex is then detected as an indication that the sample contains an anti-hnRNP antibody (such as anti- hnRNP-F). Methods for detecting antigen (the agent is the antigen in this case)-antibody complexes are well known in the art of immunology, and include techniques that utilize hnRNP-expressing cells (such as hnRNP-H1 expressing cells for detecting anti-hnRNP-H1 antibodies) as well as non-cellular methods. Cell-based detection methods include techniques such as immunohistochemistry, immunofluorescence microscopy, or flow cytometric analysis. Non-cell based assays include immunosorbent assays (e.g. ELISA and RIA) and immunoprecipitation assays. As one example, the hnRNP of this invention (e.g. hnRNP-H1) is immobilized on a substrate, a human serum sample is placed on the substrate under conditions that would allow binding of said anti-hnRNP antibodies (e.g. anti-hnRNP-H1 antibodies) to the immobilized hnRNP (e.g. hnRNP-H1). After washing, detectably labeled secondary antibody (e.g. an anti- human immunoglobulin antibody if the biological sample was derived from a human subject) is added to the substrate. The presence of detectable label (e.g. an enzyme, fluorophore, or radioisotope) on the substrate after washing indicates that the sample contained said anti-hnRNP (e.g. anti-hnRNP-H1) antibodies. As another example, antibodies contained within a biological sample are immobilized on a substrate, a detectably labeled hnRNP such as hnRNP-H1 is then placed on the substrate under conditions that would allow binding of the immobilized anti-hnRNP antibodies such as anti-hnRNP-H1 antibodies to said hnRNP such as hnRNP-H1. The presence of detectable label remaining on the substrate after washing indicates that the sample contained these anti-hnRNP antibodies, such as anti- hnRNP-H1 antibodies.

The method of the invention can be carried out as qualitative or quantitative determination. In a qualitative determination, the antibody concentrations which are above the so-called cutoff value are classified as positive. The cutoff value can be determined by calibrating the test system with positive and negative control samples. Alternatively, it is also possible to carry out a quantitative determination.

The specific test format is not in general critical. However, a test format can be preferred in which an immune complex comprising a hnRNP-antigen and the hnRNP autoantibody to be detected is bound to a solid phase. Alternatively, a competitive test format can be envisaged. Solid phases which can be employed are reaction vessels, microtiter plates, beads, biochips etc. The antigen can be immobilized on the solid phase by adsorptive interactions, covalent bonding or mediated by a high-affinity binding pair (streptavidin/biotin, hapten/anti-hapten antibody). The immobilized test reagent can be employed in a form which is already bound to a solid phase or else be immobilized only during the test.

The method can be carried out as liquid test (e.g. in a reaction vessel) or else as dry test (e.g. on a test strip).

The labeled test reagent may itself have a detectable or signal-emitting group (direct labeling) or be capable of binding to a detectable group (indirect labeling). The labeling group can be selected as desired from all labeling groups known from the prior art for immunological detection methods, for example from enzymes, metal particles or latex particles, and luminescent or fluorescent groups. It is particularly preferred for the labeling group to be selected from enzymes, e.g. peroxidase, β-galactosidase or alkaline phosphatase, and for the method to be carried out in the ELISA format.

Immunological methods to detect anti-hnRNP antibodies of the invention such as anti-hnRNP-F antibodies

The next assays are described with the hnRNP-F antigen/antibody as an example for all other antibodies/antigens and antibody/antigen combinations of this invention.

Single assays:

Dot blot assay Purified or recombinant hnRNP-F-antigen is spotted on a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. The membrane strips are firstly blocked with blocking reagent. After a washing step to remove unbound proteins, they are incubated with diluted serum of the patient, a positive and a negative control serum. They are further washed with washing buffer and treated with alkaline phosphatase labeled protein A conjugate to bind the captured antibodies. After a final washing step, 5- bromo-4-chloro-3-indolylphosphate/Nitro Blue Tetrazolium (BCIP/NBT) substrate is added to give a blue colored spot confirming the presence of anti-hnRNP-F antibodies. Dot blots assays are a qualitative evaluation method. Several antigens can be tested simultaneously by this method. Western blot

In comparison with the dot blot assay, the purified or recombinant hnRNP-F-antigen is separated on a one dimensional sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) prior to blotting on a membrane. After electro-transfer of the separated protein on a nitrocellulose or PVDF membrane, the membrane strip is blocked in blocking buffer (BSA or skim milk powder). The membrane is subsequently treated with diluted serum of the patient, anti-human IgG conjugated with horseradish peroxidase (HRP) or a fluorescent dye and a substrate to visualize the bound antibodies. Intermittent washing steps will remove the unbound proteins.

This Western blot assay can be used in combination with other proteins of different molecular weight loaded in the same lane on SDS-PAGE (cfr. line assay). This technique is labor intensive but permits to screen the sera simultaneously for the presence of different auto-antibodies. It's a qualitative method.

ELISA

An Enzyme Linked Immunosorbent Assay (ELISA) provides a quantitative in vitro assay for the detection of antibodies. Polystyrene microplate strips are coated with purified, biochemically characterized pure or recombinant hnRNP-F-antigen as solid phase. If the sample is positive, specific antibodies in the diluted serum sample attach to the antigens coupled to the solid phase. In a second step, the attached antibodies are detected with peroxidase-labeled anti-human antibodies. In a third step, the bound antibodies are visualized using a chromogen/substrate solution which is capable of promoting a color reaction. The intensity of the color produced is proportional to the concentration of antibodies in the serum sample and is measured using a spectrophotometer with filter settings of a suitable wavelength.

FEIA

A Fluoro Enzyme Immuno Assay (FEIA) provides a quantitative in vitro assay for the detection of antibodies. A carier (e.g. polystyrene microplate strips) is coated with purified, biochemically characterized pure or recombinant antigen (e.g. hnRNP-F-antigen) as solid phase. If the sample is positive, specific antibodies in the diluted serum sample attach to the antigens coupled to the solid phase. In a second step, the attached antibodies are detected with an enzyme-labeled anti-human antibodies. In a third step, the bound antibodies are visualized using a substrate solution which is capable of promoting a color reaction. The intensity of the color/fluorescence produced is proportional to the concentration of antibodies in the serum sample and is measured using a spectrophotometer with filter settings of a suitable wavelength. The fluorescence of the serum sample is than compared to a standard curve. Radio Immuno Assay (RIA)

Radio Immuno assays are immuno assays in which radioactive isotopes are used as labels for detection. These assays use gamma and beta counters for detection and are highly sensitive. The greatest disadvantage of radio immuno assays is that the amount of use and the kind of radioisotopes are limited by regulations and radioactive wastes are released in large amounts. Therefore, the RIA is becoming less popular as an immunoassay.

Multiplexed immunological assays:

Multiplex platforms allow to screen for autoantibody profiles in human serum samples. In this multiprotein assays, the presence of hnRNP-F antibodies is tested simultaneously with other anti- ENA antibodies and quantitatively expressed.

Bead-based assays: Human serum is treated with different sets of color-coded polystyrene beads, each bearing a different antigen (including hnRNP-F). Individual beads bearing interacting antibodies are detected by flow cytometry. The beads are aligned into single files as they enter a stream of sheath fluid and then enter a flow cell. Once the beads are in single file within the flow cell, each bead is individually interrogated for bead color (analyte) and assay signal strength (fluorescence intensity). This is a fast, reproducible and reliable but expensive technique. Only a limited number of antigens can be screened.

Antigen anray: Several antigens, including hnRNP-F, are printed on nitrocellulose or superaldehyde coated slides. Printed microarray chips are then washed in wash buffer and probed with diluted patient's serum. Afterwards, fluorescently labeled anti-human IgG conjugate is added and the slides are washed again. The fluorescent signal is further quantified by scanning and image analysis software. These antigen arrays allow to simultaneously screen for the presence of an unlimited amount of autoantibodies.

Anti-auto-hnRNP antibodies of the invention (such as anti-auto-hnRNP-F antibodies) for therapy

The next (therapy) section is described with the hnRNP-F antigen/antibody as an example for all other antibodies/antigens and antibody/antigen combinations of this invention:

Anti-autoimmune antibodies or inhibitor(s) of the present invention include polypeptides comprising the epitope of the antibody or biologically active fragment thereof, or polypeptide that is functional in conferring protection in the individual suffering from autoimmune disease, or functionally conserved fragments or amino acid variants thereof. Identification of the epitope is a matter of routine experimentation. Most typically, one would conduct systematic substitutional mutagenesis of the compound molecule while observing for reductions or elimination of cytoprotective or neutralizing activity. In any case, it will be appreciated that due to the size of many of the antibodies, most substitutions will have little effect on binding activity. The great majority of variants will possess at least some cytoprotective or neutralizing activity, particularly if the substitution is conservative. Conservative amino acid substitutions are substitutions from the same class, defined as acidic (Asp, Glu), hydroxy-like (Cys, Ser, The), amides (Asn, Gin), basic (His, Lys, Arg), aliphatic-like (Met, He, Leu, Val, Gly, Ala, Pro), and aromatic (Phe, Tyr, Trp).

Homologous antibody or polypeptide sequences generally will be greater than about 30 percent homologous on an identical amino acid basis, ignoring for the purposes of determining homology any insertions or deletions from the selected molecule in relation to its native sequence. The compounds discussed herein, i.e. autoimmune inhibitors for administration to the patient with autoimmune disease and/or for removal, neutralization or inhibition of the autoimmunogen(s) by extracorporeal immunosorption in accordance with the present invention, also include glycosylation variants as well as unglycosylated forms of the agents, fusions of the agents with heterologous polypeptides, and biologically active fragments of the agents, again so long as the variants possess the requisite neutralizing or cytoprotective activity.

In an embodiment of the invention, treatments involving administration of an autoimmune inhibitor to a patient, and treatments involving the extracorporeal exposure of the patient's fluid to an autoimmune inhibitor, may be performed alone or in combination. Administered autoimmune inhibitor of the invention binds to, neutralizes and/or inhibits the molecule(s) associated with or causing the autoimmune response in the patient. More specifically, administration of the autoimmune inhibitor to a patient results in suppression of pathological humoral and adaptive immunity in the patient. In other words, in accordance with the method of the present invention, treatment of a patient with the autoimmune inhibitor causes the humoral and adaptive immune response of the patient to be inhibited or neutralized over that which was, or would have been, present in the absence of treatment.

A patient is in need of treatment with an autoimmune inhibitor, when the patient is suffering from an autoimmune disease or when the patient has produced autoantibodies.

The autoimmune inhibitor antibody(ies) is also effective when immobilized on a solid support. Examples of such solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art(Weir et a/., "Handbook of experimental Immunology" 4th Ed., Blackwell Scientific Publications, Oxford, England, Chap. 10 (1986); Jacoby et a/., Meth. Enzym. 34 Academic Press, N.Y.(1974)). For therapeutic purposes, autoantibody gene products may be generated which include proteins that represent functionally equivalent gene products. For example, an equivalent hnRNP-F antibody gene product may contain deletions, including internal deletions, additions, including additions yielding fusion proteins, or substitutions of amino acid residues, but that result in a "silent" change, in that the change produces a functionally equivalent auto-hnRNP-F antibody gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Alternatively, where alteration of function is desired, deletion or non-conservative alterations can be engineered to produce altered anti-hnRNP-F antibody gene products. Such alterations can, for example, alter one or more of the biological functions of the autoantibody gene product. Further, such alterations can be selected so as to generate autoantibody gene products that are better suited for expression, scale up, etc. in the host cells chosen. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges. This applies to any autoimmune hnRNP-F molecule and allelic variants thereof, that are identified in an individual.

The autoantibody gene products, peptide fragments thereof and fusion proteins thereof, of the invention can be produced by recombinant DNA technology using techniques well known in the art. Methods that are well known to those skilled in the art can be used to construct expression vectors comprising auto- hnRNP-F antibody gene product coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook, et at., 1989, and Ausubel, et at., 1989. Alternatively, RNA capable of encoding auto-hnRNP-F antibody gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, ed., IRL Press, Oxford.

A variety of host-expression vector systems may be utilized to express the autoantibodies, such as anti- hnRNP-F gene products. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells that may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the anti- hnRNP-F gene product of the invention in situ. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner, er a/., 1987, Methods in Enzymol. 153, 516-544).

In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g. glycosylation) and processing (e.g. cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, JURKAT, Hep2, VERO, BHK, HeLa, COS, MDGK, 293, 3T3, and W138. A variety of methods can be employed for the diagnostic and prognostic evaluation autoimmune disorders such as Sjogren's syndrome and for the identification of subjects having a predisposition to such autoimmune disorders. Such methods may, for example, detect the presence of hnRNP (such as hnRNP- F) gene mutations, or the detection of either over- under- or no expression of hnRNP (such as hnRNP- F) protein, or mutants.

Mutations at a number of different genetic loci may lead to phenotypes related to autoimmune disorder, structural and synaptic abnormalities. Ideally, the treatment of patients suffering from such disorders will be designed to target the particular genetic loci comprising the mutation mediating the disorder. Genetic polymorphisms have been linked to differences in drug effectiveness. Thus, identification of alterations in hnRNP (such as hnRNP-F) molecules, such as, for example, gene or protein can be utilized to optimize therapeutic drug treatments.

In a preferred embodiment, autoimmune related molecule, such as, for example, hnRNP-F, expression levels, mutations, polymorphisms can be detected by using a microassay of for example, hnRNP-F nucleic acid sequences immobilized to a substrate or "gene chip" for detection of hnRNP-F molecules (see, e.g. Cronin, ef a/., 1996, Human Mutation 7:244-255). Preferred methods are detailed in the examples which follow.

The level of hnRNP (e.g. hnRNP-F) or any hnRNP(e.g.-F)-related molecule gene expression, can also be assayed as described in detail in the examples which follow. Additionally, it is possible to perform hnRNP (e.g. hnRNP-F ) gene expression assays "in situ", i.e. directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. For such in situ procedures (see, for example, Nuovo, G. J., 1992, "PCR In Situ Hybridization: Protocols And Applications", Raven Press, N.Y.) standard northern analysis can be performed to determine the level of mRNA expression of the hnRNP gene (e.g. the hnRNP-F gene).

To assess the efficacy of cell-based gene therapy, in vitro immunoassays can be used. Antibodies directed against hnRNP (such as hnRNP-F) gene products may be used in vitro to determine, for example, the level of hnRNP (such as hnRNP-F) antibody gene expression achieved in cells genetically engineered to produce such a gene product. In the case of intracellular gene products, such an assessment is done, preferably, using cell lysates or extracts. Such analysis will allow for a determination of the number of transformed cells necessary to achieve therapeutic efficacy in vivo, as well as optimization of the gene replacement protocol.

The tissue or cell type to be analyzed will generally include those that are known, or suspected, to express the hnRNP gene (e.g. the hnRNP-F gene). The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the hnRNP gene (e.g. the hnRNP-F gene). Preferred diagnostic methods for the detection of autoimmune molecules, such as, for example, hnRNP-F gene products, conserved variants or peptide fragments thereof, may involve, for example, immunoassays wherein the hnRNP-F gene products or conserved variants or peptide fragments are detected by their interaction with an anti-hnRNP-F gene product-specific antibody. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.

Definitions

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

The term "subject" or "patient" refers to any human or animal mammals.

The term "autoimmune disease" (AD) refers to a disease that result from an aberrant immune response of an organism against its own cells and tissues due to a failure of the organism to recognise its own constituent parts (down to the sub-molecular level) as "self. As used herein, "autoimmune disease" is intended to further include autoimmune conditions, syndromes and the like. Examples of autoimmune diseases include, but are not limiting to, Sjogren syndrome, scleroderma, dermatomyositis, polymyositis, rheumatoid arthritis, mixed connective tissue disease, insulin-dependent diabetes mellitus, hemolytic anemias, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, and systemic lupus erythematosus.

The term "antigen" as used herein refers to a structure of a macromolecule, typically protein (with or without polysaccharides) or made of proteic composition comprising one or more hapten(s) and/or comprising at least one epitope. An "autoantigen" as used herein refers to a human or animal protein present in the body, which elicits an immune response within the same human or animal body, which may in turn lead to a chain of events, including the synthesis of other autoantigens or autoantibodies. An "autoantibody" is an antibody (ab) produced by an autoimmune patient to one or more of his own constituents which are perceived to be antigenic. For example, in SLE autoantibodies are produced to DNA, while in many other types of AD autoantibodies are produced to target cells.

Patients suffering from autoimmune diseases including, e.g., rheumatoid arthritis, insulin-dependent diabetes mellitus, hemolytic anemias, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, systemic lupus erythematosus and others, are in need of treatment in accordance with the present invention. Treatment of patients suffering from these diseases by administration of autoimmune inhibitor and/or removal of compound(s) by extracorporeal immunosorption in accordance with the present invention will alleviate the clinical manifestations of the disease and/or minimize or prevent further deterioration or worsening of the patient's condition. Treatment of a patient at an early stage of an autoimmune disease including, e.g., rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, or others, will minimize or eliminate deterioration of the disease state into a more serious condition.

The term "epitope" refers to one or several portions (which may define a conformational epitope) of an antigenic protein which is/are specifically recognised and bound by an antibody or a portion thereof (Fab', Fab2", etc.) or a receptor presented at the cell surface of a B or T cell lymphocyte, and which is able, by said binding, to induce an immune response. An epitope can comprise as few as 3 amino acids in a spatial conformation which is unique to the epitope. Generally an epitope consists of at least 6 such amino acids, and more usually at least 8-10 such amino acids. Methods for determining the amino acids which make up an epitope include x-ray crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping e.g. the Pepscan method described by H. Mario Geysen et al. 1984. Proc. Natl. Acad. Sci. U.S.A. 81 :3998-4002; PCT Publication No. WO 84/03564; and PCT Publication No. WO 84/03506. It is well known to a person skilled in the art that antibodies which recognize and bind an epitope can be monoclonal or polyclonal.

The phrase "specifically (or selectively) binds" to an antibody, when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to marker "X" from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with marker "X" and not with other proteins, except for polymorphic variants and alleles of marker "X". This selection may be achieved by subtracting out antibodies that cross-react with marker "X" molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background. "Immunoassay" is an assay that uses an antibody to specifically bind an antigen (e.g. a marker). The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.

"Diagnostic" means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay, are termed "true negatives." The "specificity" of a diagnostic assay is 1 (or 100 when indicated as percentage) minus the (percentage) false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. As used herein, "molecule" is used generically to encompass any vector, antibody, protein, drug and the like which are used in therapy and can be detected in a patient by the methods of the invention. For example, multiple different types of nucleic acid delivery vectors encoding different types of genes which may act together to promote a therapeutic effect, or to increase the efficacy or selectivity of gene transfer and/or gene expression in a cell. The nucleic acid delivery vector may be provided as naked nucleic acids or in a delivery vehicle associated with one or more molecules for facilitating entry of a nucleic acid into a cell. Suitable delivery vehicles include, but are not limited to: liposomal formulations, polypeptides; polysaccharides; lipopolysaccharides, viral formulations (e.g. including viruses, viral particles, artificial viral envelopes and the like), cell delivery vehicles, and the like. Detailed description

We identified several hnRNPs as new target of auto-antibodies in patients with autoimmune diseaes such as Sjogren's syndrome. Antibodies to hnRNP-AI , -B1 , -C1 , -El , -F , -H1 , -I, -K, and -P2 were all significantly detected in patients with primary Sjogren's syndrome (see Table 1 and 2 in Example 2). Certain combinations of these hnRNP-markers are extremely relevant for the diagnosis of autoimmune diseaes such as Sjogren's syndrome. These combinations comprise the ones depicted in grey background color in Table 4. Preferred combinations of hnRNP-markers to detect Sjogren's syndrome are 'hnRNP-F and -H1 ', 'hnRNP-E1 , and -F', 'hnRNP-E1 , -F, and -B1', 'hnRNP-E1 , -F, -H1 , and -B1 ' 'hnRNP-A1 , -B1 , -F, and -H1\ 'hnRNP-E1 , -F, and -H1', and 'hnRNP-A1 , -B1 , -E1 , -F, and -HT.

To a lesser extend the combination 'hnRNP-EI , -F, and -H1' occurs in patients suffering from dermatomyositis.mixed connective tissue disease and SLE. Therefore, a method comprising the analysis of this combination of markers (hnRNP-E1 , -F, and -H1 ) can be used to diagnose patients for Sjogren's syndrome, mixed connective tissue disease (MCTD), dermatomyositis (DM) and SLE.

The combinations 'hnRNP-BI and -K', 'hnRNP-F and -H1', and 'hnRNP-B1 and -A1' do significantly occur in patients with DM. Therefore a method comprising the analysis of the combination of these sets of markers (hnRNP-B1 and -K', 'hnRNP-F and -H1', or 'hnRNP-B1 and -A1) can be used to diagnose patients for DM.

The combination 'hnRNP-B1 and -A1' does significantly occur in patients with scleroderma (SSc).

Therefore, a method comprising the analysis of this combination of markers (hnRNP-B1 and -A1) can be used to diagnose patients for SSc.

Patients with SSp had significantly high antibodies to all hnRNPs tested (except for hnRNP-Gi).

Particularly, single antibodies to hnRNP-A1 , -B1 , -E1 , -F, and -H1 are detected in more than 37% of the

SSp patients. Therefore, all combinations of these 5 markers, more particularly the combinations 'hnRNP-

F and -HT; ^* hnRNP-E1 , and -F'; 'hnRNP-E1, -F, and -B1'; 'hnRNP-EI , -F, -H1, and -BT; 'hnRNP-AI , - B1 , -F, and -HT; 'hnRNP-E1 , -F, and -HT; and 'hnRNP-A1 , -B1 , -E1 , -F, and -HT, are useful in methods for diagnosing Sjogren's syndrome.

In particular, fragments of hnRNP-H1 were analysed as markers for several autoimmuun diseases. We clearly show that fragments 1 , 2, 3 and 4 of hnRNP-H1 (SEQ ID 4 to 7, see Example 2) are useful as a marker for Sjogren's syndrome. Fragments 2, 3 and 4 show the highest positive values for Sjogren's syndrome in Table 1 indicating that most antibodies to hnRNP-H1 in Sjogren's syndrome patients recognize these fragments. Moreover, fragments 2 and 4 of hnRNP-H1 (covering RRM2 and RRM3 of hnRNP-H1 respectively) are extremely homologue to certain regions (the RRM1 en 2 regions respectively) in hnRNP-F (as shown in Fig 2), indicating that these hnRNP-H1 and -F markers and more particularly the aforementioned regions/fragments or amino acid sequences that are higly homologues to these regions/fragments (preferably more than 80, 85, 90, or 95 % homologues to the aforementioned regions/fragments in hn NP-H1 or -F) are good candidate markers for Sjogren's syndrome. Particularly fragment 4 (SEQ ID N07) or amino acid sequences that are higly homologues to this fragment (preferably more than 80, 85, 90, or 95 % homolgy) are good candidate markers for autoimmune diseases, in particular Sjogren's syndrome.

Using Western blotting, we confirmed previous observations that patients with systemic lupus erythematosus, rheumatoid arthritis, and mixed connective tissue disease have higher reactivity to hnRNP B1 than controls (Steiner). We also confirmed our previous observation (WO2009055880) that patients with Sjogren's syndrome have higher reactivity to hnRNP-H1 than controls. A striking finding was that patients with Sjogren's syndrome displayed reactivity to 9 of the 10 hnRNP's tested. Patients with Sjogren's syndrome particularly had antibodies to hnRNP-B1 , hnRNP-E1 , hnRNP-F and hnRNP-H1. Another striking finding was that the simultaneous presence of antibodies to several hnRNP's was strongly associated with Sjogren's syndrome and with some other systemic rheumatic diseases. For example, antibodies to at least 5 of the 10 antigens tested were found in none of the controls, but in 32% of Sjogren's syndrome, 16% of mixed connective tissue disease, 7% of systemic lupus erythematosus, 14% of dermatomyositis, and 7% of systemic sclerosis. The simultaneous presence of antibodies to at least 7 of the 10 antigens was found in 23% of Sjogren's syndrome and in 10% of dermatomyositis.

The high reactivity to hnRNP-B1 , hnRNP-E1 , and hnRNP-F in Sjogren's syndrome was confirmed by ELISA. ELISA, however, did not reveal significantly higher reactivity for hnRNP-H1 in Sjogren's syndrome than in controls. This might be related to differences in conformation of the protein between ELISA and Western blotting. Antibodies to hnRNP B1 were found in Sjogren's syndrome and in several other connective tissue diseases including mixed connective tissue disease and systemic lupus erythematosus. ELISA analysis further confirmed the notion that the simultaneous presence of antibodies to several hnRNPs was strongly associated with connective tissue disease in general and Sjogren's syndrome in particular. For example, reactivity to at least 2 of the 4 tested antigens was found in 1.1 % of controls, in 16% of patients with systemic lupus erythematosus, and in 18% of patients with Sjogren's syndrome. Reactivity to at least 3 of the 4 antigens was found in none of the controls and in 15% of patients with Sjogren's syndrome.

ELISA analyses were done on diagnostic samples (i.e. samples that were obtained at the time of diagnosis). We have data that there are patients that test positive for anti-hnRNP antibodies and negative for antibodies to the classical extractable nuclear antigens or dsDNA. For example, of the 34 samples from patients with Sjogren's syndrome, 19 tested positive for at least 1 of the four antigens tested (hnRNP-B1 , -E1 , -F and -H1 ). Two of these patients were negative for anti-SSA or anti-SSB antibodies. We also identified diagnostic samples from patients with systemic lupus erythematosus and with systemic sclerosis that were negative for the classical antibodies to extractable nuclear antigens and to dsDNA but positive for antibodies to hnRNPs.

Between the ten hnRNPs studied, the highest identity was found between hnRNP A1 an hnRNP B1 and between hnRNP-F and hnRNP-H1. After manual alignment (www.ncbi.nlm.nih.gov), hnRNP-A1 and -B1 showed 60.5% identity. Both proteins have two moderate homologous RRM's and a glycine rich region. HnRNP-H1 and -F have an identity of 78%. Even though the proteins are closely sequence-related, they exhibit differences, especially in their COOH terminus, where hnRNP-F is shorter and contains a deletion when compared with hnRNP-H1. These differences occur outside the RRM regions. Therefore hnRNP-F and -H1 are highly related immunologically.

In conclusion, several hnRNP's are target antigens in Sjogren's syndrome. The combined presence of antibodies to several hnRNP's was strongly associated with connective tissue disease in general and Sjogren's syndrome in particular.

In the present invention we described several new hnRNP's (the most important are hnRNP-E1 and hnRNP-F) as new targets of autoantibodies in patients with an autoimmune diseases, in particular Sjogren's syndrome. Therefore, we identified these antibodies and combinations of these antibodies as valuable diagnostic markers in autoimmune diseases, in particular Sjogren's syndrome.

The present invention will now be illustrated by means of the following examples which are provided without any limiting intention.

Examples

EXAMPLE 1 : Materials and Methods

Study population

Two groups of serum samples were included in the study.

The first group consisted of samples obtained from well-defined patients with an autoimmune connective tissue disease (n=298). This group included patients with systemic lupus erythematosus (n=71 , male/female ratio 10:61 , mean age 48.9 years, range 18-80 years), primary Sjogren's syndrome (n=56, male/female ratio 1:13, mean age 57.9 years, range 23-83 years), scleroderma (n=58 , male/female ratio 19:39, mean age 56.7 years, range 21-74 years), dermatomyositis (n=29 , male/female ratio 12:17, mean age 52 years, range 25-82 years), polymyositis (n=18 , male/female ratio 1 :5, mean age 64 years, range 49-83 years), rheumatoid arthritis (RA) (n=47 , male/female ratio 13:34, mean age 59.6 years, range 35- 82 years), and mixed connective tissue disease (n=19 , male/female ratio 5:14, mean age 48.4 years, range 20-72 years). Patients with chronic fatigue syndrome (CFS or CVS) (n=106, male/female ratio 10:43, mean age 40 years, range 18-75 years) served as controls. In these patients organic disease (such as connective tissue disorders) was excluded.

The second group of samples consisted of diagnostic samples (i.e. samples obtained at the time of diagnosis). This group included patients with systemic lupus erythematosus (n=62 , male/female ratio 11 :51, mean age 42.7 years, range 15-73 years), primary Sjogren's syndrome (n=34 , male/female ratio 5:29, mean age 49.9 years, range 21-73 years), scleroderma (n= 65, male/female ratio 17:48, mean age 51.4 years, range 17-75 years), dermatomyositis (n=22, male/female ratio 9:13, mean age 48.7 years, range 24 -77 years), polymyositis (n=11 , male/female ratio 2:9, mean age 59.5 years, range 43-78 years), rheumatoid arthritis (RA) (n=23 , male/female ratio 7:16, mean age 51.6 years, range 26-74 years), and mixed connective tissue disease (n= 11 , male/female ratio 3:8, mean age 40.4 years, range 16-66 years). Patients with chronic fatigue syndrome (CFS or CVS) (n=89, male/female ratio 14:75, mean age 40.7 years, range 20-72 years) served as controls. In these patients all organic disease (such as connective tissue disorders) was excluded. Serum samples from blood donors were also included (n= 82 , male/female ratio 42:40, mean age 45 years/range 22-61 years).

All patients with Sjogren's syndrome had disease characteristics that conformed with the American- European consensus classification criteria (15). Patients with SLE, scleroderma, and RA met the classification criteria of the American College of Rheumatology (16r18). Patients with poly- and dermatomyositis met the criteria of Bohan and Peter (19), and patients with MCTD met the criteria of Alarcon-Segovia (20). The serum samples that were used for this study were from the serum data bank. Samples were obtained from patients as part of routine screening for autoantibodies in the clinical laboratory. There was no informed consent for this study, but the study was approved by the local ethics committee.

Preparation of recombinant hnRNP's

cDNA of hnRNP A1 , H1 , F and I (obtained from Douglas Black, Howard Hughes Medical Institute, LA), hnRNP P2 (obtained from Takumi, Osaka Bioscience, Institute Japan), hnRNP E1 en K (obtained from Kent Duncan, University of Berlin), hnRNP G (obtained from Chiou, National Chung Hsing University, Taiwan) and hnRNP C1 (obtained from Chang, National Taiwan University, Taiwan) was used.

cDNA of hnRNP B1 was constructed by isolation of RNA out of HeLa cells by means of the RNeasy mini kit (Qiagen). RNA was converted to cDNA using the RevertAid H Minus First Strand synthesis kit (Fermentas).

Cloning of the PCR products was performed using the recombination-based Gateway technology (Invitrogen) according to the suppliers' instructions. The PCR product was first cloned in the plasmid pDONR-221 using a BP clonase reaction and subsequently in the expression plasmid pDEST-17 by a LR clonase reaction. The gene was extended with a hexahistidine (6x His) coding sequence at its 5' end upon cloning in the pDEST-17 vector. Thereafter, expression was induced in E. coli BL21-AI with 0.1 mM IPTG and 0.1% L-arabinose for 4 h at 37°C and the protein was purified from inclusion bodies using a ΝΪ2+-ΝΤΑ affinity column. The gateway-adapted primers used for cloning are given in Table 12.

Protein identification by MALDi-TOF/TOF

Gel pieces containing the protein of interest were washed with HPLC-grade water, dried in a Speed Vac (Savant), and digested overnight at 37 °C with 10 pL of 25 mg/L trypsin (sequence grade) in 200 mmol/L ammonium bicarbonate. The resulting peptide mixture was subjected to a ZipTip C18 cleanup and analyzed by MALDI-TOF/TOF (Applied Biosystems 4800 Proteomics Analyzer) in the presence of -cyano 4-hydrocinnamic acid (HPLC grade).

Gel electrophoresis and Western blotting

Samples were separated on 12.5% 1D SDS agarose gels. The proteins were either subjected to Western blotting or stained by Coomassie Brilliant blue. For Western blot analysis, polyvinylidene difluoride membranes with electrotransferred proteins (Hybond-P and Novablot apparatus; GE Healthcare) were consecutively treated with 5% (w/v) BSA, human serum (1 :500, overnight), goat antihuman IgG (1:5000), and horseradish peroxidase- conjugated rabbit anti-goat IgG (1 :5000) with intermittent washings in Tris- buffered saline (3x10 min). Protein-antibody interactions were visualized by use of 0.7 mmol/L 3,3'- diaminobenzidinetetrahydrochloride. Blotting signals were scanned with a CanoLiDE25 (Canon) scanner and quantified by ImageJ software (Image Processing and Analysis in Java). We included a positive and negative control serum sample in each run. The CV of the positive control (for all proteins) was <25% over 103 runs.

ELISA

Immobilizer amino plates (Nunc) were coated 2 hours at room temperature with 100 μΙ per well with 1 pg/ml hnRNP B1 , ΕΞ1 , F or M . After washing the plates four times with 1x PBS-Tween 20 (0.05%), 50 μΙ of serum sample was added (dilution 1:50 in PBS). The plates were incubated at room temperature for 30 min. Thereafter, 100 μΙ of horseradish peroxidase-conjugated goat anti-human IgG (diluted 1 :5000 in PBS) was added to each well and the plates were incubated at room temperature for 30 min. Between each step, the plates were washed four times with 1x PBS-Tween 20 (0.05%). The color reaction was developed using 3,3',5,5'-tetramethylbenzidine as substrate (100 μΙ per well). The wells were incubated in the dark at room temperature for 15 min. This enzymatic coloration was stopped by addition of 100 μΙ 0.5 H2S04 and the optical density was measured at 450 nm. A positive sample was used as standard. To this standard (six bi-fold serial dilutions starting from 1/50 to 1/1600), 100 arbitrary units were assigned. In each run, a positive and a negative control were included. The optical density of the samples was measured. Arbitrary units were calculated based on a standard curve. The coefficient of variation of the positive control (for all antigens) was <13% over 12 runs.

Statistical analysis

Kruskall Wallis analysis, ROC analysis, Mann-Whitney analysis and Fisher exact analysis was performed by using Analyze-lt for Microsoft Excel (version 2.09).

EXAMPLE 2: Results

Autoantibodies to hnRNP-H1 in sera from patients with a connective tissue disease: preparation of oligopeptide fragments

Recently, hnRNP-H1 was described as a newly recognized target of autoantibodies. These antibodies were mainly found in patients with Sjogren's syndrome, but were also present in patients with other autoimmune-mediated diseases (WO2009055880). Characterization of reactive epitopes allows us to study the mechanism of epitope spreading and to simplify the design of detection methods for the fast detection of autoantibodies. In addition we want to examine whether positivity against a specific epitope can be linked with a characteristic pathology or clinical phenotype. Previous epitope mapping studies proved that with some hnRNPs, like hnRNP A, B en D, the prominent antibody binding sites are localized at the RNA Recognition Motifs (RRMs). HnRNP-H1 was split up in five separate fragments (1-5) and these were expressed as recombinant oligopeptides. The choice of the restriction sites was made based on the location of the RRMs and the secondary structure of the hnRNP-H1 protein. The five different fragments that are generated (and His-tagged) in this study are: Fragment 1 (AminoAcid (AA) 1-110):

MMLGTEGGEGFWKVRGLPWSCSADEVQRFFSDCKIQNGAQGIRFIYTREGRPSGEAFVEL ESEDEVKL ALKKDRETMGHRYVEVFKSNNVEMDWVLKHTGPNSPDTAND (SEQ ID NO:4)

Fragment 2 (AA111-197):

GFVRLRGLPFGCSKEEIVQFFSGLEIVPNGITLPVDFQGRSTGEAFVQFASQEIAEK ALKKHKERIGHRYIE IFKSSRAEVRTHYDP (SEQ ID NO:5)

Fragment 3 (AA198-285):

PRKLMAMQRPGPYDRPGAGRGYNSIGRGAGFERMRRGAYGGGYGGYDDYNGYNDGYGFGS DRFGR DLNYCFSGMSDHRYGDGGSTFQS (SEQ ID NO:6)

Fragment 4 (AA286-386):

TTGHCVHMRGLPYRATENDIYNFFSPLNPVRVHIEIGPDGRVTGEADVEFATHEDAVAAM SKDKANMQH RYVELFLNSTAGASGGAYEHRYVELFLNSTAG (SEQ ID NO:7)

Fragment 5 (AA387-449):

ASGGAYGSQMMGGMGLSNQSSYGGPASQQLSGGYGGGYGGQSSMSGYDQVLQENSSDFQS NIA

(SEQ ID NO:8)

Fragments 1 to 4 are used to study the prevalence of antibodies recognizing these fragments in patients with autoimmune connective tissue disorders and the results are shown in Fig. 3 to 6. Fragment 3 and 4 appear to be the antigens with the largest antibody binding capacity. No antibodies to fragment 5 could be detected.

Autoantibodies to 10 different hnRNPs in sera from patients with a connective tissue disease

The objective of this study was to evaluate the value of antibodies against different hnRNP proteins in patients with a connective tissue disease and in diseased controls (DC). A total of 404 sera from patients with different systemic rheumatic diseases was tested for reactivity to 10 recombinant human hnRNP proteins (with numbers from UniProtKB/Swiss-Prot Database: hnRNP-A1 (P09651), -B1 (P22626), - C1(P07910), -E1(Q15365), -F (P52597), -Gi (P38159), -H1 (P31943), -I (Q9BUQ0), -K (P61978) and -P2 (P35637)). Recombinant proteins were produced according to the Gateway Cloning System. Constructs were transformed in E. coli BL21AI competent cells, and expressed as His-tagged fusion proteins. The identity of the proteins was confirmed by MALDI-TOF/TOF. Reactivity of serumantibodies to hnRNPs was tested by Gel electrophoresis and Western blotting. Statistical significance was evaluated by Mann- Whitney and Fisher exact testing. The results that show the prevalence of antibodies recognizing these 10 different hnRNPs in patients with autoimmune connective tissue disorders are depicted in Fig. 7 to 16. The results of this study are summarized in the next tables. Table 1 Summary of prevalences of antibodies to the hnRNP proteins and fragments 1 , 2, 3 and 4 of hnRNP-H1 in different systemic rheumatic diseases. Values are percentages with a cutoff set at 97,5 % specificity in CVS, the disease control (DC). SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. A, B, C, E, F, Gi, H, I, K, P, and 1 , 2, 3, and 4 stand for hnRNP-A1 , -B1 , -C1 , -E1 , -F, -Gi, -H1 , -I, -K, -P2, and fragment 1 , 2, 3 and 4 of hnRNP-H1 respectively. A B C E F Gi H I K P 1 2 3 4

More than 40% of patients with primary Sjogren's syndrome had antibodies to hnRNP-B1 , -E1 , -F and - H More than 20% of patients with primary Sjogren's syndrome had antibodies to fragment 2, 3, and 4 of hnRNP-H1. All tested antibodies (except hnRNP-Gi) were highly present in patients with primary Sjogren's syndrome.

Table 2 Summary of the fisher exact statistical analyses of all specific Abs tested. Statistically significant differences in prevalence were assessed by Fisher exact test for the comparison between the diseased and the control (CVS) group. The significant values (p<0.05) are indicated in grey background. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. A, B, C, E, F, Gi, H, I, K, P, and 1 , 2, 3, and 4 stand for hnRNP-A1 , -B1 , -C1 , - E1 , -F, -Gi, -H1 , -I, -K, -P2, and fragment 1 , 2, 3 and 4 of hnRNP-H1 respectively. P values are not corrected for multiple comparisons.

Patients with SSp had significantly higher Ab values to almost all hnRNPs tested.

Patients with DM had significantly higher Ab values to hnRNP A1 , B1 and F.

Patients with MCTD had significantly higher Ab values to hnRNP A1 , B1 and P2.

Patients with RA had significantly higher Ab values to hnRNP B1 and F. Table 3 This table shows the results for Mann-Whitney statistical analysis between each diseased group and the control group (CVS). The significant values (p<0.05) are indicated in grey background. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CVS: chronic fatigue syndrome. A, B, C, E, F, Gi, H, I, K, P, and 1 , 2, 3, and 4 stand for hnRNP-A1 , -B1 , -C1, - E1 , -F, -Gi, -H1 , -I, -K, -P2, and fragment 1 , 2, 3 and 4 of hnRNP-H1 respectively. P values are not corrected for multiple comparisons.

Simultaneous presence of antibodies to hnRNPs

Next, we evaluated whether the simultaneous presence of antibodies (Abs) to different hnRNPs is associated with certain connective tissue diseases (CTDs).

Table 4 Summary of the simultaneous presence of Abs to different hnRNPs used in this study. The significant values (p<0.05, Fisher-exact, see Table 5) are indicated in grey background. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis, CFS: chronic fatigue

, -E1 , -F, -H1 , -I, and -K respectively.

Table 5 Summary of the fisher exact statistical analyses of all combinations of specific Abs tested.

Statistically significant differences in prevalence were assessed by Fisher exact test: cut-off = 97,5 percentile of CFS, 2-tailed. The significant values (p<0.01) are indicated in grey background. SSp:

primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis; CFS:

chronic fatigue syndrome. A, B, E, F, H, I, and K stand for hnRNP-A1, -B1, -E1, -F, -H1, -I, and -K respectively.

The simultaneous presence of antibodies to hnRNP A1 and B1 was associated with DM, Ssc and SSp, while the simultaneous presence of antibodies to hnRNP H1 and F was associated with DM and SSp. The simultaneous presence of antibodies to hnRNP B1 and K was associated with DM and SSp, and the simultaneous presence of antibodies to hnRNP E1, F and H1 was associated with DM, MCTD and SSp. The simultaneous presence of antibodies to A1, B1, F and H1, or to E1, F, H1, 1, or to more combinations, including the combinations Ά1, B1 , E1, F and H1' and Ά1 , B1, C1, E1, F and H1' and Ά1, B1 , E1 , F, H1 and Γ, was highly associated with SSp. Here we show the combined presence of Abs to several hnRNPs in patients with SSp. More than 40% is positive for Abs to E1, F and H1 simultaneously, and 30% of patients has Abs to A1, B1, E1 , F and H1 simultaneously. Moreover, 45% is positive for Abs to F and H1 simultaneously, and 30% of the patients has Abs to A1. B1, F and H1 simultaneously.

Patients with DM have also combined presence to several hnRNPs. More than 20% has combined Abs to A1 and B1, and 17% has Abs to F and H1, and to B1 and K.

In patients with MCTD, presence of combined Abs to E1, F and H1 can occur, in our study in 16% of the MCTD-patients.

In Table 5 we show that the combined presence of antibodies to several different hnRNPs mainly occurs in patients with SSp, and thus are very specific for this pathology.

We evaluated reactivity to various members of the hnRNP family (A1 , B1, C1, E1, F, H1, Gi, I, K, P2) by Western blotting in controls (n=106) and in patients with a connective tissue disease (n=298) [(systemic lupus erythematosus (n=71), Sjogren syndrome (n=56), scleroderma (n=58), dermatomyositis (n=29), polymyositis (n=18), rheumatoid arthritis (n=47), and mixed connective tissue disease (n=19)]. A representative example of the Western blotting is given in Figure 17. Figures 18 and 9 show the reactivity to a selection of 8 hnRNP's in controls, in patients with systemic sclerosis, in patients with Sjogren's syndrome, and in patients with a connective tissue disease other than systemic sclerosis or Sjogren's syndrome. For all antigens the highest reactivity was found in Sjogren's syndrome. For all antigens, except Gi, the reactivity was statistically significantly higher in Sjogren's syndrome than controls , systemic sclerosis, and connective tissue diseases other than Sjogren's syndrome (p<0.05 Kruskall Wallis with Bonferroni correction). The reactivity in connective tissue diseases other than Sjogren's syndrome and systemic sclerosis was higher than the reactivity in controls and in systemic sclerosis for all antigens except hnRNP Gi and hnRNP K.

Subsequently, we calculated the prevalence of antibodies to the various hnRNP's for the different pathologies. A cutoff that corresponded to a specificity of 97.5% in controls was used. The data are summarized in Table 6. Antibodies to all antigens, except hnRNP Gi, were significantly more prevalent in Sjogren's syndrome than in controls. Antibodies to hnRNP B1 , E1 , F, and H1 were found in >45% of patients with Sjogrens syndrome. Antibodies to hnRNP A1 , hnRNP B1 , and hnRNP P2 were significantly more prevalent in mixed connective tissue disease than in controls, and antibodies to hnRNP A1 , hnRNP B1 , and hnRNP F were significantly more prevalent in dermatomyositis than in controls. Reactivity to at least 1 of the 10 antigens was found in 19% of controls and in 45% of dermatomyositis, 49% of rheumatoid arthritis, 38% of systemic lupus erythematosus, 53% of mixed connective tissue disease, and 73% of Sjogren's syndrome. Reactivity to at least 3 antigens was found in 2% of controls, 14% of systemic sclerosis, 27% of dermatomyositis, 14% of systemic lupus erythematosus, 26% of mixed connective tissue disease, and 48% of Sjogren's syndrome. Reactivity to at least 5 antigens was found in none of the controls, 7% of systemic sclerosis, 14% of dermatomyositis, 7% of systemic lupus erythematosus, 16% of mixed connective tissue disease, and 32% of Sjogren's syndrome (Table 7).

Table 6. Prevalence of antibodies measured by Western blotting to the various hnRNP's in controls and different pathologies [systemic sclerosis (SSc), polymyositis (PM), dermatomyositis (DM), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), Sjogren's syndrome (SS)]. A cutoff that corresponded to a specificity of 97.5% in controls was used. Values given are absolute numbers, percentages, and p-values.

*: statistically significant different from controls (Fisher exact). P values are corrected for multiple comparisons. Controls SSc PM DM RA SLE MCTD SS

(n=106) (n=58) (n=18) (n=29) in=47) (n=71) (n=19) (n=56) hnRNP

Al 3 (2.8%) 8 (13.8%) 2 (11.1%) 8 (27.6%) 6 (12.8% 10 (14.1%) 5 (26.3%) 21 (37.5%)

0.212 1 0.00395* 0.493 0.122 0.0408* 0,000001* hnRNP

Bl 3 (2.8%) 9 (15.5%) 2 (11.1%) 10 (34.5%) 8 (17.0%) 8 (11.3%) 5 (26.3%) 28 (50.0%)

0.091 1 0.00018* 0.074 0.51679 0.0408* 0,000001* hnRNP

CI 3 (2.8%) 7 (12.1%) 1 (5.6%) 5 (17.2%) 4 (8.5%) 8 (11.3%) 4 (21.1%) 15 (26.8%)

0.477 1 0.23227 1 0.51679 0.20451 0.00019* hnRNP

El 3 (2.8%) 4 (6.9%) 1 (5.6%) 5 (17.2%) 4 (8.5%) 9 (12.7%) 3 (15.8%) 28 (50.0%)

1 1 0.23227 1 0.255 0.894 0,000001* hnRNP F 3 (2.8%) 5 (8.6%) 2 (11.1%) 8 (27.6%) 8 (17.0%) 9 (12.7%) 4 (21.1%) 28 (50.0%)

1 1 0.00395* 0.074 0.255 0.20451 0,000001* hnRNP

Gi 3 (2.8%) 5 (8.6%) 1 (5.6%) 2 (6.9%) 1 (2.1%) 4 (5.6%) 1 (5.3%) 3 (5.4%)

1 1 1 1 1 1 1 hnRNP

HI 3 (2.8%) 2 (3.4%) 2 (11.1%) 5 (17.2%) 3 (6.4%) 5 (7.0%) 3 (15.8%) 26 (46.4%)

1 1 0.23227 1 1 0.894 0,000001* hnRNP I 3 (2.8%) 3 (5.2%) 2 (11.1%) 1 (3.4%) 2 (4.3%) 2 (2.8%) 1 (5.3%) 12 (21.4%)

1 1 1 1 1 1 0.00436* hnRNP K 3 (2.8%) 3 (5.2%) 1 (5.6%) 5 (17.2%) 4 (8.5%) 5 (7.0%) 1 (5.3%) 14 (25.0%)

1 1 0.23227 1 1 1 0.00056* hnRNP

P2 3 (2.8%) 7 (12.1%) 2 (11.1%) 3 (10.3%) 6 (12.8% 12 (16.9%) 6 (31.6%) 15 (26.8%)

0.477 1 1 0÷493 0.026* 0.00722* 0.00019*

Table 7. Antibodies to ten hnRNP proteins (A1 , B1 , C1 , E1 , F, H1 , Gi, I, K, P2) were determined by Western blotting. The table summarizes the combined reactivity to these antigens in various connective tissue diseases [systemic sclerosis (SSc), polymyositis (PM), deimatomyositis (DM), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), Sjogren's syndrome (SS)] and in controls. A cutoff that corresponded to a specificity of 97.5% in controls was used. *: statistically significant different from controls (Fisher exact).

Controls SSc PM DM RA SLE MCTD SSp

(n=106) (n=58) (n=18) (n=29) (n=47) (n=71) (n=19) (n=56)

Reactivity to >1 6

Ag 20 (18.9%) 15 (25.9%) (33.3%) 13 (44.8%) 23 (48.9%) 27 (38.0%) 10 (52.6%) 41 (73.2%)

0.510983 0.281129 0.010902* 0.000385* 0.008294* 0.006371* O.000001*

Reactivity to >2 3

Ag 6 (5.7%) 10 (17.2%) (16.7%) 11 (37.9%) 11 (23.4%) 15 (21.1%) 6 (31.6%) 32 (57.1%)

0.038134* 0.246444 0.00008* 0.004641** 0.004234* 0.005879* O.000001*

Reactivity to >3 2

Ag 2 (1.9%) 8 (13.8%) (11.1%) 8 (27.6%) 5 (10.6%) 10 (14.1%) 5 (26.3%) 27 (48.2%)

0.008082* 0.200613 0.000126* 0.057482 0.004233* 0.001696* <0.000001*

Reactivity to >4

Ag 1 (0.9%) 6 (10.3%) 1 (5.6%) 6 (20.7%) 4 (8.5%) 8 (11.3%) 5 (26.3%) 23 (41.1%)

0.065875 0.540519 0.00075* 0.062505 0.006292* 0.000537* O.000001*

Reactivity to >5

Ag 0 (0.0%) 4 (6.9%) 1 (5.6%) 4 (13.8%) 1 (2.1%) 5 (7.0%) 3 (15.8%) 18 (32.1%)

0.029208* 0.290322 0.003589* 0.614379 0.019041* 0.006099* <0.000001*

Reactivity to >6

Ag 0 (0.0%) 4 (6.9%) 1 (5.6%) 3 (10.3%) 1 (2.1%) 2 (2.8%) 2 (10.5%) 16 (28.6%)

0.029208* 0.290322 0.018224* 0.614379 0.31908 0.044129* O.OOOOOl*

Reactivity to >7

Ag 0 (0.0%) 1 (1.7%) 1 (5.6%) 3 (10.3%) 0 (0.0%) 1 (1.4%) 1 (5.3%) 13 (23.2%)

0.707317 0.290322 0.018224* 1 0.802259 0.304 <0.000001*

In a next step, we evaluated reactivity to hnRNP B1, hnRNP E1, hnRNP F and hnRNP H1 by ELISA (Table 8, Fig 20 and Fig 21) in controls (n=89), blood donors (n=82), and in diagnostic samples of patients with a connective tissue disease (n=228) (see materials and methods). These four antigens were selected because they displayed a high reactivity for Sjogren's syndrome in Western blotting. Differences in reactivity between controls and patients were evaluated by Kruskall Wallis statistical analysis with Bonferroni correction. For all four antigens, significantly higher antibody levels were found in Sjogren's syndrome, systemic lupus erythematosus, and rheumatoid arthritis than in controls (p<0.004). For hnRNP H1 , hnRNP F, and hnRNP B1, higher antibody levels were also found in mixed connective tissue disease than in controls (p<0.001). For hnRNP H1 and B1 , significantly higher antibody levels were also found in systemic sclerosis than in controls (p<0.05) and for hnRNP B1 significantly higher antibody levels were also found in dermatomyositis than in controls (p=0.01). Significantly lower antibody levels were found in blood donors than in controls (p<0.01).

Subsequently, we calculated the prevalence of antibodies to hnRNP B1 , hnRNP E1, hnRNP F and hnRNP H1 in various connective tissue diseases. A cutoff that corresponded to a specificity of 97.5% in controls was used. The data are summarized in Table 9. The prevalence of antibodies to hnRNP B1 was significantly higher in Sjogren's syndrome (44%), mixed connective tissue disease (45%) and systemic lupus erythematosus (37%) than in controls (3.4%). The prevalence of antibodies to hnRNP E1 and hnRNP F was significantly higher in Sjogren's syndrome (21 %) than in controls (3.4%). Reactivity to at least 1 of the 4 tested antigens was found in 11% of the controls, 35% of rheumatoid arthritis, 47% of systemic lupus erythematosus, 64% of mixed connective tissue disease, and 56% of Sjogren's syndrome. Reactivity to at least 2 of the 4 tested antigens was found in 1.1% of controls, in 16% of patients with systemic lupus erythematosus, and in 18% of patients with Sjogren's syndrome. Reactivity to at least 3 of the 4 antigens was found in none of the controls and in 15% of patients with Sjogren's syndrome. 10 000061

Table 8. Prevalence of antibodies measured by ELISA to hnRNP B1, E1, F, and H1 in controls and different connective tissue diseases [systemic sclerosis (SSc), polymyositis (PM), dermatomyositis (DM), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), Sjogren's syndrome (SS)]. A cutoff that corresponded to a specificity of 97.5% in controls was used.

*: statistically significant different from controls (Fisher exact). P values are corrected for multiple comparisons. blood

Controls donors SSc ( PM DM RA SLE MCTD SS

(n=89) (n=82) n=65) (n=ll) (n=22) (n=23) (n=62) (n=ll) (n=34)

10

Bl 3 (3.4%) 0 (0.0%) (15.4%) 1 (9.1%) 5 (22.7%) 5 (21.7%) 23 (37.1%) 5 (45.5%) 15 (44.1%)

1 0.074088 1 0.05976 0.071316 O.000001* 0.002332* <0.000001*

El 3 (3.4%) 0 (0.0%) 2 (3.1%) 1 (9.1%) 1 (4.5%) 1 (4.3%) 3 (4.8%) 0 (0.0%) 7 (20.6%)

1 1 1 1 1 1 1 0.036624*

F 3 (3.4%) 1 (1.2%) 1 (1.5%) 1 (9.1%) 1 (4.5%) 3 (13.0%) 9 (14.5%) 1 (9.1%) 7 (20.6%)

1 1 1 1 0.798836 0.118404 1 0.036624*

HI 2 (2.2%) 1 (1.2%) 1 (1.5%) 0 (0.0%) 0 (0.0.%) 0 (0.0%) 6 (9.7%) 1 (9.1%) 4 (11.8%)

1 1 1 1 1 0.414868 1 0.391052

Table 9. Antibodies to four hnRNP proteins (B1, E1, F, H1) were determined by ELISA. The table summarizes the combined reactivity to these antigens in various connective tissue diseases [systemic sclerosis (SSc), polymyositis (PM), dermatomyositis (DM), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), Sjogren's syndrome (SS)], in controls, and in blood donors. A cutoff that corresponded to a specificity of 97.5% in controls was used.

*: statistically significant different from controls (Fisher exact). blood

Controls donors SSc PM DM RA SLE MCTD SS (n=89) (n=82) (n=65) (n=l l) (n=22) (n=23) (n=62) (n=l l) (n=34)

Reactivity to >1 10 3

1 (1.2%) 12 (18.5%) 4 (18.2%) 8 (34.8%) 29 (46.8%) 7 (63.6%) 19 (55.9%) antigen (11.2%) (27.3%)

0.013754* 0.302034 0.305123 0.576406 0.021885* 0.000002* 0.000537* 0.000001*

Reactivity to >2

1 (1, 1%) 1 (1.2%) 2 (3.1%) 0 (0.0%) 1 (4.5%) 1 (4.3%) 10 (16.1%) 0 (0.0%) 6 (17.6%) antigens

0.538837 0.766622 0.219999 0.717117 0.740025 0.001255* 0.219999 0.003521*

Reactivity to >3

0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 1 (4.5%) 0 (0.0%) 2 (3.2%) 0 (0.0%) 5 (14.7%) antigens

1 1 1 0.396396 1 0.333951 1 0.002575*

Reactivity to 4

0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 3 (8.8%) antigens

1 1 1 1 1 1 1 0.039547*

Table 10. Antibodies to hnRNP proteins (E1 and H1) were determined by ELISA. The table summarizes the combined reactivity to these antigens in various connective tissue diseases [systemic sclerosis (SSc), polymyositis (PM), dermatomyositis (DM), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), Sjogren's syndrome (SSp)], in controls (CFS), and in blood donors (BD). A cutoff that corresponded to a specificity of 97.5% in controls was used.

Table 11. Antibodies to hnRNP proteins (E1 and F) were determined by ELISA. The table summarizes the combined reactivity to these antigens in various connective tissue diseases [systemic sclerosis (SSc), polymyositis (PM), dermatomyositis (DM), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), Sjogren's syndrome (SSp)], in controls (CFS), and in blood donors (BD). A cutoff that corresponded to a specificity of 97.5% in controls was used.

Table 12.

Gateway-adapted primers used for cloning of hnRNPs.

SEQ ID NO:9

GGGGACAAGTTTGTACAAAAAAGCAGGCTCGTCTAAOTCAGAGTCTCCTAAAGAGCCCGA A GWJinRNP Al Fp

SEQ ID NO: 10

GGGGACCACTrTGTACAAGAAAGCTGGGTTTAAAATCTTCTGCCACTGCCATAGCTACT GWJinRNP Al Rp

SEQ ID NO: 11

GGGGACAAGTTTGTACAAAAAAGCAGGCTCGGAGAAAACTTTAGAAACTGTTCCTTTG GWJinRNP Bl Fp

SEQ ID NO: 12

GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGTATCGGCTCCTCCCACCATAACCCCC GWJinRNP Bl Rp

SEQ ID NO:13

GGGGACAAGTTTGTACAAAAAAGCAGGCTCGGCCAGCAACGTTACCAACAAGACAGAT GWJinRNP CI Fp

SEQ ID NO: 14

GGGGACCACTTTGTACAAGAAAGCTGGGTCTATCATCCTCCATTGGCGCTGTCTCTCTG GWJinRNP CI Rp

SEQ ID NO: 15

GGGGACAAGTTTGTACAAAAAAGCAGGCTCGATGGATGCCGGTGTGACTGAAAGTGGA GWJinRNPElFp

SEQ ID NO: 16

GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGCTGCACCCCATGCCCTTCTCAGAGGA GWJinRNPElRp

SEQ ID NO: 17

GGGGACAAGT TGTACAAAAAAGCAGGCTCGATGCTGGGCCCTGAGGGAGGTGAAGGCTTT GWJinRNP F Fp

SEQ ID NO: 18

GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGTCATAGCCACCCATGCTGTTGTGCGC GWJinRNP F Rp

SEQ ID NO: 19

GGGGACAAGTTTGTACAAAAAAGCAGGCTCGAGTTATGGAGGTCCACCTCGAAGGGAACC G GWJinRNP Gi Fp

SEQ ID NO:20

GGGGACCACTTTGTACAAGAAAGCTGGGTCTAAATAGTCACGATCACGACCATATCCATC TC GWJinRNP Gi Rp

SEQ ID NO:21

GGGGACAAGTTTGTACAAAAAAGCAGGCTCGATGTTGGGCACGGAAGGTGGAGAGGGATT C GWJinRNP HI Fp

SEQ ID NO:22

GGGGACCACTTTGTACAAGAAAGCTGGGTCTATGCAATGTTTGATTGAAAATCACTGGAG TT GWJinRNP HI Rp

SEQ ID NO:23

GGGGACAAGTTTGTACAAAAAAGCAGGCTCGGACGGCATTGTCCCAGATATAGCCGTTGG T GWJinRNP I Fp

SEQ ID NO:24

GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGATGGTGGACTTGGAGAAGGAGACCCG GWJinRNP I Rp

SEQ ID NO:25

GGGGACAAGTTTGTACAAAAAAGCAGGCTCGATGGAAACTGAACAGCCAGAAGAAACC GWJinRNPKFp

SEQ ID NO:26

GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGAAAAACTTTCCAGAATACTGCTTCAC GWJinRNPKRp

SEQ ID NO:27

GGGGACAAGTTTGTACAAAAAAGCAGGCTCGGCCTGAAACGATTATACCCAACAAGCAAC C GWJinRNP P2 Fp

SEQ ID NO.28

GGGGACCACTTTGTACAAGAAAGCTGGGTTTAATACGGCCTCTCCCTGCGATCCTGTCT GWJinRNP P2 Rp