Patent Application
20190120834
The present invention relates to a novel method for identifying marker sequences for rheumatoid arthritis, the novel marker sequences discovered with the aid of the method, and diagnostic use thereof. The invention also relates to diagnostic devices containing such marker sequences for rheumatoid arthritis, in particular a protein biochip or beads, and use thereof.
Rheumatoid arthritis (RA) is an autoimmune disease affecting approximately 1% of the population of the western world. Chronic inflammatory processes within the first year of the disease (early RA) lead already to progressive, joint-destroying synovitis. If the RA is not identified in good time and treated aggressively within a certain time window (“window of opportunity”), it leads to the destruction of joints with significant physical limitations and systematic manifestations, which lead to disabilities and reduced life expectancy.
In the early phase of the disease, for patients with joint inflammation, the criteria for diagnosis of RA and differentiation between arthritis and merely arthralgia (joint pain) are often difficult. This delays the therapy management for patients with possible early RA, and therefore the joint destruction can progress if the diagnosis is unclear.
Markers for the early detection of RA or prognosis and therapy management are immensely important, in particular in patients of rheumatoid arthritis (RA) who are already receiving drug treatment.
The current classification criteria published jointly by the American College for Rheumatology (ACR) and the European League Against Rheumatism (EULAR) in 2010 (Aletaha, Neogi et al. 2010)) are intended to facilitate an early diagnosis of RA by the determination of autoantibodies (AAB) against citrullinated antigens (ACPAs), such that disease-modifying therapy is started as early as possible and irreversible consequences of the disease are prevented.
ACPA autoantibodies are positively detectable in approximately 75% of patients with established RA, but only in approximately 62% of patients with early RA.
This emphasises the need for further markers for the early diagnosis of RA, prognosis, and therapy management.
Previously, the RA was considered to be a disease in which primarily autoantibodies against citrullinated peptides are produced. In a small number of publications, autoantibodies against posttranslational unmodified proteins or peptides have also been described (Hueber, Tomooka et al. 2009; Somers, Geusens et al. 2011), however there have been no attempts to seek new autoantibodies systematically or to use these for the identification of patient subgroups.
Based on the recommendations published in 2010 of the European League Against Rheumatism (EULAR), the primary goal of the treatment of PA is to stop the disease progression and achieve disease remission (Smolen, Landewe et al. 2010).
Protein biochips are gaining increasing industrial importance in analysis and diagnosis as well as in pharmaceutical development. Protein biochips have become established as screening tools.
Here, the rapid and highly parallel detection of a multiplicity of specifically binding analysis molecules in a single experiment is made possible. To produce protein biochips, it is necessary to have the required proteins available. In particular, protein expression libraries have been established for this purpose. High-throughput cloning of defined open reading frames is one possibility (Heyman, J. A., Cornthwaite, J., Foncerrada, L., Gilmore, J. R., Gontang, E., Hartman, K. J., Hernandez, C. L., Hood, R., Hull, H. M., Lee, W. Y., Marcil, R., Marsh, E. J., Mudd, K. M., Patino, M. J., Purcell, T. J., Rowland, J. J., Sindici, M. L. and Hoeffler, J. P. (1999) Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation. Genome Res, 9, 383-392; Kersten, B., Feilner, T., Kramer, A., Wehrmeyer, S., Possling, A., Witt, I., Zanor, M. I., Stracke, R., Lueking, A., Kreutzberger, J., Lehrach, H. and Cahill, D. J. (2003) Generation of Arabidopsis protein chip for antibody and serum screening. Plant Molecular Biology, 52, 999-1010; Reboul, J., Vaglio, P., Rual, J. F., Lamesch, P., Martinez, M., Armstrong, C. M., Li, S., Jacotot, L., Bertin, N., Janky, R., Moore, T., Hudson, J. R., Jr., Hartley, J. L., Brasch, M. A., Vandenhaute, J., Boulton, S., Endress, G. A., Jenna, S., Chevet, E., Papasotiropoulos, V., Tolias, P. P., Ptacek, J., Snyder, M., Huang, R., Chance, M. R., Lee, H., Doucette-Stamm, L., Hill, D. E. and Vidal, M. (2003) C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression. Nat Genet, 34, 35-41; Walhout, A. J., Temple, G. F., Brasch, M. A., Hartley, J. L., Lorson, M. A., van den Heuvel, S. and Vidal, M. (2000) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol, 328, 575-592). However, such an approach is closely linked to the progress of the genome sequencing projects and the annotation of these gene sequences. In addition, the determination of the expressed sequence is not always clear due to differential splicing processes. This problem can be avoided by the use of cDNA expression libraries (Bussow, K., Cahill, D., Nietfeld, W., Bancroft, D., Scherzinger, E., Lehrach, H. and Walter, G. (1998) A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library. Nucleic Acids Research, 26, 5007-5008; Bussow, K., Nordhoff, E., Lubbert, C., Lehrach, H. and Walter, G. (2000) A human cDNA library for high-throughput protein expression screening. Genomics, 65, 1-8; Holz, C., Lueking, A., Bovekamp, L., Gutjahr, C., Bolotina, N., Lehrach, H. and Cahill, D. J. (2001) A human cDNA expression library in yeast enriched for open reading frames. Genome Res, 11, 1730-1735; Lueking, A., Holz, C., Gotthold, C., Lehrach, H. and Cahill, D. (2000) A system for dual protein expression in Pichia pastoris and Escherichia coli, Protein Expr. Purif., 20, 372-378). Here, the cDNA of a specific tissue is cloned into a bacterial or eukaryotic expression vector, such as yeast. The vectors used for the expression are generally characterised in that they carry inducible promoters that may be used to control the time of protein expression. In addition, expression vectors have sequences for what are known as affinity epitopes or affinity proteins, which on the one hand permit the specific detection of the recombinant fusion proteins by means of an antibody directed against the affinity epitope, and on the other hand render possible the specific purification via affinity chromatography (IMAC).
By way of example, the gene products of a cDNA expression library from human foetal brain tissue in the bacterial expression system Escherichia coli were arranged in high-density format on a membrane and could be successfully screened with different antibodies. It was possible to show that the proportion of full-length proteins is at least 66%. Additionally, the recombinant proteins from expression libraries could be expressed and purified in a high-throughput manner (Braun P., Hu, Y., Shen, B., Halleck, A., Koundinya, M., Harlow, E. and LaBaer, J. (2002) Proteome-scale purification of human proteins from bacteria. Proc Natl Acad Sci USA, 99, 2654-2659; BUssow (2000) supra; Lueking, A., Horn, M., Eickhoff, H., BUssow, K., Lehrach, H. and Walter, G. (1999) Protein microarrays for gene expression and antibody screening. Analytical Biochemistry, 270, 103-111). Such protein biochips based on cDNA expression libraries are disclosed in particular in WO 99/57311 and WO 99/57312.
Auger et al. (2009) Annals of the Rheumatic Diseases, British Medical Association, London, GB, vol. 68, no. 4, pages 591-594 discloses a method for identifying IgG autoantibodies in sera of patients with rheumatoid arthritis (RA). Here, serum samples of patients with RA are examined comparatively with those of healthy control individuals on the Invitrogen ProtoArray (8268 human proteins as GST fusion proteins, purified under native conditions and spotted onto a glass slide coated with nitrocellulose). The arrays are incubated with the serum samples and examined by Alexa Fluor 647 conjugated to anti-human IgG. A panel of measured values is evaluated by Z-score, CIP (Chebyshev Inequality Precision) and CV (Coefficient of Variation). In Auger et al. the antigens peptidylarginine deiminase 4 (PAD4), protein kinase CR1 (PKCR1), phosphatidylinositol-4-phosphate-5-kinase type II γ (PIP4K2C) and v raf murine sarcoma viral oncogene homologue B1 catalytic domain (BRAF) were identified using this method. Auger et al. does not disclose the diagnostic use of the identified antigens.
EP 1 731 608 A1 discloses a method for identifying “genes susceptible to RA” by gene mapping with the aid of microsatellite markers and PCR techniques. With the aid of the method the genes TNXB, NOTCH4 (chromosome 6), RAB6A, MPRL48, FLJ11848, UCP2 and UCP3 (chromosome 11) were discovered in human genomic DNA. EP 1 731 608 A1 claims a marker gene for an RA test consisting of a partial DNA sequence of one of the discovered marker genes and comprising at least one SNP in human genomic DNA. In addition, a method is disclosed for detecting RA comprising the steps of obtaining partial DNA sequences corresponding to one of the marker genes from a subject to be examined, determining the nucleotide sequence of the partial DNA sequence, and comparing the nucleotide sequence to the corresponding nucleotide sequence obtained from a normal individual. A test kit for RA comprising one of the marker genes or a primer derived therefrom, a polypeptide coded by one of the marker genes, and a screening method are also disclosed.
WO 2009/138408 A2 claims a diagnostic method, in which an autoantigen marker comprising the catalytic domains of BRAF or an antibody fragment thereof is used to detect RA, wherein, where appropriate, anti-PAD4 antibodies are also detected in the biological sample of the subject to be examined. WO 2009/138408 A2 also discloses a detection kit for detecting anti-BRAF autoantibodies, an array with autoantigen markers comprising BRAF and PAD4 for diagnosing RA, and the use of an autoantigen marker comprising BRAF for diagnosing RA, preferably in patients who are CCP negative (page 3, paragraph 1).
In WO 2009/138408 A2 the biomarkers were identified in that serum from RA patients and controls (patients with spondylarthropathy (AS)), systemic lupus erythematosus (SLE), systemic sclerosis (SSC) and healthy individuals) were screened with the ProtoArray Human Protein Microarray (Invitrogen), wherein the detection was performed by means of anti-human IgG conjugated to Alex Fluor 647. A panel of measured values was evaluated by Z-score, CIP and CV.
WO 2007/039280 A1 claims a method for the differential diagnosis of RA by determining the concentration of anti-CCP and anti-nuclear antibodies in a sample and correlation with the diagnosis of RA. Here, the markers CRP, SAA, IL-6, S100, osteopontin, RF, MMP-1, MMP-3, hyaluronic acid, sCD14, angiogenesis markers and products from the metabolism of bone, cartilage or synovial membrane can be used in addition. WO 2007/039280 A1 also claims the use of a panel comprising anti-CCP and ANA for the diagnosis of RA, and a test kit.
Nicaise et al. (2008) Arthritis Research Therapy, Biomed Central LTD, GB, vol. 10, no. 6, pages R142-R142.7 examines the suitability of anti-MCV (mutated citrullinated vimentin) antibodies for the diagnosis of RA in CCP-negative patients and the use for monitoring during therapy with Infliximab. Here, groups of patients with RA and CCP and with RA and without CCP are compared with patients having other rheumatic diseases (psoriatic rheumatism, primary SjOgren's syndrome, ankylosis spondylitis) and healthy control individuals. The use of an array/an arrangement is not described.
Vossenaar et al. (2004) Clinical and Applied immunology Reviews 4, 239-262 concerns the use of citrullinated autoantigens and of anti-CCP antibodies and antigens thereof as serological markers for the detection of RA. Vossenaar et al. proposes using microarray technology to analyse autoantibody profiles of RA patients (page 254, paragraph 2).
US 2007/0254300 A1 uses a yeast two-hybrid system, in order to identify anti-inflammatory compounds ((0504) to [0506]) and claims a protein complex, wherein the first protein is PAK, a fragment thereof, or a fusion protein containing this, and the second protein is ERK3, PRKAR1A, KRT23(209), PN7098, AL117237, PCNT2, PROX1, HOOKI, IGHG1, GOLGA2, KIAA0555, LRPPRC or a fragment of these proteins. US 2007/0254300 A1 also discloses a microarray comprising this protein complex and a method for discovering “modulators” of the protein complex using this microarray. A method for detecting a change in an inflammatory disease, for example RA, is also claimed, wherein the sample of a patient is examined to ascertain whether a change in the expression level of one of the proteins in Tables 1 to 82 (82 different proteins are specified here) or in the nucleotide sequence of a gene coding for the 82 proteins is determined compared with patients without this inflammatory disease.
WO 2009/030226 discloses marker sequences for rheumatoid arthritis and diagnostic use thereof as well as a method for screening potential active ingredients for rheumatoid arthritis by means of these marker sequences. A diagnostic device containing such marker sequences for rheumatoid arthritis, in particular a protein biochip, and use thereof are also disclosed.
DE 10 2007 041 656 A1 discloses the use of marker sequences for diagnosing RA, methods for diagnosing RA with use of these marker sequences, methods for stratification, an arrangement of marker sequences, an assay/protein biochip, the use of the arrangement, diagnostic agents comprising the marker sequences, a target for treatment and therapy, and the use of the marker sequences to carry out an apheresis.
The RA-specific expression clones were obtained in DE 10 2007 041 656 A1 by screening 10 or more patient samples individually against a cDNA expression library and were identified by comparison with 10 or more healthy samples. FIG. 1 shows the differential screening between two protein biochips, one from a cDNA expression bank of a patient and one from a healthy test subject. The differential clones are detected by means of fluorescence labelling and evaluated by means of bioinformatics.
There is still a pressing need for indication-specific diagnostic devices for rheumatoid arthritis.
The object of the present invention is therefore to discover improved marker sequences for rheumatoid arthritis and to specify a diagnostic use thereof.
The provision of specific marker sequences allows a reliable diagnosis and stratification of patients with rheumatoid arthritis (RA), particularly advantageously in seronegative RA patients.
In a multi-stage method comprising firstly the selection of marker sequence candidates with the aid of protein biochips and the subsequent validation thereof by means of beads, highly specific marker sequences are discovered for rheumatoid arthritis.
The invention relates to a method for identifying marker sequences for rheumatoid arthritis (RA), comprising the steps of:
In the field of microarrays flat substrates are used, to which marker sequences or sequences to be examined are bound. In protein biochips the marker sequences to be examined or the sequences binding to these marker sequences are immobilised on a solid, flat support. An alternative arrangement or panel of marker sequences or sequences to be examined is possible on beads, which therefore differ inter alia in view of their sensitivity and specificity from conventional microarrays. Bead arrays are created for example by impregnating pellets either with different concentrations of fluorescent dye or for example by barcode technology. The pellets can be addressed and can be used to identify specific binding events that occur on their surface. Bead technology is based on microscopically small spherical pellets or platelets, which are referred to as microspheres or beads. These beads can serve analogously to ELISA and Western Blot as solid phase for biochemical detection reactions. A wide range of different bead types are available, which for example differ in their fluorescence shade and each of which carries its own specific detection reagent on the surface. In this way, an accordingly large number of different detection reactions can be carried out simultaneously in a very small sample volume. With bead arrays specific interactions between two defined biochemical compounds can be detected. Compared with conventional microarrays, the bead-based validation is characterised by a particularly high sensitivity and specificity. With the method according to the invention and the use of beads for validation, marker sequences for RA can be identified that differ in terms of their sensitivity and specificity from the previously known marker sequences.
The invention also relates to a marker sequence for rheumatoid arthritis obtainable by a method according to the invention, wherein the marker sequence is selected from the group of sequences SEQ ID No. 1 to 84, partial sequences or fragments thereof, and homologues of sequences SEQ ID No. 1 to 84 with at least 90% homology.
The invention also relates to the use of one or more marker sequence(s) according to the invention for the diagnosis of rheumatoid arthritis.
One embodiment concerns the use according to the invention, wherein the marker sequence(s) is/are determined on or from a patient to be examined.
One embodiment concerns the use according to the invention, characterised in that 2 or 3, preferably 4 or 5, particularly preferably 6, 7 or 8 or more different marker sequences, for example 10 to 20 or 30 or more different marker sequences, are determined on or from a patient to be examined.
One embodiment concerns the use according to the invention, characterised in that the marker sequence(s) is/are applied to a solid support, wherein the solid support is selected from filters, membranes, wafers, for example silicon wafers, glass, metal, plastic, chips, mass spectrometry targets, matrices, and beads, for example magnetic, coated or labelled beads, such as fluorophore-labelled beads or Luminex beads.
The invention also relates to a method for diagnosing rheumatoid arthritis, wherein
a.) at least one marker sequence according to the invention is applied to a solid support, preferably to a bead and
b.) is brought into contact with bodily fluid or tissue sample of a patient and
c.) an interaction of the bodily fluid or of the tissue sample with the marker sequence from a.) is detected.
Such an interaction can be detected for example by a probe, in particular by an antibody.
The invention also relates to a method for stratification, in particular for risk stratification, or for therapy management of a patient with rheumatoid arthritis, wherein at least one marker sequence according to the invention is used in order to examine a sample from the patient.
One embodiment concerns a method according to the invention for diagnosing rheumatoid arthritis, wherein the stratification or the therapy management includes decisions regarding the treatment and therapy of the patient, in particular the hospitalisation of the patient, the use, efficacy and/or dosage of one or more drugs, a therapeutic measure or the monitoring of the course of a disease and the course of therapy, aetiology or classification of a disease, inclusive of prognosis.
The invention also relates to an arrangement or panel comprising or consisting of one or more marker sequence(s) according to the invention.
The invention also relates to an assay or protein array comprising an arrangement or panel according to the invention.
The invention also relates to the use of an arrangement/panel according to the invention or of an assay or protein array according to the invention for identifying and/or characterising a substance for rheumatoid arthritis containing means for detecting binding success, characterised in that an arrangement/panel or an assay or protein array is brought into contact with a.) at least one substance to be examined, and b.) binding success is detected.
The invention also relates to a diagnostic agent for the diagnosis of rheumatoid arthritis containing at least one marker sequence according to the invention and where appropriate further auxiliaries and additives.
The invention also relates to a target for the treatment or therapy of rheumatoid arthritis, wherein the target is selected from the marker sequences according to the invention.
The invention also relates to the use of at least one marker sequence according to the invention for identifying a subgroup of patients within the group of patients with rheumatoid arthritis, wherein the patients of the subgroup cannot be identified by means of the marker CCP and/or cannot be identified with the markers or marker sequences for rheumatoid arthritis known in the prior art.
The invention therefore relates to the use of marker sequences for the diagnosis of rheumatoid arthritis, wherein at least one marker sequence selected from the group of marker sequences SEQ ID No. 1 to 84 and/or the genomic sequences comprising one of the sequences SEQ ID No. 1 to 42 and/or a protein coded by the sequences SEQ ID No. 43 to 84, partial sequences or fragments thereof, and homologues of sequences SEQ ID No. 1 to 84 with at least 90% homology is determined on or from a patient to be examined.
A further embodiment of the invention concerns the use of the marker sequence(s) according to the invention for the diagnosis of rheumatoid arthritis, characterised in that the determination is performed by means of in-vitro diagnosis.
The marker sequences according to the invention were able to be identified by means of differential screening of samples from healthy test subjects with patient samples with rheumatoid arthritis. The marker sequences according to the invention were then expressed and, following coupling of the expressed marker sequence candidates to Luminex beads, validated with the aid of the Luminex beads, partly by comparison with known biomarkers for rheumatoid arthritis. Highly specific marker sequences could thus be identified for rheumatoid arthritis.
“Beads” (pearls, pellets, originally also referred to as latex particles) designate what are known as microspheres or microparticles, which are used as supports for biomolecules in tests and assays. Uniform (approximately equally sized) microparticles that are produced by special chemical methods are required for tests and assays. These methods are known to a person skilled in the art. Beads for different applications are also commercially available (for example from the company Progen Biotechnik GmbH). Beads may consist of different materials, for example glass, polystyrene, PMMA and different other polymers, partly also copolymers. Beads can be labelled with different dyes or dye mixtures and can be provided with coatings. Biomolecules can be coupled to the surface of beads. Different coupling methods are available for this purpose and are known to a person skilled in the art, for example adsorption or covalent coupling. The surface of the beads can be modified, such that a directed coupling of the biomolecules on the bead surface, for example in conjunction with spacers, tags or special modifications, is possible, and whereby the analytical sensitivity can be further increased.
The term “rheumatoid arthritis (RA)” is defined for example by Pschyrembel, de Gruyter, 261st edition (2007), Berlin. In accordance with the invention “juvenile idiopathic arthritis” is also included (ICD-10: M08.-. abb.: JIA. Earlier synonyms: juvenile rheumatoid arthritis, juvenile chronic arthritis, Still's disease or the popular name “child's rheumatism”) and is the collective term for a series of diseases primarily affecting the joints (arthritis) of rheumatic origin in childhood (juvenile) (definition for example according to Pschyrembel, de Gruyter, 261st edition (2007), Berlin). This is a polygenic disease that can be diagnosed particularly advantageously by means of the marker sequences according to the invention, preferably SEQ ID No. 1 to 84.
In a further embodiment of the invention the marker sequences according to the invention can also be combined, supplemented, consolidated or expanded with known biomarkers for this indication.
In a preferred embodiment the marker sequences are determined outside the human body and the determination is performed in an ex vivo/in vitro diagnosis.
In the sense of this invention, “diagnosis” means the positive determination of rheumatoid arthritis by means of the marker sequences according to the invention as well as the assignment of the patients to the indication rheumatoid arthritis. The term diagnosis includes the medical diagnostics and examinations in this regard, in particular in-vitro diagnostics and laboratory diagnostics, and also proteomics and nucleic acid blotting. Further tests may be necessary to be sure and to exclude other diseases. The term diagnosis therefore also includes the differential diagnosis of rheumatoid arthritis by means of the marker sequences according to the invention, and the prognosis in the case of determined rheumatoid arthritis.
The invention also relates to a method for the stratification, in particular risk stratification and/or therapy management of a patient with rheumatoid arthritis, for example in a patient with a very early stage of RA or RA that cannot be detected by means of the marker CCP, wherein at least one marker sequence according to the invention is determined on a patient to be examined. The stratification of the patient with rheumatoid arthritis in new or established sub-groups within the disease rheumatoid arthritis is also included, as well as the expedient selection of patient groups for the clinical development of new therapeutic agents or the selection for therapy with certain active agents. The term therapy management also includes the division of patients into responders and non-responders in respect of a therapy or the course of a therapy
In the sense of this invention, “stratification or therapy management” means that the method according to the invention renders possible decisions for the treatment and therapy of the patient, whether it is the hospitalisation of the patient, the use, efficacy and/or dosage of one or more drugs, a therapeutic measure, or the monitoring of the course of a disease and the course of therapy or aetiology or classification of RA, for example into a new or existing sub-type, or the differentiation of RA and relevant patients.
In a further embodiment of the invention, the term “stratification” in particular includes the risk stratification with the prognosis of an “outcome” of a negative health event.
Within the scope of this invention, the term “patient” is understood to mean any test subject (human or mammal), with the provision that the test subject is examined for rheumatoid arthritis.
The term “marker sequences” in the sense of this invention means that the nucleic acid sequence, for example the mRNA, cDNA or the polypeptide or protein obtainable therefrom are significant for rheumatoid arthritis. By way of example the mRNA or cDNA or the polypeptide or protein obtainable therefrom can interact with substances from the bodily fluid or tissue sample of a patient with rheumatoid arthritis (for example (auto)antigen (epitope)/(auto)antibody (paratope) interaction).
In the sense of the invention “wherein at least one marker sequence selected from the group of marker sequence SEQ ID No. 1 to 84 and/or the genomic sequences comprising one of the sequences SEQ ID No. 1 to 42 and/or a protein coded by the sequences SEQ ID No. 43 to 84, partial sequences or fragments thereof, homologues of sequences SEQ ID No. 1 to 84 with at least 90% homology is determined on or from a patient to be examined” means that an interaction between the bodily fluid or the tissue sample of a patient and the marker sequence(s) according to the invention is detected. Such an interaction is, for example, a binding, in particular a binding substance at least at one of the marker sequences according to the invention, or in the case of a cDNA is the hybridisation with a suitable substance under selected conditions, in particular stringent conditions (for example as defined typically in J. Sambrook, E. F. Fritsch, T. Maniatis (1989), Molecular cloning: A laboratory manual, 2nd Edition, Cold Spring Habor Laboratory Press, Cold Spring Habor, USA or Ausubel, “Current Protocols in Molecular Biology”, Green Publishing Associates and Wiley Interscience, N.Y. (1989)). One example for stringent hybridisation conditions is: hybridisation in 4×SSC at 65° C. (alternatively in 50% formamide and 4×SSC at 42° C.), followed by a number of washing steps in 0.1×SSC at 65° C. for a total of about one hour. One example for less stringent hybridisation conditions is hybridisation in 4×SSC at 37° C., followed by a number of washing steps in 1×SSC at room temperature.
Such substances, in accordance with the invention, are part of a bodily fluid, in particular blood, whole blood, blood plasma, blood serum, patient serum, urine, cerebrospinal fluid, synovial fluid, or a tissue sample of the patient.
In a further embodiment of the invention the marker sequences according to the invention can be present in the examined test subjects in a significantly higher or lower expression rate or concentration compared with the expression rate or concentration of the marker sequence in question in a healthy individual or in a test subject without RA. The increased or reduced expression rate or concentration is an indication of rheumatoid arthritis and the diagnosis RA. The relative expression rates diseased/healthy of the marker sequences according to the invention can be determined for example by means of proteomics or nucleic acid blotting.
The marker sequences according to the invention, in a further embodiment of the invention, have an identification signal, which is addressed to the substance to be bound (for example antibody, nucleic acid). In accordance with the invention, the recognition signal for a protein is preferably an epitope and/or paratope and/or hapten, and for a cDNA is preferably a hybridisation or binding region. In a particular embodiment of the invention the marker sequences according to the invention identify autoantibodies that are specific for RA or that are formed and/or are formed to an increased or reduced degree with the onset and the development of the RA disease. Two or more marker sequences according to the invention can be used to detect autoantibody profiles or changes in autoantibody profiles during therapy or during the course of the disease or to monitor such changes within the scope of follow-up care.
The marker sequences according to the invention SEQ ID No. 1 to 84 are specified in Table 1 and can be unambiguously identified (see RefSeq Accession or GI Accession) by the respective cited database entries (also by means of the Internet: http://www.ncbi.nlm.nih.gov/).
The invention therefore also relates to the full-length sequences of the marker sequences according to the invention and the marker sequences as defined in the tables via the known database entries and also the marker sequences specified in the accompanying sequence protocol.
The invention furthermore likewise includes analogous embodiments of the marker sequences, in particular of the nucleic acid sequences SEQ ID No. 1 to 42 and the protein sequences SEQ ID No. 43 to 84.
In a further embodiment of the invention marker sequences are preferred that have P-values less than or equal to 0.006, preferably less than or equal to 0.001 or less than or equal to 0.0001, particularly preferably less than or equal to 0.00001 (see Tables 2 and 3).
SEQ ID No. 4 and 46 (DCTN1) are very particularly preferred, wherein responsiveness is observed in all patients. SEQ ID No. 5 and 47 (GNPTG), SEQ ID No. 6 and 48 (HNRNPA1), and SEQ ID No. 7 and 49 (ITFG3) are also preferred.
An arrangement or panel containing at least one sequence selected from the group SEQ ID No. 4 and 46 (DCTN1), SEQ ID No. 5 and 47 (GNPTG), SEQ ED No. 6 and 48 (HNRNPA1) and SEQ ID No. 7 and 49 (ITFG3) and optionally further marker sequences according to the invention is also preferred.
In a further embodiment of the invention homologues of the marker sequences according to the invention are included. In particular, these are homologues having an identity of 70%, 80% or 85%, preferably 90%, 91%, 92%, 93%, 94% or 95% identity, in particular 96%, 97%, 98%, 99% or more identity, with the marker sequences according to the invention and suitable for the use according to the invention—the detection of rheumatoid arthritis (what are known as “homologues” or homologous marker sequences). Homologues can be protein sequences or nucleic acid sequences.
Partial sequences or fragments are sequences that comprise 50 to 100 nucleotides or amino acids, preferably 70-120 nucleotides or amino acids, particularly preferably 100 to 200 nucleotides or amino acids of one of the marker sequences SEQ ID No. 1 to 84.
In accordance with the invention the marker sequences also comprise modifications of the nucleotide sequence, for example of the cDNA sequence and the corresponding amino acid sequence, such as chemical modification, for example citrullination, acetylation, phosphorylation, glycosylation or polyA strand and further modifications known accordingly to a person skilled in the art.
In a further embodiment the respective marker sequence can be represented in different amounts in one or more regions on a solid support, for example a bead. This allows a variation of the sensitivity. The regions may each comprise a totality of marker sequences, i.e. a sufficient number of different marker sequences, in particular 2 to 5 or 10 or more marker sequences, and where appropriate further nucleic acids and/or proteins, in particular biomarkers. However, at least 96 to 25,000 (numerically) or more different or identical marker sequences and further nucleic acids and/or proteins, in particular biomarkers, are preferred. Furthermore, more than 2,500 different or identical marker sequences are preferred, particularly preferably 10,000 or more, and where appropriate further nucleic acids and/or proteins, in particular biomarkers.
Within the scope of this invention, “arrangement” or “panel” is synonymous with “array”, and, if this “array” is used to identify substances to be bound on marker sequences, this is to be understood to be an “assay” or a diagnostic device. In a preferred embodiment the arrangement is designed such that the marker sequences represented on the arrangement are present in the form of a grid on a solid support. Furthermore, those arrangements are preferred that permit a high-density arrangement of marker sequences, and the marker sequences are spotted. Such high-density spotted arrangements are disclosed for example in WO 99/57311 and WO 99/57312 and can be used advantageously in a robot-assisted automated high-throughput method.
Within the scope of this invention, however, the term “assay” or diagnostic device likewise comprises those embodiments of a device such as ELISA (for example individual wells of a microtitre plate are coated with the marker sequences or combinations of marker sequences according to the invention, and where appropriate are applied to the individual wells of the microtitre plate in a robot-assisted manner; examples include diagnostic ELISA kits from the company Phadia or “Searchlight” Multiplex ELISA kits from the company Pierce/Thermo Fisher Scientific), bead-based assay (spectrally distinguishable bead populations are coated with marker sequences/combinations of marker sequences. The patient sample is incubated with this bead population and bound (auto)antibodies are detected by means of a further fluorescence-labelled secondary antibody or a detection reagent by measuring the fluorescence; for example Borrelia IgG kit or Athena Multilyte from the company Multimetrix), line assay (marker sequences or combinations of marker sequences according to the invention are irrmmobilised in a robot-assisted manner on membranes, which are examined or incubated with the patient sample; example “Euroline” from the company Euroimmun AG), Western Blot (example “Euroline-WB” from the company Euroimmun AG), and immunochromatographic methods (for example what are known as lateral flow immunoassays; marker sequences or combinations of marker sequences are immobilised on test strips (membranes, U.S. Pat. No. 5,714,389 and many others); example “One Step HBsAg” test device from Acon Laboratories) or similar immunological single or multiplex detection methods.
A further object of the invention is therefore that of providing a diagnostic device or an assay, in particular a protein biochip, which allows a diagnosis or examination for rheumatoid arthritis.
In order to achieve this object, the marker sequences of the arrangement or panel according to the invention are fixed on a solid support, but preferably spotted or immobilised or imprinted, i.e. are applied reproducibly. One or more marker sequences can be present repeatedly in the totality of all marker sequences and can be present in different amounts based on a spot. Furthermore, the marker sequences on the solid support can be standardised (for example by means of serial dilution series for example of human globulins as internal calibrators for data standardisation and quantitative assessment).
The invention therefore concerns an assay or protein biochip or one or more beads (bead-based assay) consisting of an arrangement or panel containing marker sequences according to the invention.
In a further embodiment the marker sequences are present as clones. Such clones can be obtained for example by means of a cDNA expression library according to the invention (Bussow et al. 1998 (above)). In a preferred embodiment such expression libraries containing clones are obtained using expression vectors from a cDNA expression library consisting of the cDNA marker sequences. These expression vectors preferably contain inducible promoters. The induction of the expression can be carried out for example by means of an inducer, such as IPTG. Suitable expression vectors are described in Terpe et al. (Terpe T Appl Microbiol Biotechnol. 2003 January; 60(5):523-33).
Expression libraries are known to a person skilled in the art; they can be produced in accordance with standard works, such as Sambrook et al, “Molecular Cloning, A laboratory handbook, 2nd edition (1989), CSH press, Cold Spring Harbor, N.Y. Expression libraries that are tissue-specific (for example human tissue, in particular human organs) are furthermore preferable. Further, expression libraries that can be obtained by means of exon-trapping are also included in accordance with the invention. Instead of the term expression library, reference may also be made synonymously to an expression bank.
Protein biochips or beads or corresponding expression libraries that do not exhibit any redundancy (what is known as a Uniclone® library) and that can be produced in accordance with the teaching of WO 99/57311 and WO 99/57312 are furthermore preferred. These preferred Uniclone® libraries have a high proportion of non-defective fully expressed proteins of a cDNA expression library.
Within the scope of this invention the clones can also be, but are not limited to, transformed bacteria, recombinant phages or transformed cells of mammals, insects, fungi, yeasts or plants.
The clones are fixed, spotted or immobilised on a solid support.
The invention therefore relates to an arrangement, wherein the marker sequences are present as clones.
In addition, the marker sequences can be present in the respective form of a fusion protein, which for example contains at least one affinity epitope or “tag”. The tag may be or may contain one such as c-myc, his tag, arg tag, FLAG, alkaline phosphatase, V5 tag, T7 tag or strep tag, HAT tag, NusA, S tag, SBP tag, thioredoxin, DsbA, a fusion protein, preferably a cellulose-binding domain, green fluorescent protein, maltose-binding protein, calmodulin-binding protein, glutathione S-transferase or lacZ.
A marker sequence can be composed of a number of individual marker sequences. This may include the cloning of individual fragments to form a large common fragment and the expression of this combined fragment.
In all embodiments, the term “solid support” includes embodiments such as a filter, a membrane, a magnetic or fluorophore-labelled pellet, a silicon wafer, glass, metal, plastic, a chip, a mass spectrometry target or a matrix. However, a filter and beads are preferred in accordance with the invention.
Furthermore, PVDF, nitrocellulose or nylon is preferred as a filter (for example Immobilon P Millipore, Protran Whatman, Hybond N+ Amersham).
In a further preferred embodiment of the arrangement according to the invention, this corresponds to a grid with the dimensions of a microtiter plate (8-12 well strips, 96 wells, 384 wells or more), a silicon wafer, a chip, a mass spectrometry target or a matrix.
In a further preferred embodiment pellets or what are known as beads are used as support. Here, bead-based multiplex assays are preferably used. The analysis and evaluation of the bead-based assays can be performed for example with a Luminex analysis system, which is performed on the basis of the method of flow cytometry with use of two different lasers.
Whereas the measurements on planar protein arrays offer merely a dynamic range of 1.5-2 magnitudes (powers of 10), a dynamic range of 3.5-4 magnitudes can be covered by the use of Luminex beads. The measurements in the low response ranges also provide very good coefficients of variation (CVs), i.e. no more than 10%.
Whereas the measurements on planar protein arrays offer merely coefficients of variation (CVs) from 10 to 25% (intra-array comparison) or 10 to 50% (inter-array comparison), the CVs of the Luminex measurements are located between 3 to 10%. An assay quality not generally achieved by commercial ELISAs is thus provided. The known disadvantages (limited plexing rate by interference of different detection antibodies) for Luminex-based analysis and diagnostic methods do not occur with the UNIarray concept, since merely a single fluorescence-labelled anti-human IgG from goat, sheep or mouse is used as detection probe. Due to the transfer of the UNIarray concept to Luminex (i.e. bead-based protein arrays), a number of apparatuses can additionally be saved, i.e. protein printers, hybridisation machines and array readers, and can be replaced by one apparatus. Here, the UNIarray concept is not bound to Luminex, but can also be used on other platforms, such as Randox, VBC Genomics, etc. The high measurement accuracy and the low CVs of the individual measurements allow the use of better and new statistical methods for the identification of potent individual markers and also for rapid sorting of false positives.
In a further embodiment the invention relates to an assay or protein biochip for identifying and characterising a substance for rheumatoid arthritis, characterised in that an arrangement or assay according to the invention is brought into contact with a.) at least one substance to be examined, and b.) binding success is detected. The substance to be examined may be any native or non-native biomolecule, a synthetic chemical molecule, a mixture, or a substance library. Once the substance to be examined contacts a marker sequence, the binding success is evaluated, this being performed for example with use of commercially available image analysing software (GenePix Pro (Axon Laboratories), Aida (Raytest), ScanArray (Packard Bioscience).
Protein-protein interactions (for example protein at the marker sequence, such as antigen/antibody) or corresponding “means for detecting the binding success” can be visualised for example by means of fluorescence labelling, biotinylation, radio-isotope labelling or colloidal gold or latex particle labelling in the conventional manner. Bound antibodies are detected with the aid of secondary antibodies, which are labelled using commercially available reporter molecules (for example Cy, Alexa, Dyomics, FITC or similar fluorescent dyes, colloidal gold or latex particles), or with reporter enzymes, such as alkaline phosphatase, horseradish peroxidase, etc., and the corresponding colorimetric, fluorescent or chemoluminescent substrates. A readout is performed for example by means of a microarray laser scanner, a CCD camera or visually.
Patient groups were screened individually against a cDNA expression library. The identity of the marker sequences was determined by DNA sequencing.
Differential screening was performed between two protein biochips, one from a cDNA expression bank of a patient and one from a healthy test subject, and the differential clones were detected by means of fluorescence labelling and evaluated by means of bioinformatics.
6,000 proteins that were detected as antigens for inflammatory diseases were included in tests with protein biochips.
These 6,000 proteins were then produced in relatively large quantities (several mg), purified, and coupled on Luminex beads. In addition, biomarkers already known (public domain), such as CCP for rheumatoid arthritis, Aquaporin 4 for Neuromyolitis Optica, various cytokines, typical autoimmune markers, etc., were produced and measured together with the 6,000 proteins. The inclusion of known autoantigens in screening and validation is important insofar as the best new candidates can be selected very quickly as a result.
Multiplex Bead-Based Autoantibody Detection:
The 6,000 antigens were expressed recombinantly in E. coli and purified. To this end, 5 cDNA banks (oligo(dT)primed, his tag) from human tissue were used (inter alia intestine, lung, liver) (BUssow (1998), supra) and cloned into the expression vector pQE30-NST (ORFeome) (Rual J-F, Hirozane-Kishikawa T, Hao T, Bertin N, Li S, Dricot A, Li N, Rosenberg J, Lamesch P, Vidalain P-O, Clingingsmith T R, Hartley J L, Esposito D, Cheo D, Moore T, Simmons B, Sequerra R, Bosak S, Doucette-Stamm L, Le Peuch C, Vandenhaute J, Cusick M E, Albala J S, Hill D E, Vidal M: Human ORFeome version 1.1: a platform for reverse proteomics. Genome Res 2004, 14:2128-2135). Recombinant gene expression was performed in E. Coli SCS1 by means of pSE111 for improved expression of human genes (Brinkmann U, Mattes R E, Buckel P: High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product. Gene 1989, 85:109-114). Proteins were obtained from harvested and lysed cells (Overnight Express auto-induction medium, Novagen®, 6 M guanidinium-HCl, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 8.0) and purified (Protino® Ni-IDA 1000 Funnel Column (Macherey-Nagel®) and washed and eluted (6 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, 0.5% (w/v) trehalose pH 4.5) and stored at −20 degrees C.
The bead-based assay is performed by means of MagPlex™ microspheres, Luminex Corporation in accordance with Luminex protocol, wherein for each individual coupling reaction up to 12.5 μg antigen and 8.8×105 MagPlex™ beads from a colour region (ID) were used. The beads were evaluated in a Flexmap3D apparatus from Luminex Corp. (DD gate 7,500-15,000; sample size: 80 μl; 1000 events per bead ID; timeout 60 sec.) and the median fluorescence intensity (MFI) was determined.
Statistical Evaluation:
The statistical evaluation was performed by the Mann-Whitney test under consideration of the p-values and the absolute values of the fold-change of the patient groups (below); see also Tables 2 and 3.
The data were standardised by the method of Bolstad B M, Irizarry R A, Astrand M, Speed T P: A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinforma Oxf Engi 2003, 19:185-193, wherein the “statistical software R” (version 2.14.2 (2012-02-29)) [http://www.r-project.org] was used for all analyses and the reactome algorithm [http://www.reactome.com].
Patient groups were as follows: Group A: 84 consecutive patients with RA according to the American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) 2010 criteria (age 56.1±13.3 years, 73.6% female, Disease Activity Score for 28 joints (DAS28) 3.5±2.3, therapy: methotrexate 40%, leflunomide 12.5%, tumour necrosis factor alpha (TNF)-blockade 18%), Heinrich-Heine-University DUsseldorf, compared with 71 healthy controls (age 54.6±11.3 years, 73.2% female).
Group B (early RA): 116 patients with early RA from the HIT HARD study (Detert J, Bastian H, Listing. J, WeiB A, Wassenberg S, Liebhaber A, Rockwitz K, Alten R, KrUger K, Rau R, Simon C, Gremmelsbacher E, Braun T, Marsmann B, Hdhne-Zimmer V, Egerer K, Buttgereit F, Burmester G-R: Induction therapy with adalimumab plus methotrexate for 24 weeks followed by methotrexate monotherapy up to week 48 versus methotrexate therapy alone for DMARD-naive patients with early rheumatoid arthritis: HIT HARD, an investigator-initiated study. Ann Rheum Dis 2013, 72:844-850) (age 49.8±13.8 years, 71.3% female, DAS28 6.1±1.0, all therapy naive), compared with 116 healthy controls (age 49.8±12.8 years, 71.6% female).
Group C (seronegative RA group): 184 patients with ACPA-negative RA according to 2010 ACR/EULAR criteria (age 60.2±13.8 years, 62.5% female, all therapy naive) compared with 343 healthy controls (age 47.7±11.7 years, 58.3% female).
Table 1 summarises the identified sequences SEQ ID No. 1 to 84. The details of the sequence data can be found in the accompanying sequence protocol.
The statistical results for the validated marker sequence candidates (marker sequences according to the invention for rheumatoid arthritis) are specified in Tables 2 and 3.
| Number | Date | Country | Kind |
|---|---|---|---|
| 16163611.3 | Apr 2016 | EP | regional |
| 16170028.1 | May 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/057896 | 4/3/2017 | WO | 00 |
