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• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/99—Isomerases (5.)
• • 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
  • G01N2800/104—Lupus erythematosus [SLE]

Definitions

• the present invention relates to novel diagnostic methods for systemic sclerosis and systemic lupus erythematosus, uses of anti-topoisomerase I and fragments thereof for such diagnosis and diagnostic kits.
• SSc Systemic sclerosis
• Topo I is a 765 amino acid (AA) long DNA-relaxing enzyme which contains five distinct regions: the N-terminal domain (AA 1-215), core subdomains I— II (AA 216-435), core subdomain III (AA 436- 636), the linker domain (637-713) and the C-terminal domain (AA 714-765).
• dcSSc is characterized by extensive fibrosis of the skin, lungs and other internal organs, while in IcSSc vascular abnormalities are dominating and fibrosis is limited [5].
• topo-I and topo-I peptides have been proposed in detection of autoantibodies.
• topo-I polypeptide In US 5070192 cloning of a topo-I polypeptide is described and detection of autoantibodies in patient samples by binding to said topo-I polypepetide is suggested. In US 5849503 a mutant topo-I polypeptide is described which is thought to be useful for detecting autoantibodies e.g. in an immunoassay.
• the present invention relates to a use of one or more isolated peptide(s) for diagnosis of a systemic autoimmune disorder, said one or more peptide(s) separately or simultaneously comprising at least one of the following epitopes: a) an epitope having the immunological property of an at least 20 amino acids long segment of the portion of a DNA Topoisomerase I spanning amino acids 5 to 30, b) an epitope having the immunological property of an at least 20 amino acids long segment of the portion of a DNA Topoisomerase I spanning amino acids 350 to 400, said one or more peptide(s) having an amino acid sequence which is at least 70%, 75%, 80%, 85% or 90%, 92%, 94%, 96% or 98% identical with the amino acid sequence of a peptide fragment of a wild type DNA Topoisomerase I, wherein - if binding of autoantibodies of a sample from a patient to epitope according to a) is detected, it is considered as indicative of diffuse cutane systemic
• SLE systemic lupus erithematosus
• one or more isolated peptide(s) also comprise c) an epitope having the immunological property of the at least 20 amino acids long segment of the portion of said DNA Topoisomerase I spanning amino acids 451 to 593, and wherein
• dsSSC diffuse cutane systemic scleroderma
• the epitope of said peptide is different in not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids from the respective epitope of the wild type sequence.
• - epitope according to a) is an at least 20 amino acids long segment of the portion of said DNA
• Topoisomerase I spanning amino acids 5 to 30, and/or - epitope according to b) is an at least 20 amino acids long segment of the portion of said DNA Topoisomerase I spanning amino acids 350 to 400, and/or, preferably
• - epitope according to c) is an at least 20 amino acids long segment of the portion of said DNA Topoisomerase I spanning amino acids 451 to 593. In a preferred embodiment all the epitopes are present on separate peptides.
• peptides epitopes a) and b) are present on different peptides.
• peptides may be used wherein epitopes according to a) and c) are present on the same peptide and epitope according to b) is present on a different peptide, or epitopes according to b) and c) are present on the same peptide and epitope according to a) is present on a different peptide. If binding of an antibody to an epitope can be detected specifically, in an alternative embodiment, epitopes according to a) and c) may be present on one peptide and epitopes according to b) and c) may be present on another peptide.
• the invention relates to a kit, preferably a diagnostic kit, for use in the diagnosis of a systemic autoimmune disorder, said kit comprising one or more peptides as defined in any of the previous claims, said one or more peptide(s) separately or simultaneously comprising at least two of the following epitopes: a) an epitope having the immunological property of an at least 20 amino acids long segment of the portion of a DNA Topoisomerase I spanning amino acids 5 to 30, b) an epitope having the immunological property of an at least 20 amino acids long segment of the portion of a DNA Topoisomerase I spanning amino acids 350 to 400, and preferably c) an epitope having the immunological property of an at least 20 amino acids long segment of the portion of a DNA Topoisomerase I spanning amino acids 451 to 593.
• a preferred kit of the invention comprises
• - at least one peptide selected from a) a peptide comprising an epitope having the immunological property of an at least 20 amino acids long segment of the portion of a DNA Topoisomerase I spanning amino acids 5 to 30, b) a peptide comprising an epitope having the immunological property of an at least 20 amino acids long segment of the portion of a DNA Topoisomerase I spanning amino acids 350 to 400, and
• a reference peptide comprising an epitope having the immunological property of an at least 20 amino acids long segment of the portion of a DNA Topoisomerase I spanning amino acids 451 to 593; and optionally - means for detecting binding of patient autoantibodies to a peptide epitope.
• a reference peptide comprising an epitope having the immunological property of an at least 20 amino acids long segment of the portion of a DNA Topoisomerase I spanning amino acids 451 to 593; and optionally - means for detecting binding of patient autoantibodies to a peptide epitope.
• the peptide according to a) is a peptide fragment of said DNA Topoisomerase I spanning amino acids
• the peptide according to b) is a peptide fragment of said DNA Topoisomerase I spanning amino acids 350 to 400, and
• the peptide according to c) is a peptide fragment of said DNA Topoisomerase I spanning amino acids 451 to 593.
• the invention relates to a diagnostic method useful for differential diagnosis of systemic autoimmune disorders comprising the steps of i) contacting a sample obtained from a patient with one or more peptide(s) comprising at least one of the following epitopes: a) an epitope having the immunological property of an at least 20 amino acids long segment of the portion of said DNA Topoisomerase I spanning amino acids 5 to 30, b) an epitope having the immunological property of an at least 20 amino acids long segment of the portion of said DNA Topoisomerase I spanning amino acids 350 to 400 said one or more peptide(s) having an amino acid sequence which is at least 70%, 75%, 80%, 85% or 90%, 92%, 94%, 96% or 98% identical with the amino acid sequence of a peptide fragment of a wild type DNA Topoisomerase I, wherein - if binding of autoantibodies of said sample to epitope according to a) is detected, it is considered as indicative of diffuse cutane systemic s
• SLE systemic lupus erithematosus
• the one or more isolated peptide(s) also comprise c) an epitope having the immunological property of the at least 20 amino acids long segment of the portion of said DNA Topoisomerase I spanning amino acids 451 to 593, and wherein
• dsSSC diffuse cutane systemic scleroderma
• the one or more peptide(s) as defined above are used in the methods, uses and or kits of the invention.
• the sample is a blood sample, preferably a serum sample.
• An epitope having the immunological property of a segment of a portion of DNA Topoisomerase F is understood herein as an epitope capable of binding an antibody also capable of binding to said segment, preferably a patient antibody, more preferably a patient antibody of a patient having a systemic disease, preferably a systemic sclerosis or systemic lupus erythematosus.
• DNA Topoisomerase F in accordance with the present invention can be a wild type DNA Topoisomerase I (EC 5.99.1.2.) from eukaryotic, preferably vertebrate, preferably mammalian, more preferably human source, or having the sequence identical with such a wild type DNA Topoisomerase
• DNA Topoisomerase I is maintained or one or more antibody capable of binding to the wild type protein is capable of binding to the mutant protein as well.
• Said DNA Topoisomerase I can be isolated from a natural source or recombinantly prepared.
• peptide denotes any sequence of amino acids having an epitope for autoantibodies to eukaryotic, preferably vertebrate, preferably mammalian, more preferably human topoisomerase I (topo I).
• sequence of amino acids of said peptide is encoded by all or part of a polynucleotide, e.g. a cDNA, encoding said topo I or the amino acids sequence of said peptide having a sequence identity of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 97%, 98%, 99% or 100% therewith.
• “Autoantibodies” are antibodies of a subject raised against or capable of binding to a protein of said subject.
• Patient refers to an animal or human subject who is treated, diagnosed or a body sample of whom is analyzed, or to be treated, diagnosed or analyzed.
• FIG. 1 The pattern of recognized topoisomerase I (topo I) epitopes is different between dcSSc, IcSSc and SLE patients.
• Deduced amino acid sequences of phage clones selected with IgG purified from 5 diffuse cutaneous systemic sclerosis (dcSSc), 6 limited cutaneous systemic sclerosis (IcSSc) and 4 systemic lupus erythematosus (SLE) patients are plotted along the human topo I sequence.
• FIG. 3 Immunoblots using topoisomerase I (topo I) fusion proteins F4 and Fl as antigens.
• Purified recombinant fusion proteins panel A: F4, panel B: Fl
• MBP maltose binding protein
• the membranes were cut into strips and probed with serial serum samples (obtained at dates indicated) of patients or with an anti-MBP antibody (a-MBP).
• MW molecular weight marker (kDa).
• anti-topo I antibodies in pathogenesis of SSc is not fully understood, however, it can be assumed based on the present invention that immune response against topo I may differ among anti- topo I positive patients leading to production of anti-topo I autoantibodies with different epitope specificity.
• the present inventors have chosen a different strategy and have constructed an antigen fragment library of topo I displayed on bacteriophage lambda and screened this library with sera of dcSSc, IcSSc and SLE patients. Regions of topo I selected from the library were expressed as recombinant fusion proteins and were further tested with patients' sera. Longitudinal analysis of epitope specificities has been performed and compared with clinical findings.
• fragment F4 (AA 451-593) detected in all patient sera tested
• fragment Fl (AA 5-30) was specifically recognized by a subset of dcSSc patients' sera
• fragment F8 (350-400) was recognized by SLE patients, indicating that these fragments could represent characteristic epitopes for dcSSc and SLE, respectively.
• fragment F4 In addition to an immunodominant part of topo I (fragment F4), two new regions have been identified which were previously not shown to be targeted by anti-topo I antibodies. dcSSc patients recognized several short fragments (spanning AA 5-145) at the N-terminal part of the molecule. Specifically, fragment Fl (AA 5-30) was recognized by a subset of dcSSc patients' sera, while fragment F8 was recognized by SLE patients.
• fragment Fl contains an experimentally proven granzyme B cleavage site [22].
• fragment Fl contains an experimentally proven granzyme B cleavage site [22].
• in vivo cleavage of topo I by granzyme B released during T cell mediated cytotoxic responses results in the formation of a neo-antigenic determinant represented by fragment Fl.
• In vitro assays using the full length antigen or the full length N-terminal domain may fail to detect antibodies recognizing these short epitopes.
• any of the methods below are applicable: an immunoassay, e.g. ELISA, RIA, lateral flow, immunoprecipitation, a binding assay, e.g. Biacore, fluorescence quenching, a spectrophotometric method, e.g. FT-IR, circular dichroism, NMR, a physico-chemical method, e.g. calorimetry, ultracentrifugation etc.
• an immunoassay e.g. ELISA, RIA, lateral flow
• immunoprecipitation e.g. Biacore
• a binding assay e.g. Biacore
• fluorescence quenching e.g. Biacore
• a spectrophotometric method e.g. FT-IR, circular dichroism, NMR
• a physico-chemical method e.g. calorimetry, ultracentrifugation etc.
• the diagnostic method is carried out in the form of an immunoassay, like RIA or DELPHIA or preferably in an immunosorbent assay, like ELISA.
• an immunoassay like RIA or DELPHIA
• an immunosorbent assay like ELISA.
• a detailed teaching can be found e.g. in the book Autoantibodies, by Yehuda Shoenfeld, Pier-Luigi Meroni, M. Eric Gershwin, Elsevier, second edition.
• the object may be to provide an assay which selectively differentiates among patient autoantibodies which a related to the disorder and other antibodies. Setting the sensitivity of the assay is well within the skills of a person skilled in the art, who will be able to find appropriate control peptides. Examples for such peptides are provided herein.
• a positive inner control is in fact F4 fragment itself.
• As a negative control other, non-immunogenic fragments or other proteins, like MBP can be used.
• IgM or IgG are measured as autoantibodies, provided that the patients are not deficient in any of these types.
• kits for performing the diagnostic methods as outlined above may comprise the parts as mentioned above or as useful in carrying out the methods outlined above.
• a kit necessarily comprises peptides carrying the epitope and at least instructions for use.
• topo I antigen fragment library Construction and affinity selection of topo I antigen fragment library
• the coding region of full length human topo I was amplified by PCR from cDNA reverse transcribed from total RNA.
• the PCR product was cloned into a T/A vector using the InsT/Aclone PCR Product Cloning Kit (Fermentas, Vilnius, Lithuania).
• Library construction was done using the lambdaD-bio phage display vector [9] with minor modifications as described previously [10].
• the primary topo I library contained 2xlO 7 insert bearing independent clones; titer of the amplified library was 3x10 1 VmI.
• topo I antigen fragment library with 5 dcSSc, 6 IcSSc and 4 SLE patient derived IgG purified on protein G sepharose (Amersham Pharmacia, Uppsala, Sweden) was performed essentially as described [H]. After the third round of selection individual clones were picked up for further propagation and DNA sequencing.
• MBP maltose binding protein
• Fusion proteins were purified from bacterial lysates with affinity chromatography on amylose resin according to the manufacturer's instruction (New England Biolabs, Ipswich, UK), and integrity of purified proteins was verified by SDS-PAGE on a 10% gel followed by Coomassie brilliant blue staining.
• 96-well polystyrene plates (Nunc, Roskilde, Denmark) were coated with recombinant topo I fragments or with MBP in PBS at a concentration of 10 ⁇ g/ml. Plates were washed with wash buffer (PBS, 0.05% Tween-20) and blocked with 3% non-fat dry milk in wash buffer for 1 h. Serum samples were incubated in triplicates at 1 :250 dilutions in wash buffer containing 2% non-fat dry milk for 1 h. Finally, the plate was incubated with HRP conjugated anti-human-IgG secondary antibody (Dako, Glostrup, Denmark) for 60 min.
• HRP conjugated anti-human-IgG secondary antibody Dako, Glostrup, Denmark
• the reaction was developed with o-phenylenediamine (Sigma-Aldrich, Budapest, Hungary), and optical density (OD) was measured at 492 nm.
• OD optical density
• sera of 146 healthy controls previously tested negative for anti-topo I antibody with a commercial ELISA kit
• Reactivity of healthy controls' sera with topo I fragments and MBP was shown to be minimal (OD 492 0.019-0.032), and a cut off value of 0.1 have been chosen for further measurements.
• MBP fusion proteins or MBP (40 ⁇ g/ml) diluted 1 :1 with SDS sample buffer were boiled for 10 minutes, separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. After blocking with 5% non-fat dry milk (Bio-Rad, Budapest, Hungary) in wash buffer (10OmM NaCl, 1OmM Tris-base pH 7.4, 0.1% Tween 20) for 1 h, membranes were incubated for 1 h with sera diluted 1 :500 in 2% non-fat dry milk in wash buffer. After washing, HRP-conjugated anti-human-IgG diluted at 1 :2000 was added for 1 hour.
• membrane strips were first incubated with rabbit anti-MBP antibody (New England Biolabs, Ipswich, UK) (1 :5000), followed by incubation with HRP-conjugated goat anti-rabbit antibody (1 :2000). Membranes were developed with SuperSignal West Pico Chemiluminescent (Pierce, Rockford, USA) substrate and exposed to x-ray films.
• Categorical data were analyzed by the Chi-square test. To investigate the possible differences between patient groups, frequency and mean values of continuous variables were tested by Student's t test. Spearman's rank correlation coefficient was used to examine the relationship between the values of optical density and continuous variables. A p value less than 0.05 was considered statistically significant. Statistical analyses were conducted using SPSS statistical software package.
• the present diagnostic method may not be applicable in each case, if the patient has topo-I reactive antibodies, it can safely differentiate between at least dcSSc and SLE.
• topo I antigen fragment library displayed on bacteriophage lambda, and subsequently screened this library with individual IgGs purified from sera of anti-topo I positive patients (5 dcSSc, 6 IcSSc and 4 SLE patient). After the third round of affinity selection inserts of 60 clones (30 from each patient group) were sequenced. Alignment of deduced amino acid sequences with human topo I showed that the pattern of recognized epitopes is different between dcSSc, IcSSc and SLE patients (fig 1).
• topo I antigen fragment library In order to verify results obtained by affinity selection of the topo I antigen fragment library we have constructed recombinant topo I-MBP fusion proteins. On the basis of fragments identified by library selection nine fusion proteins have been constructed and expressed (fig 2). Recognition of these fusion proteins was tested with sera of 67 anti-topo I antibody positive patients (34 dcSSc, 25 IcSSc and 8 SLE) including those which have been used for library selection by ELISA. The results are summarized in table 1.
• topo I topoisomerase I
• Fragment F4 (AA 450-600) was recognized by all of the 67 patients' sera.
• Fragment Fl (AA 5-30) was recognized by 9 of 34, 1 of 25 and 0 of 8 dcSSc, IcSSc and SLE patients, respectively.
• Fragment F8 (AA 350-400) was recognized by 4 of 8 SLE patients and none of the SSc patients.

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