Detection of autoantibodies for diagnosing degenerative diseases of the skeletal system

Abstract

Methods, kits, and active ingredients for diagnosing or treating arthritis or a degenerative disease of the skeletal system, or for selection of subjects for therapy. The methods for diagnosing arthritis involve the detection of an autoantibody, which is associated with arthritis, or excluding the presence of an autoantibody against collagen II. The methods for diagnosing a degenerative disease of the skeletal system, involve the detection of an autoantibody against thrombospondin-4 or COMP. The kits contain a detection agent for an autoantibody and can be used for diagnosing arthritis or a degenerative disease of the skeletal system. The active ingredient can be used for treating or preventing autoimmune-associated arthritis.

Claims

1. A method of treating arthrosis in a subject, the subject not having rheumatoid arthritis, the method comprising: (a) obtaining a sample from the subject, wherein the sample is a serum, blood, or plasma sample, and wherein the subject is a human; (b) detecting whether an autoantibody against thrombospondin-4 (TSP-4), a degradation product or a fragment thereof, is present in the sample; (c) diagnosing the subject as having arthrosis or a risk of developing arthrosis by detecting the autoantibody against thrombospondin-4 (TSP-4), the degradation product or the fragment thereof, in the sample; and (d) administering an immunotherapy to the diagnosed subject of step (c).

2. The method of claim 1, wherein the method comprises the exclusion of rheumatoid arthritis in the subject, wherein the subject is tested for rheumatoid arthritis before detecting autoantibody against TSP-4.

3. The method of claim 1, further comprising the exclusion of the presence of an autoantibody against collagen II or a degradation product or a fragment thereof in the sample.

4. The method of claim 3, further comprising detecting the absence of an autoantibody against collagen II or a degradation product or a fragment thereof in the sample.

5. The method of claim 4, wherein the autoantibody against collagen II or a degradation product or a fragment thereof is detected using a detection agent.

6. The method of claim 1, wherein the method comprises the detection of at least one further autoantibody in the subject's sample.

7. The method of claim 6, wherein the at least one further autoantibody is an autoantibody against cartilage oligomeric matrix protein (COMP) or a degradation product or a fragment thereof.

8. The method of claim 1, wherein the detection is carried out using a detection agent.

9. The method of claim 1, wherein the course of arthrosis is monitored by detecting the autoantibody at various time points; or wherein the stage of arthrosis is diagnosed.

10. The method of claim 8, wherein the detection agent is capable of binding to the antigen-binding region of the autoantibody.

11. The method of claim 6, wherein the at least one further autoantibody is an autoantibody against C-type lectin domain family 3 member A (CLEC3A) or a degradation product or a fragment thereof.

12. The method of claim 6, wherein the at least one further autoantibody is an autoantibody against cartilage oligomeric matrix protein (COMP) or a degradation product or a fragment thereof and an autoantibody against C-type lectin domain family 3 member A (CLEC3A) or a degradation product or a fragment thereof.

13. The method of claim 1, wherein the immunotherapy comprises administering to the subject an antibody against B cells and/or a Wnt signaling inhibitor.

14. The method of claim 1, wherein the immunotherapy comprises administering to the subject rituximab, fluoxetine, and/or SM04690.

Description

MATERIAL AND METHODS

Selection of Patient and Control Groups

(1) The study population is composed of two patient groups and one healthy control group (HD=healthy donors). The test groups are either arthrosis patients or rheumatoid arthritis (RA) patients. The inclusion criteria for the arthrosis group are a clinically established osteoarthritis (OA) of the large joints. Exclusion criteria are indications of a rheumatoid joint disease or other autoimmune or malignant underlying diseases. Inclusion criteria for the RA group are an established RA in accordance with the ACR/EULAR classification criteria. Exclusion criteria are an arthrosis or other autoimmune or malignant underlying diseases. Inclusion in the control group requires that the subjects be symptom-free in relation to the joints. Exclusion criteria are diagnostic indications of an arthrosis, RA, and autoimmune or malignant underlying diseases. The serum from 10 arthrosis patients, 10 RA patients and 10 healthy controls was tested. All the samples were provided by our cooperation partner Prof. Pongratz of Rheumatologie der Universitätsklinik Düsseldorf [Düsseldorf university hospital, rheumatology].

Cloning, Recombinant Expression and Protein Purification

(2) The human CLEC3A gene was cloned into a modified pCEP-Pu vector and transfected into HEK-293 EBNA cells. The recombinant protein was purified from the supernatant of the cells by affinity chromatography. Human collagen II was purified from human cartilage (PMID: 6439184). Recombinant human TSP-4 (R&D) and recombinant human COMP (R&D) were ordered from the manufacturer in question.

SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblotting

(3) To detect autoantibodies in the serum from the subjects, the matrix proteins were resolved by means of SDS-PAGE and detected by means of immunoblotting. To this end, the proteins (1 μg in each case per well) were transferred to a PVDF membrane (0.45 μm, Invitrogen) after carrying out the SDS-PAGE (4-12% bis-Tris gels: 12 wells, 1 mm thickness, MOPS buffer, 200 V for 50 min), free binding sites were saturated with 5% milk powder and 1% bovine serum albumin in TBS-T (Tris-buffered saline solution, 0.1% Tween), and were successively incubated with 50 μl of patient serum in 10 ml of blocking solution, overnight at 4° C.) and with an HRP-conjugated (horseradish peroxidase-conjugated) anti-human IgG antibody (SantaCruz, 1:200 000 in blocking solution for 1 h at room temperature). Serum from a healthy person was used as reference. The concentration of IgG in the serum was determined and 0.3, 3 and 30 ng of IgG were loaded onto the SDS-PAGE, each in a reference lane. Signals were detected using the ChemiDoc XRS+ (BioRad) western blot imager after incubation with ECL Plus (Amersham Pharmacia Biotech) and the image sequences were evaluated using ImageLab (BioRad) software.

Results and Discussion

(4) Detection of TSP-4, CLEC3A, COMP and collagen II autoantibodies for the diagnosis of an arthrosis—In our study, we tested the serum from 10 arthrosis (OA) patients, 10 rheumatoid arthritis (RA) patients and 10 healthy subjects (Healthy Donors, HD) for IgG isotype antibodies against TSP-4, CLEC3A, COMP and collagen II by means of SDS-PAGE and immunoblotting (Table 1). The detection of TSP4, CLEC3A, COMP and collagen II autoantibodies in the blood and/or in synovial fluid can be used for the diagnosis, specifically also for an early diagnosis, of an arthrosis or an activated arthrosis and for the diagnosis of the stage of arthrosis, the monitoring of the course of arthrosis and the monitoring of the therapy of an arthrosis (Table 1).

(5) TABLE-US-00007 TABLE 1a Number of positive antibody reactivities against the various matrix proteins in the comparison of OA and HD. Frequencies were each reported in relation to the group size. Significant differences are marked by an *. (source: http://www.socscistatistics.com/tests/fisher/Default2.aspx). Positive Negative Antibody prediction prediction against: OA HD value value Sensitivity Specificity p value TSP-4 * 5/10 0/10 1 0.67 0.5 1 0.0325 COMP 4/10 1/10 0.8 0.67 0.4 0.9 0.303 CLEC3A 2/10 0/10 1 0.56 0.2 1 0.474 Collagen II 0/10 7/10 0 0.23 0 0.3 0.003

(6) TABLE-US-00008 TABLE 1b Individual antibody intensities against various antigens. Patients 1 to 10 represent the OA group, patients 11 to 20 the healthy control group. The mean values and standard deviations were calculated for the corresponding group in which there were ≥ two values for the respective antigen. Signals of the immunoblot were evaluated by densitometry and quantified in relation to the associated 0.3 ng IgG reference band (=1) (x-fold IgG) or reported in relation to the group mean value (x-fold MV). If no values were entered in a box, it was not possible to detect a band in the immunoblot. MV = mean value, StdDev = standard deviation. TSP-4 COMP CLEC3A Collagen II x-fold TSP-4 x-fold COMP x-fold CLEC3A x-fold Collagen II IgG x-fold IgG x-fold IgG x-fold IgG x-fold reference MV reference MV reference MV reference MV MV 0.41 1 0.27 1 0.27 1 0.40 1 MV −/+ −0.32 to 1.13 — −0.22 to 0.77 — 0.12 to 0.42 — 0.27 to 0.53 — 2x Std Dev Patient 1 0.96 2.36 Patient 2 0.15 0.55 0.19 0.72 Patient 3 0.15 0.37 Patient 4 0.09 0.22 Patient 5 0.72 1.77 Patient 6 0.2 0.73 Patient 7 0.05 0.18 Patient 8 Patient 9 0.11 0.27 0.69 2.53 Patient 10 0.34 1.28 Patient 11 0.33 0.83 Patient 12 0.42 1.05 Patient 13 Patient 14 Patient 15 0.53 1.33 Patient 16 0.35 0.88 Patient 17 0.34 0.85 Patient 18 0.39 0.98 Patient 19 0.44 1.10 Patient 20

(7) By combining the antibodies, it is possible to additionally distinctly increase the diagnostic informative value (Table 2).

(8) TABLE-US-00009 TABLE 2 Number of positive antibody reactivities, accumulated, against multiple proteins in groups OA and HD. Frequencies were each reported in relation to the group size. For the cumulative comparisons, the differences in the allocation of the absolute frequencies to the two groups are highly significant. Collagen II autoantibodies could only be detected in the HD group. OA patients showed no collagen II autoantibodies. The lack of detection of collagen II autoantibodies therefore serves as a positive result for OA. Positive Negative Antibody prediction prediction group OA HD value value Sensitivity Specificity P value TSP-4 5/10 0/10 1 0.67 0.5 1 0.033 TSP-4/ 8/10 1/10 0.89 0.82 0.8 0.9 0.005 COMP TSP-4/ 9/10 1/10 0.9 0.9 0.9 0.9 0.001 COMP/ CLEC3A TSP-4/ 10/10  3/10 0.77 1.0 1.0 0.7 0.003 COMP/ CLEC3A/ Collagen II

(9) Detection of TSP-4, CLEC3A and COMP antibodies for therapy selection in the case of arthrosis—In various studies, it has been shown that the immunization of animals with collagen II (CIA, collagen II-induced arthritis) or COMP (COMPIA) leads to a severe, chronic arthritis in the animals. In these (RA) animal models, antibodies against matrix proteins are of pathophysiological significance. This shows that human autoantibodies against matrix proteins can also exhibit a pathophysiological effect and that patients having autoantibodies against matrix proteins are therefore amenable to an immunosuppressant therapy. In this connection, an important criterion for pathogenicity appears to be the number of different autoantibodies and/or the autoantibody concentration (autoantibody titer) in the blood. In our study, there are multiple autoantibodies in two OA patients and the antibody intensities against the different antigens also differ.

(10) Detection of anti-TSP-4 and anti-COMP antibodies in the case of rheumatoid arthritis—In our study population, we found, in three RA patients, concentrations of TSP-4 and COMP autoantibodies (RA patient No. 21, 22, 29) that were higher by a factor of 30-fold to 130-fold compared with the intensities from the other RA patients (Table 3).

(11) TABLE-US-00010 TABLE 3 Mean values of the antibody intensities in the RA group. To calculate the mean values, patients 21, 22, 29 were excluded, since they were, statistically speaking, significant outliers (Grubbs' outlier test). MV = mean value, StdDev = standard deviation. Signals of the immunoblot were evaluated by densitometry and quantified in relation to the associated 0.3 ng IgG reference band (=1) (x-fold IgG) or reported in relation to the group mean value (x-fold MV). TSP-4 COMP x-fold TSP-4 x-fold COMP IgG x-fold IGg x-fold reference MV] reference MV] MV 0.95 1 0.63 1 MV −/+ 2x StdDev −0.60 to 2.50 — −0.05 to 1.32 — Patient 21 1.84 1.93 82.9 130.67 Patient 22 30.7 32.27 0.9 1.42 Patient 23 1.3 1.37 0.81 1.28 Patient 24 0.81 0.85 1.29 2.03 Patient 25 0.52 0.55 0.45 0.71 Patient 26 No band No band 0.13 0.20 Patient 27 0.19 0.20 0.28 0.44 Patient 28 0.57 0.60 0.45 0.71 Patient 29 51.8 54.45 0.53 0.84 Patient 30 2.38 2.50 0.87 1.37

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