EP2235540A2 - Neues verfahren zur diagnose des sjögren-syndroms - Google Patents

Neues verfahren zur diagnose des sjögren-syndroms

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Publication number
EP2235540A2
EP2235540A2 EP08845893A EP08845893A EP2235540A2 EP 2235540 A2 EP2235540 A2 EP 2235540A2 EP 08845893 A EP08845893 A EP 08845893A EP 08845893 A EP08845893 A EP 08845893A EP 2235540 A2 EP2235540 A2 EP 2235540A2
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EP
European Patent Office
Prior art keywords
hnrnp
antibodies
syndrome
biological sample
sjogren
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP08845893A
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English (en)
French (fr)
Inventor
Xavier Bossuyt
Karolien Van Den Bergh
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Katholieke Universiteit Leuven
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Katholieke Universiteit Leuven
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Application filed by Katholieke Universiteit Leuven filed Critical Katholieke Universiteit Leuven
Publication of EP2235540A2 publication Critical patent/EP2235540A2/de
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis

Definitions

  • the present invention relates to a method of diagnosing Sjogren's syndrome. More particularly the present invention relates to methods and compositions for detecting anti-heterogeneous nuclear ribonucleoprotein-H1 (hnRNP-H1) autoantibodies for diagnosing Sjogren's syndrome.
  • hnRNP-H1 anti-heterogeneous nuclear ribonucleoprotein-H1
  • 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.
  • 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.
  • 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).
  • the present invention shows hnRNP-H1 as a new target of autoantibodies in patients with Sjogren's syndrome. More in particular, the present invention identifies anti-hnRNP-H1 antibodies as a valuable diagnostic marker in Sjogren's syndrome.
  • the present invention provides for a method for diagnosing
  • the present invention provides methods and compositions for detecting anti-hnRNP-H1 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 hnRNP- H1 is determined in an isolated biological sample derived from a subject.
  • the present invention further relates to a method for detecting hnRNP-H1 antibodies 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 hnRNP-M.
  • said method is characterized in that hnRNP-H1 proteins, peptides, variants or fragments thereof are used for determining the presence of the hnRNP-H1- antibodies.
  • said method is characterized in that a hnRNP- H1-antigen, more specific an immobilized hnRNP-H1-antigen is used for determining the presence of the hnRNP-M -antibodies.
  • said analysis or determination of the presence of autoantibodies against hnRNP-H1 is performed in an immunoassay.
  • said immunoassay is selected from the group consisting of ELISA, FEIA, western blot, dot blot, bead-based assay, antigen array and Radio lmmuno Assay.
  • 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-H1 proteins, peptides, variants or fragments thereof to detect the presence of autoantibodies against hnRNP-H1 in an isolated biological sample derived from a subject.
  • the present invention further relates to the use of hnRNP-H1 proteins, peptides, variants or fragments thereof to detect the presence of autoantibodies against hnRNP-H1 in an isolated biological sample derived from a patient for diagnosing an autoimmune disease, more particularly Sjogren's syndrome.
  • said hnRNP-H1 proteins, peptides, variants or fragments thereof are used for carrying out the methods 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.
  • 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.
  • said isolated biological sample is an antibody-containing isolated biological sample.
  • test kit for detecting the presence of antibodies against hnRNP-H1 in an isolated biological sample.
  • the test kit additionally comprises a solid phase onto which said antigen is or can be bound.
  • the test kit additionally comprises a labeling group which is bound to said antigen or can be bound thereto.
  • 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 ivention 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-H1-autoantibodies of said patient, in an amount effective to treat the autoimmune disease, more particularly Sjogren's syndrome.
  • said inhibitor is selected from the group consisting of hnRNP-H1 proteins, peptides, variants, fragments, or epitopes thereof and an antibody.
  • the present invention further relates to the use of a compound which specifically binds to hnRNP-H1 autoantibodies for the production of a medicine.
  • said medicine is used for the treatment of an auto-immunedisease, more particularly Sjogren's syndrome.
  • 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.
  • FIG. 1 Sequence alignments of hnRNP-H1 from mouse, rat and humans.
  • the three sequences 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), whereas sequence Q499R8_RAT (SEQ ID NO:4) has 72 % homology with HNRH1_HUMAN (SEQ ID NO:2) and HNRH1_MOUSE (SEQ ID NO:3) and 30 % with Q8VHV7_RAT (SEQ ID NO:1). Amino acids in bold were identified by MALDI-TOF ⁇ TOF mass spectrometry.
  • Figure 2 Reactivity to hnRNP-H1 on Western blotting analysis.
  • Rat hnRNP-H1 was subjected to SDS-PAGE and transferred onto a PVDF membrane.
  • the membrane was incubated with purified antibody or with serum, as described in example 1.
  • Lanes 1 , 2, and 3 show Western blotting analyses after incubation of the membranes with purified anti-hnRNP- H1 from rabbit, serum from a diseased person, and serum from a control person, respectively.
  • Lane 4 shows the gel after SDS-PAGE and staining with Coomassie Brilliant Blue. The most left and most right lane shows the molecular weight marker.
  • Figure 3 Indirect immunofluorescence pattern of anti-hnRNP-H1 antibodies. Effect of pre-incubation with recombinant hnRNP-H1.
  • Panel A HEp-2 cells were incubated with anti-hnRNP-H1 antibodies from rabbit (0.25 ⁇ g/ml).
  • Panels B-E HEp-2 cells were incubated with a serum sample that reacted with hnRNP-H1 (on Western blotting) after pre-incubation with phosphate buffered saline (Panel B) or with increasing concentrations of recombinant rat hnRNP-H1 (24 ⁇ g/mL, 63 ⁇ g/mL, or 126 ⁇ g/mL) (PanelC, D, and E, respectively).
  • the figures show the indirect immunofluorescence analysis (magnification 400 X).
  • 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 from rat.
  • SSp primary Sjogren's syndrome
  • SLE systemic lupus erythematosus
  • MCTD mixed connective tissue disease
  • SSc scleroderma
  • DM dermatomyositis
  • PM polymyositis
  • RA rheumatoid arthritis
  • * statistically significantly different from all other groups (Mann Whitney). For each group, the median 'peak x area' value is indicated.
  • Figure 5 Prevalence of anti-hnRNP-H1 antibodies in patients with autoimmune connective tissue disorders by performing ELISA using recombinant hnRNP-H1 from rat.
  • SSp primary Sjogren's syndrome
  • SLE systemic lupus erythematosus
  • MCTD mixed connective tissue disease
  • SSc scleroderma
  • DM dermatomyositis
  • PM polymyositis
  • RA rheumatoid arthritis
  • * statistically significantly different from all other groups (Mann Whitney). For each group, the median absorbance value is given.
  • the invention provides a method of detecting anti-hnRNP-H1 antibodies in a patient suffering from or susceptible to an autoimmune disease such as Sjogren's syndrome.
  • the method of detecting anti-hnRNP-H1 antibodies of the invention includes the step of analyzing a biological sample for the presence of an antibody that specifically binds a hnRNP-H1.
  • a biological sample that would normally be expected to contain immunoglobulins might be used.
  • 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.
  • 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).
  • an agent to which an anti-hnRNP-H1 antibody specifically binds is used.
  • agents might include a native (i.e. naturally occurring) hnRNP-H1 or fragments, mutants or variants thereof; or a non-hnRNP-H1 test reagent such as an anti-idiotypic antibody.
  • native hnRNP-H1s have been characterized (e.g. amino acid sequenced), including those from human, mouse and rat.
  • those hnRNP-H1s 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.
  • rat hnRNP-H1 can be used to detect human anti-hnRNP-H1 antibodies.
  • test reagent that specifically binds to these proteins, a number of different methods might be used.
  • 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-H1 antibody.
  • hnRNP-H1 is immobilized on a substrate, a human serum sample is placed on the substrate under conditions that would allow binding of anti-hnRNP-H1 antibodies to the immobilized hnRNP-H1.
  • detectably labeled secondary antibody e.g. an anti-human immunoglobulin antibody if the biological sample was derived from a human subject
  • detectable label e.g. an enzyme, fluorophore, or radioisotope
  • antibodies contained within a biological sample are immobilized on a substrate, a detectably labeled hnRNP-H1 is then placed on the substrate under conditions that would allow binding of the immobilized anti-hnRNP-H1 antibodies to the hnRNP-H1.
  • the presence of detectable label remaining on the substrate after washing indicates that the sample contained anti-hnRNP-H1 antibodies.
  • the method of the invention can be carried out as qualitative or quantitative determination.
  • hnRNP-H1 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.
  • test format is not in general critical. However, a test format can be preferred in which an immune complex comprising a hnRNP-H1 -antigen and the hnRNP-H1 autoantibody to be detected is bound to a solid phase.
  • 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, ⁇ - galactosidase or alkaline phosphatase, and for the method to be carried out in the ELISA format.
  • Purified or recombinant hnRNP-H1-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.
  • the purified or recombinant hnRNP-H1-antigen is separated on a one dimensional sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) prior to blotting on a membrane.
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel
  • the membrane strip is blocked in blocking buffer (BSA or skim milk powder).
  • BSA blocking buffer
  • 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.
  • An Enzyme Linked Immunosorbent Assay provides a quantitative in vitro assay for the detection of antibodies.
  • Polystyrene microplate strips are coated with purified, biochemically characterized pure or recombinant hnRNP-H1 -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.
  • the attached antibodies are detected with peroxidase-labeled anti-human antibodies.
  • 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.
  • a Fluoro Enzyme lmmuno Assay provides a quantitative in vitro assay for the detection of antibodies.
  • a carier e.g. polystyrene microplate strips
  • a carier e.g. polystyrene microplate strips
  • purified, biochemically characterized pure or recombinant antigen e.g. hnRNP-H1-antigen
  • the attached antibodies are detected with an enzyme-labeled anti-human antibodies.
  • 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 lmmuno 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.
  • Bead-based assays Human serum is treated with different sets of color-coded polystyrene beads, each bearing a different antigen (including hnRNP-H1). 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 array Several antigens, including hnRNP-H1, 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.
  • 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, GIu) 1 hydroxy- like (Cys, Ser, The), amides (Asn, GIn), basic (His, Lys, Arg), aliphatic-like (Met, He, Leu, VaI, GIy, 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.
  • 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.
  • treatments involving administration of an autoimmune inhibitor to a patient 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.
  • 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 al., "Handbook of experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chap. 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, N.Y.(1974)).
  • autoantibody gene products may be generated which include proteins that represent functionally equivalent gene products.
  • an equivalent hnRNP-H1 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-H1 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.
  • 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.
  • deletion or non-conservative alterations can be engineered to produce altered anti-hnRNP-H1 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.
  • cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges. This applies to any autoimmune hnRNP-H1 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-H1 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 a/., 1989, and Ausubel, et a/., 1989.
  • RNA capable of encoding auto-hnRNP-H1 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.
  • host-expression vector systems may be utilized to express the autoantibodies, such as anti-hnRNP-H1 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-H1 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, et a/., 1987, Methods in Enzymol. 153, 516-544).
  • 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, MDCK, 293, 3T3, and W138.
  • a variety of methods can be employed for the diagnostic and prognostic evaluation of 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-H1 gene mutations, or the detection of either over-, under-, or no expression of hnRNP-H1 protein, or mutants.
  • Mutations at a number of different genetic loci may lead to phenotypes related to autoimmune disorder, structural and synaptic abnormalities.
  • 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.
  • identification of alterations in hnRNP-H1 molecules, such as, for example, gene or protein can be utilized to optimize therapeutic drug treatments.
  • autoimmune related molecule such as, for example, hnRNP-H1, expression levels, mutations, polymorphisms
  • a microassay of for example, hnRNP-H1 nucleic acid sequences immobilized to a substrate or "gene chip" for detection of hnRNP- H1 molecules see, e.g. Cronin, ef a/., 1996, Human Mutation 7:244-255. Preferred methods are detailed in the examples which follow.
  • hnRNP-H1 or any hnRNP-H1 -related molecule gene expression can also be assayed as described in detail in the examples which follow. Additionally, it is possible to perform hnRNP-H1 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-H1 gene.
  • Antibodies directed against hnRNP-H1 gene products may be used in vitro to determine, for example, the level of hnRNP-H1 antibody gene expression achieved in cells genetically engineered to produce such a gene product.
  • 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-H1 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-H1 gene.
  • Preferred diagnostic methods for the detection of autoimmune molecules may involve, for example, immunoassays wherein the hnRNP-H1 gene products or conserved variants or peptide fragments are detected by their interaction with an anti-hnRNP-H1 gene product-specific antibody.
  • immunoassays wherein the hnRNP-H1 gene products or conserved variants or peptide fragments are detected by their interaction with an anti-hnRNP-H1 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.
  • autoimmune disease 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.
  • autoimmune disease is intended to further include autoimmune conditions, syndromes and the like.
  • antigen 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 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.
  • 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.
  • 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 1 , 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • hnRNP-H1 as a new target of auto-antibodies in patients with primary and secondary Sjogren's syndrome.
  • the specificity of anti-hnRNP-H1 antibodies 91.7% - 94.4%) tended to be higher than the specificity of anti-SSA antibodies (78.6% - 89%).
  • the low specificity of anti-SSA antibodies is mainly related to the high prevalence of anti-SSA antibodies in patients with SLE.
  • anti-hnRNP-H1 antibodies were not very much prevalent in patients with SLE.
  • Anti-hnRNP-H1 antibodies are an interesting diagnostic marker in anti-SSA and/or anti-SSB seronegative patients with primary Sjogren's syndrome. We found that 5/11 (45%) anti-SSA/anti-SSB seronegative patients had anti-hnRNP-H1 antibodies.
  • the negative predictive value is higher for anti-SSA antibodies than for anti-hnRNP-H1 antibodies.
  • the analysis of the 188 consecutive (random) samples revealed relevant information on the specificity of anti-hnRNP-H1 antibodies and showed that these antibodies can be found, to a lesser extent in a number of other autoimmune diseases.
  • One explanation is that in some of these patients, the detection of hnRNP-H1 antibodies is indicative for the development of secondary Sjogren's syndrome since we have shown (in example 2) that hnRNP-H1 auto-antibodies are an early marker for Sjogren's syndrome.
  • anti-SSA antibodies are found, to a bigger extent compared with hnRNP-H1 antibodies, in a wide range of medical conditions with a possible autoimmune pathogenesis.
  • Anti-hnRNP-H1 antibodies were not only found in samples with a speckled ANA pattern, but also in samples with a homogeneous staining (nuclear dots) pattern.
  • Five of the 18 patients with anti-hnRNP- H1 antibodies received Infliximab, a TNF- ⁇ blocking drug, and displayed a homogeneous staining on ANA indirect immunofluorescence analysis. It is known that Infliximab can induce the formation of auto-antibodies, especially anti-dsDNA antibodies. We assumed that in such cases the typical speckled pattern caused by anti-hnRNP-H1 antibodies was masked by other antibodies.
  • hnRNP-H1 we described hnRNP-H1 as a new target of autoantibodies in patients with Sjogren's syndrome. Therefore, we identified anti-hnRNP-H1 antibodies as a valuable diagnostic marker in Sjogren's syndrome.
  • Recombinant his-tagged hnRNP-H1 protein from rat was prepared as described (Fijak M et al., J Pathol. 2005;207: 127-38).
  • Recombinant GST-tagged hnRNP-H1 protein from human was prepared from cDNA according to Devogelaere et al. (Biochem. J. 2007;407:303-311). o
  • PVDF Polyvinylidene difluoride
  • Hybond-P Hybond-P
  • IPG strips pH-range 3-10, ImmobilineTM DryStrip gels
  • Rabbit thymus, goat anti-human IgG, and HPLC grade water were obtained from Sigma-Aldrich.
  • Horseradish peroxidase conjugated rabbit anti- goat IgG was from DakoCytomation.
  • C18 clean-up (ZipTip) was from Millipore Corporation.
  • Purified rabbit anti-hnRNP-H1 antibodies were from Acris Antibodies.
  • a second group of samples consisted of 188 consecutive serum samples submitted to the clinical laboratory for ANA testing (titer ⁇ 160).
  • Antinuclear antibody testing identification of antibodies to extractable nuclear antigens, counterimmunoelectrophoresis
  • ANA were determined by indirect immunofluorescence using HEp-2000 ® cells (Immunoconcepts) with an Axioplan 2 fluorescence microscope (Carl Zeiss Microimaging) and Cytovision 3.6 software (Applied Imaging Corporation). The presence of antibodies to ENAs was assayed by ENA dot blot (BioMedical Diagnostics), UniCaplOO and/or UniCap250 system (Phadia Diagnostics), ANA3 test (anti-SSA 52/anti-SSA 60 status, Euroimmun), or counterimmunoelectrophoresis with rabbit thymus as antigenic source.
  • PVDF polyvinylidene difluoride
  • Blotting signals were scanned using a Typhoon 9400 scanner set on green laser light (532 nm), filter 526 SP, PMT 640V at normal sensitivity and quantified by ImageQuant TL software (both from GE Healthcare).
  • a positive and negative control serum sample were included in each run. The coefficient of variation of the positive control was 23.8% over 13 runs.
  • Serum from the patient and from a control individual were used for Western blotting after 2D gel separation of rabbit thymus extract. Proteins to which there was reactivity were excised and identified by MALDI-TOF/TOF analysis, and for one of the proteins identified here, a significant score was obtained for the protein hnRNP-H1 from mouse (Mus musculus) mining the SwissProt database (Hn ⁇ pM, 035737) or Ratsgi from rat (Rattus norvegicus) screening the NCBI database ⁇ Hnrnphi, Q8VHV7) (see Fig.1).
  • hnRNP-H1 heterogeneous nuclear ribonucleoprotein
  • hnRNP-H1 heterogeneous nuclear ribonucleoprotein
  • HnRNP-HI from rat has 99% similarity with human hnRNP-H1 (P31943
  • anti-hnRNP-H1 antibodies give a speckled fluorescence pattern on indirect immunofluorescence analysis by two complementary approaches. First, incubating HEp-2 cells with a commercial rabbit anti-hnRNP-H1 antibody resulted in a fine speckled antinuclear antibody pattern (Fig. 3A).
  • Anti-hnRNP-H1 antibodies are associated with Sjogren's Syndrome
  • serum samples from 246 patients with well-defined autoimmune connective tissue disorders for reactivity to hnRNP-H1 in Western blot analysis. The results are shown in Fig. 4.
  • a receiver operating characteristics analysis was performed for anti-hnRNP-H1 antibodies as a marker of primary Sjogren's syndrome.
  • the sensitivity of anti-hnRNP-H1 antibodies for primary Sjogren's syndrome was 48.4% when a cutoff ('peak x area' 1.90) was used that corresponded to a specificity of 94.8% in diseased controls.
  • a cutoff ('peak x area' 0.71) was used that maximized the sum of sensitivity and specificity, then the sensitivity of anti- hnRNP-H1 antibodies for primary Sjogren's syndrome was 74.2% and the specificity 81.7% (Youden index 0.559). In all subsequent analyses, the cutoff based on a specificity of 95% was used.
  • Anti- hnRNP-H1 antibodies were found in 48% (45/93) of patients with primary Sjogren's syndrome, in 2.4% (1/41) of patients with SLE, in 22% (2/9) of patients with MCTD, in 7.7% (3/39) of patients with scleroderma, in 0% (0/15) of patients with dermatomyositis, in 0% (0/7) of patients with polymyositis, in 4.8% (2/42) of patients with RA, and in 5% (2/41) of healthy individuals.
  • the sensitivity and specificity of anti-SSA antibodies for primary Sjogren's syndrome in the same patient cohort was 88.2% and 76.3%, respectively.
  • Anti-SSA antibodies were found in 33% of MCTD, in 7.7% of patients with scleroderma, and in 63.4% of patients with SLE.
  • the sensitivity of anti-hnRNP-H1 antibodies for Sjogren's syndrome was 43.7% and the specificity 94.4%.
  • the area under the curve for anti-SSA antibodies was 0.82.
  • the sensitivity and specificity of anti-SSA antibodies in the same patient group was 85.3% and 78.6%, respectively.
  • the specificity of anti-hnRNP-H1 antibodies for Sjogren's disease was higher than the specificity of anti-SSA antibodies. This is mainly related to the high prevalence of anti-SSA antibodies in patients with SLE.
  • the likelihood ratios were 7.81, 0.60, 3.98, and 0.19 for, respectively, hnRNP-H1 positive, hnRNP-H1 negative, SSA positive, and SSA negative.
  • Eleven out of 93 patients with Sjogren's syndrome were seronegative for anti-SSA (both anti-SSA 52 and anti-SSA 60 kDa) and anti-SSB antibodies.
  • Anti-hnRNP-H1 antibodies were found in 5 of these 11 seronegative patients (45%) (Table 1).
  • FIG. 1 Another method for the evaluation of anti-hnRNP-H1 antibodies in a study cohort of 246 serum samples of patients with autoimmune connective tissue diseases and 41 healthy subjects is an ELISA using recombinant His-tagged rat hnRNP-H1. Using this method, the prevalence of anti-hnRNP-H1 antibodies was 22.5% for patients with primary Sjogren's disease.
  • Figure 5 shows the reactivity of patient's serum samples to hnRNP-H1 by performing an ELISA using recombinant His-tagged hnRNP- H1 from rat.
  • ELISA detected anti-hnRNPH1 antibodies in 2.4% (1/41) of patients with SLE 1 in 22% (2/9) of patients with MCTD, in 7.7% (3/39) of patients with scleroderma, in 13.3% (2/15) of patients with dermatomyositis and in 2.5% (1/40) of healthy individuals.
  • No anti-hnRNP-H1 antibodies were detected in patients with RA (0/42) or polymyositis (0/7).
  • a statistically significant difference between the prevalence of anti-hnRNP-H1 antibodies in patients with primary Sjogren's syndrome, healthy controls and diseased controls was found (p 0.0063, Fisher Exact Test).
  • the sensitivity of anti-hnRNP-H1 antibodies for Sjogren's syndrome was 19.4% and the specificity 94.4% (cutoff 110, based on 95% specificity for diseased controls).
  • Three of the 18 patients had secondary Sjogren's syndrome (one in combination with SLE and 2 in combination with RA [treated with anti-TNF- ⁇ ]).
  • Primary Sjogren's syndrome was assumed in this patient, but he didn't fulfill the EU-US classification criteria.
  • the sensitivity and specificity of anti-hnRNP-H1 for Sjogren's syndrome was 42.9% and 91.7%, respectively.
  • the sensitivity and specificity were, respectively, 71.4% and 89.0%.
  • the likelihood ratios were 5.16, 0.62, 6.46, and 0.32 for, respectively, hnRNP-H1 positive, hnRNP-H1 negative, SSA positive, and SSA negative.

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