Patent Application
20180017554
The present invention relates to methods for identifying markers for systemic sclerosis (SSc; also scleroderma or synonymously Progressive Systemic Sclerosis (PSS)) and to the markers identified with the aid of this method, which can differentiate between SSc and other autoimmune diseases on the one hand and between different SSc subgroups on the other hand. The invention also relates to panels, diagnostic devices and test kits which comprise these markers, and to the use and application thereof, for example for the diagnosis, prognosis and therapy control of SSc. The invention also relates to methods for screening and for validating active substances for use in SSc subgroups.
SSc is a chronic, inflammatory, rheumatic disease, which counts among the classic immunological connective tissue diseases (collagenoses).
SSc is a heterogeneous disease with excessive fibrosis of the skin. Further organ systems, such as the lungs, gastrointestinal area, kidneys, heart and blood vessels can also be affected. In addition, joint symptoms (arthritis) also occur.
SSc is a very rare disease. The incidence is approximately 0.5-1.5/100,000 individuals/year. It mostly occurs between the ages of 30 and 50. Women are 10-15 times more likely to be affected than men (LeRoy et al. 1988).
Clinically, a distinction can be made between limited and diffuse SSc in accordance with LeRoy et al. (1988). In earlier phases of the disease, it is often difficult to classify patients unambiguously, with this being referred to as undifferentiated SSc. If, in addition to scleroderma, fundamental symptoms of other rheumatic diseases also occur, reference is made to scleroderma overlap syndrome or overlap syndrome.
The limited form of SSc occurs at a frequency of up to 60%. This is characterised by fibrosis of the hands and feet, which spreads to below the elbows and knee joints. The Raynaud phenomenon often exists already for many years prior to the appearance of skin fibrosis. Gastrointestinal changes (difficulty in swallowing) and pulmonary arterial hypertony (PAH) also often occur. The limited form also includes CREST syndrome: calcinosis cutis, Raynaud phenomenon, oesophageal dysmotility, slerodactyly, and telangiectasia.
The diffuse form is the quicker and more severe form of SSc. In this case the fibrosis spreads past the elbows over the body and face. In contrast to the limited form, skin fibroses occur already 1-2 years after the appearance of the Raynaud phenomenon.
In the case of scleroderma overlap syndrome, symptoms of further non-organ-specific autoimmune diseases, such as myositis, lupus erythematodes/SLE, Sjögren's syndrome, and rheumatoid arthritis/RA, also occur in addition to the skin symptoms of scleroderma.
Patients with undifferentiated SSc have Raynaud's syndrome and have the swollen fingers typical for SSc and pulmonary arterial hypertony. Only some of the patients later actually develop diffuse or limited SSc.
The diagnosis of SSc can be provided on the basis of the clinical picture with the typical skin changes. This can be difficult, however, in the early stages of the disease. In addition, the detection of antinuclear antibodies (ANAs) is used. ANAs can be detected in approximately 90% of SSc patients. However, the ANA test is not specific for SSc, since other collagenoses and up to 20% of healthy individuals will test positively. The three most important autoantibodies in the case of SSc are anti-topoisomerase I (Sc1-70), anti-centromere (CENP), and anti-RNA polymerase III (anti-RNAP III). These autoantibodies have a high specificity for SSc and are often associated with a subform of SSc. However, these 3 autoantibodies are suitable only to a limited extent for subtyping of SSc, since they do not occur exclusively in one subtype and their frequency can deviate distinctly in different ethnicities. Anti-topomerase antibodies have a high specificity for SSc and are detectable in approximately 30% of patients having diffuse SSc. Anti-centomere antibodies are, by contrast, detectable in approximately 50-60% of patients having limited SSc and in 10% of patients having diffuse SSc. Both autoantibodies are mutually exclusive and are detectable jointly in patients only in very rare cases. Anti-RNAP III antibodies are detectable more frequently in the diffuse form and constitute a risk factor for renal crisis. On the whole, only approximately 70% of SSc patients can be identified diagnostically using the autoantibodies against anti-topoisomerase, anti-centromere and anti-RNAP (Mierau et al. 2011; Mehra et al. 2013).
Antibodies against U1-RNP and PM-Scl antibodies also occur more rarely. These, however, have only a low specificity for SSc: anti-PM-Scl antibodies are often detected in patients having polymyositis/SSc overlap syndrome. Antibodies against U1-RNP are detectable both in the case of SSc and in the case of mixed connective tissue diseases (MCTD) and SLE. In approximately one third of patients, antibodies against typical collagenosis antigens, such as Rho52/SS-A, Ro60/SS-B, and citrullinated peptide (ACPA) and rheumatoid factors are also detected.
In clinical practice, the diagnosis of an early form of SSc and classification thereof into the subgroups constituted by diffuse, limited or overlap syndrome is often difficult, since not all symptoms are yet present or approximately 10-30% of patients carry symptoms of a different collagenosis (connective tissue disease). Since the various subforms have a very different prognosis, there is a substantial need for biomarkers for improved diagnosis of SSc and for a classification into SSc subgroups. There is also a great need for prognostic and predictive biomarkers.
A further problem of the currently used diagnostic methods is that the suitability of the previously tested autoantigens for the diagnosis of organ involvement and complications is disputed, and partly conflicting data has been published.
There is thus also a need for new markers for SSc and also a need to improve the sensitivity and specificity of the previously most frequent diagnostically used autoantigens by the use of new autoantigens or markers.
The object has been achieved in accordance with the invention in that a differential method comprising a multiplicity of steps has been developed, in which serum samples of a large number of healthy individuals and patients with various autoimmune diseases were examined by comparison in respect of their reactivity with a multiplicity of potential antigens and these results were statistically evaluated. The selection of the serum samples and the sequence of the steps surprisingly made it possible to identify highly specific markers for SSc which are also suitable for identifying SSc subgroups and complications and for providing a differential diagnosis in respect of other autoimmune diseases, such as rheumatoid arthritis (RA), in particular early stages of RA (“early RA”), and ankylosing spondylitis or Bekhterev's disease (SPA).
The invention relates to a multi-stage method for identifying specific markers for SSc and also the markers for SSc identified with the aid of the method, and the use and/or specific therapeutic application of these markers for the diagnosis and/or differential diagnosis of systemic sclerosis and/or distinguishing of clinical subgroups of SSc.
One subject of the present invention is therefore a method for identifying markers for systemic sclerosis (SSc), said method comprising the following steps:
SEQ ID No. 1 to 955, homologues of sequences SEQ ID No. 1 to 955 with at least 95% homology, and subsequences of SEQ ID No. 1 to 955 and subsequences of homologues of SEQ ID No. 1 to 955 with at least 95% homology, and sequences coded by SEQ ID No. 1 to 319.
The beads used in the method according to the invention in steps a) to e) are preferably fluorescence-labelled.
The terms systemic sclerosis (SSc), RA or early RA, SLE, and SPA are defined for example in Pschyrembel, Clinical Dictionary, de Gruyter, 261st edition (2011).
In a preferred embodiment of the method, the markers are selected after univariate statistical analysis in that they have a threshold value of p less than 0.05 and a reactivity in the SSc group modified 1.5 times with respect to the control group. The control group comprises or consists of patients with SLE and/or patients with early RA and/or patients with SPA and/or healthy individuals. Healthy individuals are individuals in which no SSc, no SSc subform, no early RA, and no SPA has been detected or can be detected.
The invention also relates to a marker for SSc or one or more SSc subforms obtainable by the method according to the invention.
The invention also relates to a marker for SSc or one or more SSc subgroups selected from the sequences SEQ ID No. 1 to 955, homologues of sequences SEQ ID No. 1 to 955 with at least 95% homology, and subsequences of SEQ ID No. 1 to 955 and subsequences of homologues of SEQ ID No. 1 to 955 with at least 95% homology, and sequences coded by SEQ ID No. 1 to 319. The SSc subgroups are, for example, diffuse SSc (dSSc for short), limited SSc (1SSc for short) and/or overlap syndrome SSc (SSc-OS for short).
The invention also relates to a marker for SSc selected from the sequences SEQ ID No. 1, 3-5, 8-33, 320, 322-324, 327-352, 639, 641-643, 646-671, homologues of sequences SEQ ID No. 1, 3-5, 8-33, 320, 322-324, 327-352, 639, 641-643, 646-671 with at least 95% homology, and subsequences of SEQ ID No. 1, 3-5, 8-33, 320, 322-324, 327-352, 639, 641-643, 646-671, and subsequences of homologies of SEQ ID No. 1, 3-5, 8-33, 320, 322-324, 327-352, 639, 641-643, 646-671 with at least 95% homology, and sequences coded by SEQ ID No. 1, 3-5, 8-33.
The invention also relates to a marker for dSSc selected from the sequences SEQ ID No. 6, 7, 34-103, 325, 326, 353-422, 644, 645, 672-741, homologues of sequences SEQ ID No. 6, 7, 34-103, 325, 326, 353-422, 644, 645, 672-741 with at least 95% homology, and subsequences of SEQ ID No. 6, 7, 34-103, 325, 326, 353-422, 644, 645, 672-741, and subsequences of homologues of SEQ ID No. 6, 7, 34-103, 325, 326, 353-422, 644, 645, 672-741 with at least 95% homology, and sequences coded by SEQ ID No. 6, 7, 34-103.
The invention also relates to a marker for 1SSc selected from the sequences SEQ ID No. 2, 104-171, 321, 423-490, 640, 742-809, homologues of sequences SEQ ID No. 2, 104-171, 321, 423-490, 640, 742-809 with at least 95% homology, and subsequences of SEQ ID No. 2, 104-171, 321, 423-490, 640, 742-809, and subsequences of homologues of SEQ ID No. 2, 104-171, 321, 423-490, 640, 742-809 with at least 95% homology, and sequences coded by SEQ ID No. 2, 104-171.
The invention also relates to a marker for SSc-OS selected from the sequences SEQ ID No. 173-291, 492-610, 811-929, homologues of sequences SEQ ID No. 173-291, 492-610, 811-929 with at least 95% homology, and subsequences of SEQ ID No. 173-291, 492-610, 811-929, and subsequences of homologues of SEQ ID No. 173-291, 492-610, 811-929 with at least 95% homology, and sequences coded by SEQ ID No. 173-291.
The invention also relates to a panel (arrangement) of markers for SSc or SSc subgroups comprising at least two or three different markers selected independently of one another from the sequences SEQ ID No. 1 to 955, homologues of sequences SEQ ID No. 1 to 955 with at least 95% homology, and subsequences of SEQ ID No. 1 to 955 and subsequences of homologues of SEQ ID No. 1 to 955 with at least 95% homology and coded by the sequences SEQ ID No. 1 to 319. Preferred panels are also presented in the examples.
On account of the high clinical and serological heterogeneity of the SSc disease, it is difficult to diagnose SSc unambiguously using just one biomarker. It is therefore often necessary to combine (where possible) uncorrelated autoantigens to form what are known as panels of markers (“biomarker panels for SSc”). By way of example, within the scope of individualised medicine, corresponding panels of markers for SSc can be composed individually for the relevant SSc subtype (subgroup) for individual patients or patient groups. It is therefore also necessary to have available a multiplicity of potential markers for SSc in order to select suitable subgroups or subtypes of specific markers for SSc for the individual case in question. A corresponding panel can be embodied for example in the form of an arrangement, an array, or also one or more beads. The invention thus relates to an arrangement comprising one or more markers according to the invention, a protein array comprising one or more markers according to the invention, and a bead (pellet or platelet) comprising one or more markers according to the invention.
The invention also relates to diagnostic device or a test kit comprising at least one marker for SSc or SSc subgroups selected from the sequences SEQ ID No. 1 to 955, homologues of sequences SEQ ID No. 1 to 955 with at least 95% homology, and subsequences of SEQ ID No. 1 to 955 and subsequences of homologues of SEQ ID No. 1 to 955 with at least 95% homology and coded by the sequences SEQ ID No. 1 to 319.
The invention also relates to the use of at least one marker selected from the sequences SEQ ID No. 1 to 955, homologues of sequences SEQ ID No. 1 to 955 with at least 95% homology, and subsequences of SEQ ID No. 1 to 955 and subsequences of homologues of SEQ ID No. 1 to 955 with at least 95% homology and coded by the sequences SEQ ID No. 1 to 319 or at least one panel of markers or a diagnostic device or test kit for identifying subgroups of SSc patients, for diagnosis of SSc, for differential diagnosis of SSc or SSc subgroups, in particular for distinguishing SSc from other autoimmune diseases or rheumatic diseases, for diagnosis of dSSc, 1SSc or SSc-OS, for prognosis of SSc, for therapy control in SSc, for active substance selection in SSc, for therapy monitoring in SSc, and for aftercare in SSc.
The invention also relates to the use of at least one marker selected from the sequences SEQ ID No. 1, 3-5, 8-33, 320, 322-324, 327-352, 639, 641-643, 646-671, homologues of sequences SEQ ID No. 1, 3-5, 8-33, 320, 322-324, 327-352, 639, 641-643, 646-671 with at least 95% homology, and subsequences of SEQ ID No. 1, 3-5, 8-33, 320, 322-324, 327-352, 639, 641-643, 646-671, and subsequences of homologues of SEQ ID No. 1, 3-5, 8-33, 320, 322-324, 327-352, 639, 641-643, 646-671 with at least 95% homology, and sequences coded by SEQ ID No. 1, 3-5, 8-33 for diagnosis of SSc.
The invention also relates to the use of at least one marker selected from the sequences SEQ ID No. 6, 7, 34-103, 325, 326, 353-422, 644, 645, 672-741, homologues of sequences SEQ ID No. 6, 7, 34-103, 325, 326, 353-422, 644, 645, 672-741 with at least 95% homology, and subsequences of SEQ ID No. 6, 7, 34-103, 325, 326, 353-422, 644, 645, 672-741, and subsequences of homologues of SEQ ID No. 6, 7, 34-103, 325, 326, 353-422, 644, 645, 672-741 with at least 95% homology, and sequences coded by SEQ ID No. 6, 7, 34-103 for diagnosis of dSSc, for differential diagnosis of dSSc, in particular for distinguishing dSSc from other autoimmune diseases or rheumatic diseases or from 1SSc or SSc-OS, for prognosis of dSSc, for therapy control in dSSc, for active substance selection in dSSc, for therapy monitoring in dSSc, and for aftercare in dSSc.
The invention also relates to the use of at least one marker selected from the sequences SEQ ID No. 2, 104-171, 321, 423-490, 640, 742-809, homologues of sequences SEQ ID No. 2, 104-171, 321, 423-490, 640, 742-809 with at least 95% homology, and subsequences of SEQ ID No. 2, 104-171, 321, 423-490, 640, 742-809, and subsequences of homologues of SEQ ID No. 2, 104-171, 321, 423-490, 640, 742-809 with at least 95% homology, and sequences coded by SEQ ID No. 2, 104-171 for diagnosis of 1SSc, for differential diagnosis of 1SSc, in particular for distinguishing 1SSc from other autoimmune diseases or rheumatic diseases or from dSSc or SSc-OS, for prognosis of 1SSc, for therapy control in dSSc, for active substance selection in 1SSc, for therapy monitoring in 1SSc, and for aftercare in 1SSc.
The invention also relates to the use of at least one marker selected from the sequences SEQ ID No. 173-291, 492-610, 811-929, homologues of sequences SEQ ID No. 173-291, 492-610, 811-929 with at least 95% homology, and subsequences of SEQ ID No. 173-291, 492-610, 811-929, and subsequences of homologues of SEQ ID No. 173-291, 492-610, 811-929 with at least 95% homology, and sequences coded by SEQ ID No. 173-291 for diagnosis of SSc-OS, for differential diagnosis of SSc-OS from other autoimmune diseases or rheumatic diseases or from dSSc or 1SSc, for prognosis of SSc-OS, for therapy control in SSc-OS, for active substance selection in SSc-OS, for therapy monitoring in SSc-OS, and for aftercare in SSc-OS.
The invention also relates to a method for the early detection, diagnosis, differential diagnosis, prognosis, therapy control and/or aftercare of SSc or SSc subgroups, in which
The invention also relates to a composition, preferably a pharmaceutical composition for specific application in the case of SSc or SSc subgroups, comprising at least one of the sequences SEQ ID No. 1 to 955, the homologues of sequences SEQ ID No. 1 to 955 with at least 95% homology, subsequences of SEQ ID No. 1 to 955 or the subsequence of the homologues of SEQ ID No. 1 to 955 with at least 95% homology, or the sequences coded by SEQ ID No. 1-319.
The invention also relates to a target for the therapy of SSC selected from the sequences SEQ ID No. 1 to 955, the homologues of sequences SEQ ID No. 1 to 955 with at least 95% homology, the subsequences of SEQ ID No. 1 to 955 and the subsequences of the homologues of SEQ ID No. 1 to 955 with at least 95% homology, and also the proteins coded by the sequences.
The invention also relates to a method for screening active substances for SSc or SSc subgroups, in which
The large clinical heterogeneity of SSc currently constitutes a big problem both for diagnosis and for active substance development.
The identification of specific antibody signatures in SSc patient subgroups therefore constitutes an important step for the improved definition of patient groups in clinical studies. By way of example, specific autoantibodies for dSSc, 1SSc or SSc-OS could be used to recruit this subgroup for drug studies.
The invention also relates to the use of one or more markers according to the invention for SSc or SSc subgroups, of an arrangement according to the invention (panel of markers for SSc), of a protein array according to the invention, of a bead according to the invention, of a diagnostic device according to the invention, or of a test kit according to the invention for the individually tailored diagnosis and/or therapy in individual patients, patient groups, cohorts, population groups, variants of SSc, and stages of SSc.
The invention also relates to the use of one or more markers according to the invention for SSc or SSc subgroups, of an arrangement according to the invention (panel of markers for SSc), of a protein array according to the invention, of a bead according to the invention, of a diagnostic device according to the invention, or of a test kit according to the invention for detecting and/or determining the amount of one or more autoantibodies associated with SSc or SSc subgroups, for example in bodily fluids such as serum, tissue or tissue samples of the patient.
The invention also relates to the use of one or more markers according to the invention, of an arrangement according to the invention, of a protein array according to the invention, of a bead according to the invention, of a diagnostic device according to the invention, or of a test kit according to the invention for the analysis of autoantibody profiles of patients, in particular for the qualitative and/or quantitative analysis of autoantibodies and/or for the monitoring of changes of autoantibody profiles associated with SSc or SSc subgroups, for example in bodily fluids such as serum, tissue or tissue samples of the patient.
A particular embodiment of the invention relates to methods for the early identification and diagnosis of SSc or SSc subgroups, in which the detection of an interaction of the bodily fluid or the tissue sample with the one or more markers indicates an SSc- or SSc-subgroup-associated autoantibody profile of the patient or of a cohort or of a population group or of a certain course of disease (prognosis) or of a certain response to a therapy/drug.
The invention therefore includes the use of at least one marker for SSc selected from the sequences SEQ ID No. 1 to 955, the homologues of sequences SEQ ID No. 1 to 955 with at least 95% homology, the subsequences of SEQ ID No. 1 to 955 or the subsequences of homologues of SEQ ID No. 1 to 955 with at least 95% homology, and the proteins coded by the sequences for the analysis of autoantibody profiles of patients, in particular for the quantitative analysis and/or for the monitoring of changes of autoantibody profiles of SSc patients.
An interaction of the bodily fluid or the tissue sample with the one or more SSc markers can be detected for example by a probe, in particular by an antibody.
In a preferred embodiment at least 2, for example 3, 4, 5, 6, 7, 8, 9, 10, preferably 15 to 20 markers for SSc or SSc subgroups or 30 to 50 or 100 or more markers are used together or in combination (what are known as “panels”), either simultaneously or in succession, wherein the markers for SSc are selected independently of one another from the sequences SEQ ID No. 1 to 955, the homologues of sequences SEQ ID No. 1 to 955 with at least 95% homology, the subsequences of SEQ ID No. 1 to 955 or the subsequences of homologues of SEQ ID No. 1 to 955 with at least 95% homology, and the proteins coded by the sequences. This embodiment is implemented preferably in the form of a panel according to the invention.
A particular embodiment of the invention relates to a method according to the invention, wherein the stratification or therapy control includes decisions relating to the treatment and therapy of the patient, in particular hospitalisation of the patient, use, efficacy and/or dosage of one or more drugs, a therapeutic measure, or the monitoring of the course of the disease and course of therapy, aetiology, or classification of a disease inclusive of prognosis. The invention also relates to a method for stratification, in particular for risk stratification and/or therapy control of a patient with SSc.
The stratification of the patient with SSc into new or established SSc subgroups as well as the expedient selection of patient groups for the clinical development of new therapeutic agents is also included. The term therapy control likewise includes the division of patients into responders and non-responders with regard to a therapy or course thereof.
The invention in particular also relates to the detection and determination of the amount of at least two different autoantibodies in a patient by means of the SSc markers according to the invention, wherein at least two different SSc markers are preferably used. The invention also relates to a use according to the invention of one or more SSc markers, wherein at least 2, for example 3 to 5 or 10, preferably 30 to 50, or 50 to 100 or more SSc markers or the relevant autoantibodies on or from a patient to be tested are determined.
The invention comprises the SSc markers on a solid substrate, for example a filter, a membrane, a small platelet or ball, for example a magnetic or fluorophore-labelled ball, a silicon wafer, a bead, a chip, a mass spectrometry target, or a matrix, or the like. Different materials are suitable as substrates and are known to a person skilled in the art, for example glass, metal, plastic, filter, PVDF, nitrocellulose, or nylon (for example Immobilon P Millipore, Protran Whatman, Hybond N+ Amersham).
The substrate for example can correspond to a grid with the dimensions of a microtitre plate (8-12 well strips, 96 wells, 384 wells or more), of a silicon wafer, of a chip, of a mass spectrometry target, or of a matrix.
In one embodiment of the invention markers for SSc are present in the form of clone sequences or clone(s).
The markers according to the invention can be combined, supplemented or extended with known biomarkers for SSc or biomarkers for other diseases. With a combination of this type, a proportion of markers for SSc according to the invention of preferably at least 50%, preferably 60%, and particularly preferably 70% or more is comprised.
In a preferred embodiment the use of the SSc markers is implemented outside the human or animal body, for example the diagnosis is performed ex vivo/in vitro.
In the sense of this invention, the term “diagnosis” means the positive determination of SSc with the aid of the markers according to the invention and the assignment of the patients or symptoms thereof to the disease SSc. The term “diagnosis” includes the medical diagnosis and tests in this respect, in particular in vitro diagnosis and laboratory diagnosis, and also proteomics and nucleic acid blots. Further tests may be necessary for assurance and in order to rule out other diseases. The term “diagnosis” therefore includes in particular the differential diagnosis of SSc by means of the markers according to the invention.
In the sense of this invention, “stratification or therapy control” means that, for example, the methods according to the invention allow 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 a disease, for example into a new or existing sub-type, or the differentiation of diseases and patients thereof. 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.
“Prognosis” means the prediction of the course of a disease.
In accordance with the invention, “therapy control” means, for example, the prediction and monitoring of the response to a drug or a therapy as well as aftercare.
Within the scope of this invention, the term “patient” is understood to mean any test subject, any individual (human or mammal), with the provision that the test subject or individual is tested for SSc.
The term “marker for SSc” in the sense of this invention means that the nucleic acid, for example DNA, in particular cDNA or RNA or the coded amino acid sequence or the polypeptide or protein are significant (specific) for SSc and/or the autoantibody profiles associated with SSc. Markers according to the invention are nucleic acid sequences and/or amino acid sequences according to the definition in the appended sequence protocol (SEQ ID No. 1 to SEQ ID No. 955), homologues and subsequences thereof, wherein modified nucleic acid and amino acid sequences are also included. Here, marker for SSc means, for example, that the cDNA or RNA or the polypeptide or protein obtainable therefrom interacts with substances from the bodily fluid or tissue sample from a patient with SSc (for example antigen (epitope)/antibody (paratope) interaction). In a particularly preferred embodiment of the invention the marker for SSc is an (auto)antigen or part of an antigen or codes for an antigen or for part of an antigen.
The substances from the bodily fluid or tissue sample occur either only in an amplified manner or at least in an amplified manner in the case of SSc or are expressed, whereas these substances are not present in patients without SSc or healthy individuals, or at least are present to a lesser extent (smaller amount, lower concentration). Markers for SSc can also be characterised in that they interact with substances from the bodily fluid or tissue sample from patients with SSc, because these substances no longer occur or are no longer expressed or occur or are expressed at least in a much lower amount/concentration in the case of SSc, whereas these substances are present or are at least present to a much higher extent in patients without SSc. Markers for SSc can also be present in healthy test subjects, however the amount (concentration) thereof changes for example with the development, establishment and therapy of SSc. One or more markers can in this way map a profile of substances from bodily fluid and tissue sample, for example an SSc-associated autoantibody profile of the patient in question. Markers according to the invention are biomarkers for SSc.
Autoantibody profiles comprise the amount of one or more autoantibodies of which the occurrence/expression accompanies the development and/or establishment of SSc. Autoantibody profiles therefore include on the one hand the composition, i.e. one or more autoantibodies is/are expressed only in the case of SSc for example, and also the amount/concentration of individual autoantibodies, i.e. the amount/concentration of individual autoantibodies changes with the development and establishment of SSc. These changes can be detected with the aid of the marker (sequences) according to the invention.
In a particularly preferred embodiment the SSc marker identifies/binds to autoantibodies which are present (intensified) or are present to a lower extent (or no longer) during the course of the development, establishment and therapy of SSc. Autoantibodies are formed by the body against endogenous antigens which are formed for example in the case of SSc. Autoantibodies are formed by the body against different substances and pathogens. Within the scope of the present invention, the autoantibodies which are formed with the occurrence and during the course of the development of SSc and/or of which the expression is up-regulated or down-regulated are detected in particular. These autoantibodies can be detected with the aid of the methods and markers according to the invention, and the detection and monitoring (for example of the amount) thereof can be used for the early identification, diagnosis and/or therapy monitoring/therapy control and the prognosis and prediction of the risk of the re-occurrence of SSc within the scope of the aftercare.
The autoantibody profiles can be sufficiently characterised with use of just a single SSc marker. In other cases, two or more SSc markers are necessary in order to map an autoantibody profile which is specific for SSc.
In one embodiment of the invention autoantibodies which derive from another individual and which for example originate from a commercial cDNA bank can be detected using SSc markers.
In another embodiment of the invention these autoantibodies can be detected using SSc markers which derive from the same individual and which for example originate from a cDNA bank produced individually for the patient or a group of patients for example within the scope of individualised medicine. By way of example, homologues of the specified SSc markers with the sequences SEQ ID. No. 1 to 955 or subsequences thereof can be used.
Autoantibodies can be formed by the patient already many years prior to the occurrence of the first symptoms of disease. An early identification, diagnosis and also prognosis and preventative treatment or lifestyle change and other possibilities for prevention are therefore possible even years prior to the visible outbreak of the disease. The devices, means and methods according to the invention enable a very early intervention compared with known methods, which significantly improves the prevention, treatment possibilities and effects of SSc.
Since the SSc-associated autoantibody profiles change during the establishment and treatment/therapy of SSc, the invention also enables the detection and monitoring of SSc at any stage of the development and treatment and also monitoring within the scope of SSc aftercare. The means according to the invention, for example a corresponding diagnostic device or a test kit, also allow simple handling at home by the patient and an economical routine precautionary measure for early identification.
In particular due to the use of antigens as specific markers for SSc which derive from sequences already known, for example from commercial cDNA banks, test subjects can be tested and any present SSc-associated autoantibodies can be detected in these test subjects, even if the corresponding autoantigens are not (yet) known in these test subjects.
Different patients can have different SSc-associated autoantibody profiles, for example different cohorts or population groups can differ from one another. Here, any patient can form one or more different SSc-associated autoantibodies during the course of the development of SSc and the progression of the SSc disease, that is to say even different autoantibody profiles. In addition, the composition and/or the amount of formed autoantibodies can change during the course of the SSc development and progression of the disease, such that a quantitative evaluation is necessary. The therapy/treatment of SSc leads to changes in the composition and/or the amount of SSc-associated autoantibodies. The large selection of SSc markers according to the invention which are provided with this invention enables the individual compilation of SSc markers in an arrangement, i.e. a panel, for individual patients, groups of patients, certain cohorts, population groups and the like. In one individual case, the use of one SSc marker may therefore be sufficient, whereas in other cases at least two or more SSc markers must be used together or in combination in order to create a conclusive autoantibody profile.
Compared with other biomarkers, the detection of SSc-associated autoantibodies for example in the serum or plasma of patients has the advantage of high stability and storage capability and good detectability. The presence of autoantibodies also is not subject to a circadian rhythm, and therefore the sampling is independent of the time of day, food intake, and the like.
In addition, the SSc-associated autoantibodies can be detected with the aid of the corresponding antigens/autoantigens in known assays, such as ELISA or Western Blot, and the results can be checked in this way.
In the sense of the invention, an interaction between the SSc marker and the serum in question, for example an autoantibody of the patient, is detected. Such an interaction is, for example, a bond, in particular a binding substance on at least one SSc-specific marker, or, in the case that the SSc-specific marker is a nucleic acid, for example a cDNA, the hybridisation with a suitable substance under selected conditions, in particular stringent conditions (for example as defined conventionally 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 of 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 approximately one hour. An example of 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. The interaction between the bodily fluid or tissue sample from a patient and the markers for SSc is preferably a protein-protein interaction.
In accordance with the invention, such substances, for example antigens, autoantigens and SSc-associated autoantibodies, 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 from the patient. The invention in particular relates to the use of these bodily fluids and tissue samples for early detection, diagnosis, prognosis, therapy control and aftercare.
The SSc-specific markers, in a further embodiment of the invention, have a recognition signal that 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.
Homologues of the markers according to the invention SEQ ID No. 1 to 955, as presented in the claims for example, are also included. Within the sense of the invention, homologues are those with homology of the amino or nucleic acid sequence and those in which the corresponding sequence is modified, for example the protein variants, which indeed have the same amino acid sequence, but differ with regard to the modification, in particular the post-translational modification.
In accordance with the invention, modifications of the nucleic acid sequence and of the amino acid sequence, for example citrullination, acetylation, phosphorylation, glycosylation, ethylation, or polyA strand extensions and further modifications known as appropriate to a person skilled in the art are included.
Homologues also include sequence homologues of the markers and subsequences thereof. Sequence homologues are, for example, nucleic acid sequences and/or protein sequences that have an identity with the SSc markers of the sequences SEQ ID No. 1 to 955 of at least 70% or 80%, preferably 90% or 95%, particularly preferably 96% or 97% or more, for example 98% or 99%. In a particularly preferred embodiment of the invention, for the case in which the SSc markers are antigens, the homology in the sequence range in which the antigen-antibody or antigen-autoantibody interaction takes place, is at least 95%, preferably at least 97%, particularly preferably at least 99%. For example, mutations such as base exchange mutations, frameshift mutations, base insertion mutations, base loss mutations, point mutations and insertion mutations, are included in accordance with the invention.
The invention also relates to subsequences of the SSc markers with the sequence SEQ ID No. 1 to 955. Subsequences also include nucleic acid or amino acid sequences that are shortened compared with the entire nucleic acid or the entire protein/peptide. Here, the deletion may occur at the end or the ends and/or within the sequence. For example, subsequences and/or fragments that have 50 to 100 nucleotides or 70-120 nucleotides of the sequence SEQ ID No. 1 to 955 are included. Homologues of subsequences are also included in accordance with the invention. In a particular embodiment, the SSc markers are shortened compared with the sequences SEQ ID No. 1 to 955 to such an extent that they still consist only of the binding point(s) for the SSc-associated autoantibody in question. In accordance with the invention, SSc markers are also included that differ from the sequences SEQ ID No. 1 to 955 in that they contain one or more insertions, wherein the insertions for example are 1 to 100 or more nucleotide/amino acids long, preferably 5 to 50, particularly preferably 10 to 20 nucleotides/amino acids long and the sequences are otherwise identical however or homologous to sequences SEQ ID No. 1 to 955. Subsequences that have at least 90%, preferably at least 95%, particularly preferably 97% or 98%, of the length of the SSc markers according to the invention with sequences SEQ ID No. 1 to 955 are particularly preferred.
In a further embodiment, the respective SSc marker can be represented in different quantities in one or more regions in the arrangement or on the substrate or in a panel. This allows a variation of the sensitivity. The regions may each have a totality of SSc markers, that is to say a sufficient number of different SSc markers, in particular 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different SSc markers. By way of example, 20 to 50 (numerically) or more, preferably more than 100, particularly preferably 150 or more, for example 25,000 or 5,000 or 10,000 different or same SSc marker sequences and where applicable further nucleic acids and/or proteins, in particular other biomarkers can be represented on the substrate or in the panel.
Within the scope of this invention, “arrangement” is synonymous with “array” and “panel”. If this “array” is used to identify substances on SSc markers, this is to be understood preferably to be an “assay” or a bead or a diagnostic device or a screening assay. In a preferred embodiment, the arrangement is designed such that the markers represented on the arrangement are present in the form of a grid on a substrate. Furthermore, those arrangements are preferred that permit a high-density arrangement of SSc markers. The markers are preferably 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-supported automated high-throughput method.
Within the scope of this invention, however, the term “assay” or diagnostic device likewise comprises those embodiments such as ELISA, bead-based assay, line assay, Western Blot, and immunochromatographic methods (for example what are known as lateral flow immunoassays) or similar immunological single or multiplex detection methods.
A “protein array” in the sense of this invention is the systematic arrangement of SSc markers on a solid substrate, wherein the substrate can have any shape and/or size, and wherein the substrate is preferably a solid substrate.
The SSc markers of the arrangement are fixed on the substrate, preferably spotted or immobilised, printed on or the like, in particular in a reproducible manner. One or more SSc markers can be present multiple times in the totality of all SSc markers and may be present in different quantities based on a spot. Furthermore, the SSc markers can be standardised on the substrate (for example by means of serial dilution series of, for example, human globulins as internal calibrators for data normalisation and quantitative evaluation). A standard (for example a gold standard) can also be applied to the substrate where necessary.
In a further embodiment, the SSc markers are present as clones. Such clones can be obtained for example by means of a cDNA expression library according to the invention. In a preferred embodiment, such expression libraries are obtained using expression vectors from a cDNA expression library comprising the cDNAs of the SSc-specific 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 Jan; 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, New York. 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.
Protein arrays or corresponding expression libraries that do not exhibit any redundancy (what is known as a Uniclone® library) and that can be produced for example 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.
In addition, the SSc markers can be present in the respective form in the form of a fusion protein, which for example contains at least one affinity epitope or “tag”, wherein the tag is selected for example from 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, or the fusion protein has one or more additional domains for example, such as a cellulose-binding domain, green fluorescent protein, maltose-binding protein, calmodulin-binding protein, glutathione S-transferase or lacZ.
In a further embodiment the invention relates to an assay, for example a multiplex assay, a bead-based assay, or protein array for identifying and characterising a substance, for example a hit, a lead substance, or an active substance for SSc. Here, a substance to be tested is used. This can be any native or non-native biomolecule, a (synthetic) chemical molecule, a natural substance, a mixture or a substance library. Once the substance to be tested has contacted an SSc marker, the binding success is evaluated, for example with use of commercially available image-analysis software (GenePix Pro (Axon Laboratories), Aida (Raytest), ScanArray (Packard Bioscience).
Binding according to the invention, binding success, interactions, for example protein-protein interactions (for example protein to SSc marker, 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 colloid 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.
In a further embodiment, the invention relates to a drug or an active substance or prodrug for SSc, developed and obtainable by the use of an SSc marker according to the invention.
The invention also relates to the use of an SSc marker selected from sequences SEQ ID No. 1 to 955 and subsequences of SEQ ID No. 1 to 955 with at least 90%, preferably at least 95% of the length of SEQ ID No. 1 to 955 and homologues of SEQ ID No. 1 to 955 and subsequences thereof with an identity of at least 95%, preferably at least 98% or more, to the corresponding sequences and proteins/peptides coded by the sequences SEQ ID No. 1 to 638, coded by the subsequences thereof and homologues as affinity material for carrying out an apheresis or blood washing for patients with SSc, i.e. apheresis of SSc autoantibodies. The invention thus relates to the use of the markers according to the invention, preferably in the form of an arrangement, as affinity material for carrying out an apheresis or a blood washing in the broader sense, wherein substances from bodily fluids from a patient with SSc, such as blood or plasma, bind to the markers according to the invention and consequently can be removed selectively from the bodily fluid. The application in blood washing is a special case of use of the SSc markers as a target.
The following examples and drawings explain the invention, but do not limit the invention to the examples. In the following drawings, systemic sclerosis is denoted by PPS (progressive systemic sclerosis).
Selection of the patient groups to be tested: Blood samples were analysed from 100 SSc patients, 100 patients with SLS, 537 patients with early rheumatoid arthritis (“RA”; period of disease less than 6 months) and 82 patients with ankylosing spondylitis (SPA) or Bekhterev's disease. 343 blood samples from the Bavarian Red Cross (BRC) were used as control group. An informed consent of the Ethics Commission of the clinical partners and of the biobank of the BRC was received from all test subjects.
Five cDNA libraries that had been produced from different human tissues (foetal brain, intestine, lung, liver and T-cells) were used for the production of the recombinant antigens. All cDNAs were expressed in E. coli under the transcriptional control of the lactose-inducible promoter. The resultant proteins carry, at their amino terminus, an additional sequence for a hexahistidine purification tag (His6 tag). Target antigens which were not present in the cDNA library were produced by chemical synthesis (Life Technologies) and cloned into the expression vector pQE30-NST, which already codes an amino-terminal His6 tag.
Following recombinant expression of the proteins, these were isolated in denaturising conditions and purified by means of metal affinity chromatography (IMAC). The proteins were lyophilised and stored at −20° C. until further use.
The production of BBAs was adapted to a microtitre plate format, such that 384 coupling reactions could be assessed in parallel using automated pipette systems (Starlet, Hamilton Robotics, Evo Freedom 150, Tecan). For the use of automated pipette systems, the individual bead regions were transferred into coupling pates (96 well Greiner) and the antigens were transferred into 2D barcode vessels (Thermo Scientific). For each coupling reaction, 0.6 to 2.5 million beads and, depending on the antigen, 1 to 100 μg protein were used.
All washing and pipetting steps of the coupling reaction were carried out in coupling plates which were fixed on magnets. The beads were washed twice with 100 μl L×AP buffer (100 mM NaH2PO4, pH 6.2) and then received in 120 μl L×AP buffer. For the activation, 15 μl 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; 50 mg/ml) and 15 μl N-hydroxysulfosuccinimide (sulfo-NHS; 50 mg/ml) were added by pipette to form a bead suspension, and these suspensions were then incubated for 20 minutes in the shaker (RT, 900 rpm, protected against light). The beads were then washed 3× with 150 μl L×KPT buffer and then the protein solution was added. Following an incubation period of two hours in the shaker (RT, 900 rpm, protected against light), the beads were then washed three times with 150 μl LxKPT buffer. To block free binding points, 100 μl L×CBSP buffer (PBS, 1% BSA, 0.05% ProClin300) were added, and these mixtures were then incubated for 20 min in the shaker (RT, 900 rpm, protected against light). This was followed by incubation over night at 4-8° C. The BBA was produced by the combination of beads coupled to antigens and was stored at 4-8° C., protected against light, until use.
For application, BBAs were incubated with sera and all IgG-based autoantibodies bonded to antigens were detected with the aid of a secondary antibody. In order to enable a high throughput of measurements, the application of BBAs was adapted to a microtitre plate format so that either an 8-channel (Starlet, Hamilton Robotics) or a 96-channel (Evo Freedom 150, Tecan) automated pipetting system could be used. The sera to be examined were transferred into 2D barcode vessels and then diluted 1:100 with assay buffer (PBS, 0.5% BSA, 10% E. coli lysate, 50% low-cross buffer (Candor Technologies)). In order to neutralise human antibodies directed against E. coli, a pre-incubation of the sera dilutions was performed for 20 min. In this time, 500 beads per bead region were distributed in the assay plate. 50 μl of diluted serum were added to the beads in the coupling plate, and the reaction mixtures were incubated for 18-22 h in the shaker (4-8° C., 900 rpm, protected against light). After three washing steps in each case with 100 μl L×WPT buffer, 5 μg/ml of the detection antibody goat anti-human IgG-PE (Dianova) were added to the reaction mixtures and incubated for 1 h in the shaker (RT, 900 rpm). The beads were then washed three times with 100 μl L×WPT and incorporated in 100 μl carrier liquid (Luminex). The fluorescence signal of the beads was detected with the aid of the FlexMAP3D instrument. Here, the bead count on the one hand and the MFI value (median fluorescence intensity) on the other hand were measured.
The biostatistical analysis comprised univariate and multivariate methods for describing the statistical properties of individual antigens and of groups of antigens. In order to discover interesting candidates for panels, the key property was a good separation between the groups of samples based on the MFI values. In order to find antigen candidates for panel generation, univariate testing, receiver operating characteristic (ROC) analyses, correlation profiles, powered partial least squares discriminant analysis (PPLS-DA) and random forests were used as methods. Biostatistical analyses were subject to expert assessment in order to define final antigen panels.
Before the statistical analysis, the MFI values of log2-transformed antigens in which more than 20% of the values were missing were excluded from the analysis, and missing values were replaced by median imputation. A quantile normalisation was carried out under consideration of the reference sera in order to normalise, per BBA set, all measured samples on individual plates.
Besides descriptive standardisation for MFI values, non-parametric tests were also carried out with the aid of the two-sided Mann-Whitney-U test in order to uncover differences in the median values of the groups. The test level for multiple testing was corrected in accordance with the Bonferroni-Holm procedure. In addition, the Benjamin-Hochberg procedure inclusive of the determination of the False Discovery Rate (FDR, q-value) was applied. In addition, fold-change and effect size were determined. In order to assess the classification quality, an ROC analysis was carried out, within the scope of which sensitivity, specificity and the area under the ROC curve (AUC) were calculated, in each case inclusive of the 95% confidence interval on the basis of the bootstrap method. Boxplots and volcano plots were used for graphical representation. A scoring system was implemented on the basis of the univariate results.
By means of the application of a PPLS-DA, it was attempted to maximise the correlation between the components and the response matrix. A linear discriminant analysis with the latent component as predictors was used for the final classification. A random forest was applied, in which binary decision trees are combined. The decision trees were formed on the basis of a number of bootstrap samples of a training sample and by random selection of a subgroup of explaining variables at each node. The number of input variables, which was selected randomly with each division step, was determined as the square root of the total number of variables, and the number of trees in the random forest was set to 1000. A cross validation with 500 times throughput was implemented for both multi-variant approaches.
In a first screening the antigen reactivities of 100 SSc patients, 537 patients with early RA, 82 patients with SPA, and 343 healthy controls categorised in accordance with age and sex were differentially tested. For this purpose, the autoantibody reactivities of these blood samples were tested on 5857 antigens coupled to Luminex beads.
In order to identify antigens with which the group of all SSc patients can be distinguished from different control groups consisting of healthy samples and patients with various rheumatic diseases, univariate statistical tests were carried out. The result of the statistical test is illustrated as a volcano plot for all 5857 antigens. In the volcano plot, the x-axis shows the relative change of the antigen reactivity in SSc patients compared with healthy controls (
Table 2 comprises all autoantigens identified in SSc patients (Table 2)).
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Following univariate statistical evaluation, a threshold value of p<0.05 and a reactivity modified 1.5 times compared with the control group were applied.
Group 1 comprises antigens which in the group of all SSc patients fall short of a threshold value of p<0.05 compared to healthy controls and/or other rheumatic diseases and achieve a reactivity modified by 1.5 times compared to the control group: KDM6B, NSUN5, RTEL1, MYST2, TRIM21, ABCB8, AVEN, VEGFB, C19orf60, CENPA, CENPC1, CENPT, CHRM3, GORASP1, GYS1, HDAC5, ILKAP, LSM1, MARVELD2, NFIX, OSBP2, PIM3, PLXNB1, PMF1, PPIE, PRDM16, TTLL3, USP48, AZGP1, AZGP1, ZYX.
Table 3 summarises the results of the statistical tests for 36 antigens from Table 2 which have a p-value of <0.05 compared to healthy samples.
What are specified are the p-value, the increase in reactivity compared to the control group (Fold-Change), the area under the curve (AUC), and the confidence interval (CI), and also sensitivity (Sens.) and specificity (Spec.).
In order to analyse the frequency of the newly identified antigens from Table 3 in comparison with known antigens, a threshold value of 3 standard deviations (SD) above the mean value of the healthy samples was defined.
Astonishingly, at least 6 additional antigens were identified of which the frequency under consideration of all SSc patients is greater than or equal to 10%. These are KDM6B, (21%), BICD2 (19%), RTEL1 (14%), NSUN5 (13%), SSRP1 (10%) and SPEG (10%). In addition, two further antigens with a frequency greater than or equal to 5% in SSc were found: VEGFB(9%) and PSAT1 (7%).
Only approximately 62.5% of the analysed patients with limited form have anti-Sc170 antibodies. A further 12.5% of patients showed anti-centromere antibodies. In order to identify further autoantibodies in patients with limited SSc, the serum samples of patients with diffuse SSc were compared with various control groups. These consisted of patients with limited SSc and patients with overlap syndrome. Autoantibodies which in the group of SSc patients with diffuse form have a p-value of less than 0.05 and a fold-change greater than 1.5 were selected. The result of the statistical tests is summarised in Table 2.
Group 2 comprises 72 additional antigens which are suitable for the identification of patients with diffuse SSc.
Group 2 antigen gene symbols: PSAT1, TEX264, ABCF3, ACAT2, TCEAL4, ACTN4, ACTR5, ALDH1A1, ALPK1, APBA2, ARHGEF16, ASMTL, ATP13A2, C11orf30, C11orf60, C17orf48, C19orf52, C20orf43, CDK5RAP2, CMPK1, CORO2B, COX5A, CTBP2, CYP4F2, DENND4B, DHRS4, DTNBP1, EEF1D, EIF2B3, EPS8L2, FEZ1, FOXL1, FUCA2, GAL3ST4, GEMIN8, GLOD4, GSN, HMGCS1, HSP90AA1, INPP5B, IRAK1, ISCU, LARS, LTF, MGEA5, MPND, MSH3, NCSTN, NEU1, P4HB, PERI, PEX14, PGCP, PPL, PRKAR1B, PTPN5, RAB11B, RAB11FIP4, RUNDC3A, STMN3, STMN4, STXBP3, SYCE1, TBCD, TMF1, TSFM, TXNRD1, UBE2Q1, UBE2Q2, UBE2Z, YTHDF1, ZNF217.
Patients with diffuse SSc more often have antibodies for the antigens PSAT1 (12.5%), VEGFB (12.5%), CMPK1 (9.4%), TEX264 (9.4%), WDR44 (9.4%), PPL (6.3%) and MYST2 (6.3%).
Table 4 summarises the results of the statistical tests for selected antigens of group 1 and 2. These antigens are suitable for identification and distinction of the limited and diffuse SSc subgroups.
Only approximately 60% of the analysed patients with limited form have anti-centromere antibodies. A further 18% of the patients showed anti-Sc170 antibodies. In order to identify further antibodies in patients with limited SSc, the serum samples of the patients with limited SSc were compared with different control groups. These consisted of patients with diffuse SSc and patients with overlap syndrome. Autoantibodies which in the group of SSc patients with limited form had a p-value of less than 0.05 and a fold-change of greater than 1.5 were selected. The result of the statistical tests is summarised in Table 2.
Table 4 summarises the results of the statistical tests of the limited and diffuse SSc subgroups.
Group 3 comprises 69 additional antigens which are suitable for the identification of patients with limited SSc.
Group 3: antigen gene symbols:
BICD2, SPEG, ZFYVE19, AATF, ABI2, ACBD6, AIP, ANK1, ARHGDIA, AZIN1, C1orf131, C9orf16, CBX3, CCDC102A, CCNDBP1, CENPH, CORO7, DDX1, DTX4, DYNC1LI1, EIF4E, EML3, ENAH, EOMES, FAM50A, FCHSD1, GAB1, GCSH, GIPC1, GIT1, GPBP1, GTF2F1, HIST1H1A, IKBKB, ISY1, KDR, KIFAP3, MAPT, MED21, MOCOS, MRPL34, NSFL1C, PDIA6, PLK1S1, PLXNB2, RGS16, RNF4, RPL5, S100B, SET, SFPQ, SH3GL2, CENPB, SPIRE2, SPTAN1, SSB, SYF2, TAF9, TDRKH, TTC1, TTC28, TWF2, UBAP2, UNC119, WIZ, XBP1, YLPM1, ZCCHC17, ZCCHC9
Besides anti-centromere and anti-Ro52 (TRIM21) antibodies, antibodies against the antigens KDM6B (38%), BICD2 (26%), RTEL1 (22%), NSUN5 (20%), SSRP1 (14%), AVEN (12%), PMF1 (10%), NFKBIL2 (10%), SPEG (10%) and PLK1S1 (10%) were found with a frequency of greater than or equal to 10% in patients with limited SSc.
A common progressive form of SSc is what is known as overlap syndrome. In clinical practice, the delimitation of this form with respect to other collagenoses is often difficult.
Group 4 comprises 119 antigens which are suitable for the identification of patients with overlap syndrome:
Group 4 antigen gene symbols: ACOT7, ACTR1B, ADD1, AIFM1, AK1, AKAP1, AKT1, ANAPC4, ANKRD16, ANKS6, APC, ARHGDIB, ASB6, ATP5D, ATP5H, AVPI1, B3GNTL1, BCKDHA, BRPF3, C11orf1, C11orf84, C14orf105, C20orf20, C3orf1, CASA, CALB2, CAPG, CBLC, CC2D1A, CCDC137, CCDC40, CCDC43, CDC2L2, CEP76, CNDP2, CNTROB, COIL, CRYAB, DES, DHX29, DIP2C, DR1, EID1, ERLIN1, FAM104B, FAM60A, FASN, FCGR1A, FKBP15, FKBP8, FUBP3, FXC1, GSTZ1, H2AFV, HNRNPA1, HNRPLL, HSBP1L1, HSPA8, IRAK1BP1, KARS, KCNK5, LASS5, LIPE, LOC100129119, LOC643733, LSM14A, LSM14B, LSR, LUC7L2, MAOA, MAPK8IP2, MBD3, MRPL10, MRPL11, MSN, MXD3, MXI1, NFE2L2, NHSL1, NNAT, NRBP1, NUP35, ORC6L, PANK4, PBXIP1, PCBP4, PPP1R14A, PRDM8, PTN, RPLP0, RPS3, SCARB2, SDCCAG8, SDR39U1, SDS, SEC31A, SERPINA6, SH3KBP1, SMAD1, SNX22, SOX13, SRP68, TCEAL2, TCEB3, TCERG1, TCOF1, TIMM13, TRAK1, TRIM10, TRIM56, UBTD2, UGT1A8, UHRF1BP1L, USP47, VAT1, WDR47, ZMYND8, ZNF431, ZNF563.
Group 5 in Table 2 contains further statistically significant antigens which can be used for the diagnosis and differential diagnosis of SSc compared to healthy test subjects and other autoimmune diseases.
Group 5: antigen gene symbols: HIST1H2BA, NEFL, STMN1, STMN1, CDR2, PPP1R2, RBM28, SSRP1, C4orf27, CACNB3, CASP9, CHD1, DFNA5, FEN1, GTF2E2, MMP2, MRPL40, NAPA, PLA2G4B, PPP2CA, PVRL2, RECQLS, FAF1, WDR44.
On account of the clinical and serological heterogeneity of the SSc disease, it is not possible to diagnose this disease using just one biomarker. According to Mierau et al. (2011), only approximately 35.9% of SSc patients have autoantibodies against centromere proteins and only 30.1% have autoantibodies against anti-topomerase I (anti-Sc170). There is thus also a great need for specific diagnostic and prognostic markers.
In order to develop an improved diagnostic antigen panel for SSc, antigens from Tables 3 and 4 were selected.
The narrower selection initially included five of the antigens represented most frequently in SSc: KDM6B, BICD2, RTEL1 and NSUNS) (
In addition, the frequency of autoantibodies in diffuse SSc, which progresses particularly severely, was also taken into consideration. Here, two of the most frequent reactive antigens BICD2 (12.5) and PSAT1 (12.5) were selected.
In addition, antigens to which anti-Sc170-negative and anti-centromere-negative patients react were selected.
In particular, the antigens MARVELD2 (19%), MYST2 (19%), VEGFB (19%).PSAT1 (14%). NSUN5 (14%) and TEX264 (14%) have a frequency of more than 10% in anti-centromere- and anti-Sc170-negative SSc patients. These antigens are therefore particularly suitable for improving the diagnosis.
Four of the most frequently reactive antigens in doubly negative SSc patients (MYST2, PSAT1, NSUN5 and TEX264) were therefore selected for the development of an improved diagnostic test.
The final composition of the panel is illustrated in Table 5.
The first 7 antigens in Table 2 include the most important antigens used for the compilation of biomarker panels for the diagnosis of SSc: KDM6B, NSUN5, BICD2, RTEL1, MYST2, PSAT1 and TEX264
For validation of the antigens from panel I and panel II specified in table 5, 100 SSc patients and 100 healthy samples were measured using antigen-coupled Luminex beads.
The MFI values of the antigens TOP1, CENPB, KDM6B, NSUN5, BICD2, RTEL1, MYST2, PSAT1 and TEX264 were used to calculate the area under the curve (AUC), sensitivity, and specificity.
It was possible to increase the sensitivity by 7% from 72% to 79% due to the inclusion of the 5 antigens KDM6B, NSUN5, BICD2, RTEL1, MYST2, PSAT1 and TEX264.
On the basis of the example, it is clear how the prediction quality of a biomarker model can be increased with inclusion of the additional markers so as to achieve a better classification of patients.
The inclusion of additional markers besides the known markers improves the prediction quality in the applied method significantly with approximately 7% ROC.
The markers specified in table 2 were tested in a second independent sample collective (SSc cohort II). In total, 180 serum samples from SSc patients were analysed, of which the demographic and clinical data were taken from the EUSTAR database. EISTAR is a multi-centre, prospective cohort of the European League Against Rheumatism (EULAR) Scleroderma Trials and Research (EUSTAR) group. As control groups, n=99 serum samples from healthy controls and n=110 serum samples from patients with rheumatic autoimmune diseases (ADs) were used.
These were composed of serum samples from patients with a diagnosis of SLE, myositis, arthritis and Sjögren's syndrome.
For this purpose, the human proteins specified in Table 2 were coupled to Luminex beads and the protein-coupled beads were measured in a multiplex assay with the patient samples. The binding of autoantibodies was measured by means of a PE-conjugated autoantibody in a Luminex instrument.
A univariate statistical evaluation was carried out using the Wilcoxon rank sum test. The predefined significance level was set at 0.05. Table 7 contains 41 antigens, which, both in SSc cohort I, which was used for discovery of the markers (Example 6), and in SSc cohort II, achieved a p-value less than the set significance level of 0.05. For the comparison of all SSc samples against healthy controls (HC), 31 markers achieved a p-value of less than 0.05. For the comparison of all SSc samples against a combined group of serum samples of various rheumatoid diseases (ADs), 24 markers achieved a p-value of less than 0.05.
From the markers contained in Table 2 and Table 7, various marker combinations were tested by means of logistic regression models. Here, the specificity compared to the control group was set to at least 95% as a requirement. An overview of the antigens used in panels 1 and 3-8 is contained in Table 8. In addition, a diagnostic panel 9 was compiled with use of all markers from Table 2. The panels 1, 3, 4, 5 and 9 are preferably suitable for the diagnosis of all SSc patients, regardless of the particular subtype. The AUC (area under the curve) of panels 1, 3, 4, 6 and 9 is specified in Table 8 for 2 independent SSc sample collectives (cohort I and cohort II).
Panel 1 consists of the antigens Sc170(Topl) and CENPB used conventionally for diagnosis and achieves an AUC of 0.81 in cohort I and 0.85 in cohort II for the comparison SSc versus healthy test subject (HC).
Panel 3 consists of two diagnostic antigens CEPNB and SC170, and two new SSc antigens KDM6B and BICD2. Compared to the usually used markers of panel 1, a logistic model for panel 3 shows a much higher AUC for the comparison SSc versus healthy test subject (HC): 0.88 for cohort I compared to 0.81, and 0.87 for cohort II compared to 0.85.
Panel 4 is based on panel 3 and contains two further new markers NSUNS and PPP1R2. For the diagnosis of SSc compared to healthy test samples, a logistic regression model for panel 4 achieves an AUC of 0.9 for cohort I and an AUC of 0.85 for cohort II.
Panel 5 is based on panel 4 and, in addition to the diagnostic markers CENPB and Sc170, also contains the markersKDM6B, BICD2, NSUNS, PPP1R2, RTEL1, TRIM21, ABCB8, AVEN, CENPA,CENPC1, GYS1, MARVELD2, PMF1, TTLL3, ATP13A2, LTF, STMN4, ACBD6, GTF2F1, KDR, DIP2C, FXC1, HNRNPA1, HSBP1L1, KARS, PBXIP1, ZNF431, STMN1 and SSRP1.
As presented in Table 6, only approximately 41% of the SSc patients from the SSc cohort II tested positively for anti-CENPB antibodies, and 25.3% tested positively for anti-Sc170 antibodies. Since very few patients with doubly positive reactivity have been described in the literature, the proportion of anti-CENPB- and anti-Sc170-negative patients was specified at approximately 30%. In order to close these diagnostic gaps, a logistic regression model was created for panel 6, which consists of 7 markers.
Panel 6 contains the markers KDM6B, TRIM21, GYS1, ALPK1, STMN4, HNRNPA1 and STMN1.
As summarised in Table 6, an AUC of 0.91 was achieved in cohort I, and an AUC of 0.84 was achieved for cohort II with the markers of panel 6.
As presented in table 6, 36.8% of the SSc patients with diffuse SSc (dSSc) and 25.3% of the patients with limited form were positively tested for autoantibodies against Sc170 (Top1). This shows that anti-Sc170 antibodies are indeed associated with the diffuse form, but are not specific. Additional markers are therefore required for diagnosis of the diffuse form. Panel 7 comprises 6 markers which are particularly suitable for the diagnosis of diffuse SSc.
Panel 7 contains the markers KDM6B, TOP1, TRIM21, CENPC1, ISY1 and HNRNPA1.
For panel 7 an AUC of 0.81 for cohort I and 0.87 for cohort II were calculated by means of a logistic regression.
As presented in Table 6, 41% of SSc patients with limited SSc (1SSc) and 8.8% of patients with diffuse form tested positively for autoantibodies against CENPB. This shows that anti-CENPB antibodies are indeed associated with the limited form, but are not specific. Additional markers are therefore required for diagnosis of the limited form. Panel 8 comprises 27 markers which are particularly suitable for the diagnosis of limited SSc.
Panel 8 comprises the markers KDM6B, BICD2, NSUNS, CENPB, TOP1, RTEL1, TRIM21, ABCB8, AVEN, CENPA, CENPC1, CENPT, GYS1, C19orf52, STMN4, ACBD6, CBX3, CENPH, EIF4E, GTF2F1, MAPT, SPTAN1, DIP2C, PBXIP1, NEFL, STMN1 and SSRP1.
For panel 8, an AUC of 0.97 for cohort I and 0.99 for cohort II were calculated by means of a logistic regression.
In accordance with the invention, recombinantly produced and improved KDM6B protein (Seq ID 315) comprising amino acids 42-421 of the Uniprot Database Entry 015054 was used. KDM6B is purified over a number of stages by means of nickel chelate affinity chromatography, size exclusion chromatography, and ion exchanger chromatography. The purified KDM6B was then applied in a concentration of 1 μg per millilitre in an Na carbonate buffer pH 9.0 to a flat-bottom ELISA plate (for example NUNC) with 100 μl per cavity, and was incubated for 4h at room temperature (RT).
After incubation, the excess buffer was removed and the free binding points of the ELISA plate were blocked using an inert protein (cold water fish gelatin) in PBS for lh at room temperature.
Serum or plasma samples were diluted 1:101 in HBS-T buffer, applied to the cavities and incubated for 30 min at room temperature. During this time, the KD6MB specific IgG autoantibodies from the serum or plasma sample bind to the bound protein.
Non-specifically-bound antibodies are removed after the incubation by washing twice with a PBS Tween buffer. Incubation is then performed for half an hour with an HRP (horseradish-peroxidase-conjugated) anti-human IgG detection antibody. After the incubation, non-specific binding partners are again removed by washing twice with PBS tween buffer.
The colorimetric detection reaction is performed by addition of a TMB (tetramethylbenzidine) substrate. This reaction is stopped after 15 minutes by addition of a 1M sulphuric acid and is evaluated in an ELISA measuring device (TECAN) at a wavelength of 450 nm vs 620 nm. The colour intensity (optical density, OD) of the detection reaction is directly proportional to the concentration of the KDM6B-specific IgG autoantibodies in the serum or plasma sample.
The test is evaluated semi-quantitatively via one-point calibration.
The OD values of the anti-KDM6B ELISA were used for the calculation of the p-values by means of Wilcoxon rank sum test and the calculation of a logistic regression model with ROC analysis for the comparison of SSc against HC. Since the group of SSc patients is greater than that of the control group HC, a random under-sampling is carried out for the logistic regression, such that the SSc cohort II has groups of identical size. The ROC analysis was calculated by using 75% of this reduced data set of SSc cohort II for training and 25% for testing the model. This approach is repeated 100 times.
Table 10 shows the p-values for the comparison of SSc versus healthy controls and SSc versus autoimmune controls calculated from samples of SSc cohort II.
Table 11 shows the empirically calculated area under the curve (AUC), sensitivity, and specificity of the anti-KDM6B ELISA for the diagnosis of SSc (SSc cohort II) compared to healthy samples.
In accordance with the invention, recombinantly produced BICD2 protein was purified over a number of stages by means of nickel chelate affinity chromatography, size exclusion chromatography, and ion exchanger chromatography. The purified BICD2 was then applied in a concentration of 1.7 μg per millilitre in an Na carbonate buffer pH 9.0 to a flat-bottom ELISA plate (for example NUNC) with 100 μl per cavity, and was incubated for 4 h at room temperature (RT).
After incubation, the excess buffer was removed and the free binding points of the ELISA plate were blocked using an inert protein (cold water fish gelatin) in PBS for 1 h at room temperature.
Serum or plasma samples were diluted 1:101 in HBS-T buffer, applied to the cavities and incubated for 30 min at room temperature. During this time, the BICD2-specific IgG autoantibodies from the serum or plasma sample bind to the bound protein.
Non-specifically-bound antibodies are removed after the incubation by washing twice with a PBS Tween buffer. Incubation is then performed for half an hour with an HRP (horseradish-peroxidase-conjugated) anti-human IgG detection antibody. After the incubation, non-specific binding partners are again removed by washing twice with PBS tween buffer.
The colorimetric detection reaction is performed by addition of a TMB (tetramethylbenzidine) substrate. This reaction is stopped after 15 minutes by addition of a 1M sulphuric acid and is evaluated in an ELISA measuring device (TECAN) at a wavelength of 450 nm vs 620 nm. The colour intensity of the detection reaction is directly proportional to the concentration of the BICD2-specific IgG autoantibodies in the serum or plasma sample.
The OD values of the BICD2 antigen were used for the calculation of the p-values by means of Wilcoxon rank sum test and the calculation of the receiver operator curves (ROC).
The OD values of the anti-BICD2 ELISA were used for the calculation of the p-values by means of Wilcoxon rank sum test and the calculation of a logistic regression model with ROC analysis for the comparison of SSc against HC. Since the group of SSc patients is greater than that of the control group HC, a random under-sampling is carried out for the logistic regression, such that the SSc cohort II has groups of identical size. The ROC analysis was calculated by using 75% of this reduced data set of SSc cohort II for training and 25% for testing the model. This approach is repeated 100 times.
Table 10 shows the p-values for the comparison of SSc versus healthy controls (HC) and SSc versus autoimmune controls (AD) calculated from samples of SSc cohort II.
Table 11 shows the empirically calculated sensitivity and specificity of the anti-KDM6B and anti-BICD2 ELISA for SSc cohort II for the diagnosis of SSc compared to healthy controls
| Number | Date | Country | Kind |
|---|---|---|---|
| 14167807.8 | May 2014 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/060396 | 5/11/2015 | WO | 00 |
