HK1124345A - Method for diagnosing rheumatic diseases - Google Patents

Description

Method for diagnosing rheumatic diseases

The present invention relates to polypeptides reactive with antibodies associated with rheumatism. The invention also relates to a diagnostic agent comprising one of these polypeptides, a diagnostic kit comprising said diagnostic agent, and a method for the in vitro detection of rheumatic diseases. Furthermore, the invention relates to medicaments comprising one of these polypeptides, as well as to the use of said polypeptides for the production of medicaments for the prophylaxis and/or treatment of rheumatic diseases.

Rheumatic diseases, in particular pain in the range of joints and motor systems, are among the most common diseases in germany. Laboratory tests that enable the pain to be classified as harmless muscle spasm, joint disease or the most common and most serious disease (rheumatoid arthritis (RA)) are not known to date.

Rheumatoid arthritis is an autoimmune disease in which the body's defense mechanisms mistakenly treat endogenous articular cartilage as foreign and hostile and attack the cartilage. One of approximately 100 people in western european countries has rheumatoid arthritis. The disease progresses particularly rapidly in the first few months.

Thus, a fundamental key strategy in modern rheumatology is the early use of biopharmaceuticals that alter the course of the disease. Numerous clinical studies have shown that very good therapeutic effects and response rates can be achieved with suitable active substances, such as TNF antagonists, if the compounds are already used in patients at an early stage. Rheumatologists try to exploit the narrow time window between the onset of disease and the appearance of structural joint damage. However, reliable and sensitive detection of rheumatoid joints within said time window has not been disclosed in the prior art to date.

Rheumatoid arthritis was diagnosed according to the classification criteria of ACR (american college of rheumatology). According to the criteria of ACR, rheumatoid factor is currently the basic serological indicator for the diagnosis of Rheumatoid Arthritis (RA). Rheumatoid factor is a small group of immunoglobulins characterized by immunological cross-reactivity to the Fc-region of immunoglobulin g (igg).

However, the presence of rheumatoid factors is not limited to diseases of the rheumatic form group (Formenkreis), the basis of differential diagnosis, and is also found in the serum of patients with infectious diseases, hypercholesterolaemia, lymphoproliferative B-cell diseases, and generally in the elderly population.

Generally, elevated concentrations of rheumatoid factor are associated with a more severe course of the disease. Here, the concentration and activity (C:) And not the therapeutic effect. The presence of rheumatoid factor-based concentrations does not allow a sensitive and clear prognosis of rheumatoid arthritis development. Healthy people have elevated concentrations of rheumatoid factor without disease, in contrast to patients without rheumatoid factor who have a very aggressive form of rheumatoid arthritis.

Other serological markers such as anti-citrulline antibody (CCP), or initial HAQ-scores for assessing daily living ability, or X-ray or Computed Tomography (CT) images provide little information in an early form, and each is not sufficiently effective to be able to assess how a patient's prognosis is.

To optimize the existing classification criteria of ACR, the american college of rheumatology proposed 7 classification criteria representing poor prognosis:

1. joint morning stiffness lasting more than 1 hour,

arthritis of 2.3 or more joints,

3. joint inflammation in at least 3 joint areas at the same time,

4. the hand joints or finger joints are also affected,

5. bilateral tenderness of the metacarpophalangeal joints,

erosion on the X-ray photograph,

7. detection of specific rheumatoid factors and anti-perinuclear factor positivity (APF).

Autoantibodies against so-called "anti-perinuclear factors" were described for the first time by Young et al in patients with rheumatoid arthritis (Young, b.j.j. et al, antibodies in rhematoid arthritis, b.m.j., 2(1979), 97-99). Keratin proteins have long been recognized as corresponding antigens due to their specific response to the keratinized epithelium of the stratum corneum on the rat esophageal segment (Vincent, C.H. et al; High diagnostic value in rhematoid arthritis of the stratum corneum of Oesophagous epiphyllum, so-called "anti-inflammatory antibodies", Ann. Rheum at. Dis.48(1989), 712-. Thus, such Antibodies are also referred to up to now as anti-keratin Antibodies (AKA) (Vincent, C.H. et al, Natural IgG to epidermal Cytomerins vs IgG to the Stratum Corneuum of the Rat Oesophaga epidermidis, so-called "Antibodies" in Rheomatous arthritis and other Rheomatic Diseases; J.of Autoimmitunity 4(1991), 493 505; Paimela, L. et al, Antibodies: diagnostic and diagnostic markers for early rhematic Antibodies, Ann. Rheoma. Dis., 51(1992), 743 (746)).

In addition, later studies demonstrated that AKA or APF were also recognized by anti-filaggrin antibodies. Thus, the basic protein filaggrin was identified as the target antigen. The 40 kDa Protein aggregates into Cytokeratin microfilaments and helps to form The extracellular fibrous matrix of keratinocytes (Simon, M. et al, The cytokine refining Protein Filaggrin resistant Target of The So-called "infected" Antibodies, Autoantibodies Specific for rhematoid Arthritis, J.Clin. invest., 92(1993), 1387-93).

These antibody systems appear to be identical because sera containing APF, AKA and anti-filaggrin antibodies react in the same way. Anti-filaggrin antibodies of the IgG type with a specificity of more than 99% are highly specific markers for rheumatoid arthritis. In principle, the antibodies can be detected early and before clinical symptoms appear. In several studies, a positive correlation with the severity and activity of the disease can be found. Anti-filaggrin antibodies are independent of age, sex, or duration of disease. They can be detected in approximately 34% of patients who are rheumatoid factor negative and are valuable diagnostic aids here.

However, by using current methods in general, the antibodies are found in serum only in about 40% of cases.

It is therefore an object of the present invention to provide novel polypeptides for the detection of antibodies associated with rheumatic diseases, in particular with rheumatoid arthritis, which enable a sensitive and specific diagnosis, classification and prognosis of rheumatic diseases, in particular pain in the range of the joints and the locomotor system.

When analyzing the binding of antibodies to native vimentin, i.e., either APF positive or anti-Sa reactivity (e.r. vosseenaar et al; rhematoid Arthritis specific anti-Saantobiies targetwitriented view; Arthritis res. The.The.6 (2), (2004), 142-150), we have now found that native vimentin known in the art to be non-immunoreactive (c.a. Hitchon et al; Immune deficiencies of and serositive Arthritis in early syndromes; curr. Ophesin.14 (4), (2002), 348-353) exists as a mutated immunoreactive variant. This finding was surprising, since it was believed according to the prior art to date that in order to be immunoreactive, vimentin had to be citrullinated. By differential immunoaffinity chromatography (differenzielle)) Enrichment of immunoreactive vimentin variants with mutated sequences from human monocytes we were able to demonstrate this belief to beIs erroneous. These mutant variants of native vimentin differ from native vimentin in the presence of an additional arginine residue, and optionally, further sequence differences. They react with human RA-related antibodies and surprisingly have a higher specificity and sensitivity than citrullinated peptides known in the prior art.

Thus, the object of the present invention is a polypeptide derived from the native vimentin protein having SEQ ID No.1, which polypeptide differs from the native sequence by the presence of at least one additional arginine residue.

The additional arginine residues are preferably inserted into the sequence by substitution of other amino acid residues of native human vimentin. The polypeptide preferably has an arginine residue at least one of positions 16, 17, 19, 41, 58, 59, 60, 68, 76, 140, 142, 147, 363, 406 or 452. Particularly preferred positions are 41, 58, 59, 60 and/or 68. For example, the polypeptide has additional arginine residues at least 1, 2, 3, or 4 positions.

In another embodiment, the polypeptide further has an additional leucine residue in at least one of positions 3, 20, 33, 36, 37, 94, 165, 361, 399 or 426, preferably in positions 33, 36 and/or 37, compared to the native sequence. For example, the polypeptide has additional leucine residues at least 1, 2, 3, or 4 positions.

In another embodiment the polypeptide has an additional proline residue in at least one position compared to the native sequence, e.g. in one of positions 21, 41, 43, 50, 54, 62, 64 or 89, preferably in position 41, 43, 50, 54, 62 and/or 64. For example, the polypeptide has a proline residue in at least 1, 2, 3 or 4 positions.

In another embodiment, the polypeptide has an additional threonine residue in at least one position compared to the native sequence, for example in one of positions 24, 35 or 99. For example, the polypeptide has threonine residues at least 1, 2, or 3 positions.

In another embodiment, the polypeptide has an additional tyrosine residue in at least one position compared to the native sequence, for example in one of positions 25, 39, 42, 49, 55 or 67. For example, the polypeptide has tyrosine residues at least 1, 2, 3 or 4 positions.

In another embodiment, in the polypeptide, at least one arginine residue is present in the form of a citrulline residue, for example at least one of positions 4, 12, 23, 28, 36, 45, 50, 64, 71, 100, 320, 364 or 378. For example, the polypeptide has citrulline residues at least 1, 2, 3 or 4 of said positions. However, in another aspect, the polypeptide may also be a polypeptide that does not contain citrulline.

Preferred examples of muteins of human vimentin have a sequence comprising SEQ ID No.2, 3, 4, 5, 6, 7, 8 or 9.

Another object of the invention is a fragment of any of the above polypeptides, said fragment being derived from a native vimentin having SEQ ID No.1, comprising at least one region containing at least one arginine residue, and exhibiting reactivity to autoantibodies associated with rheumatoid disease. The fragments are preferably located in the region of positions 10-145. Particularly preferably, the fragments are located in the region of positions 30-70. Preferred examples of fragments are fragments 51-65 (C2). The length of the fragments is preferably at least 6, particularly preferably at least 8 amino acids to 120, preferably 100, particularly preferably 50 amino acids.

Another object of the present invention is a peptide derivative of the above polypeptide or fragment. For example, the peptide derivative may be a Retro (Retro-)/trans (Inverso-) polypeptide, i.e., an inverted polypeptide of the above-mentioned polypeptide prepared from a D-amino acid according to the mirror image of the polypeptide (inverssespoypeptid), a Retro polypeptide having "reverse" sequence (Retro-Polypeptid), and a Retro-trans polypeptide which is the mirror image of the above-mentioned polypeptide and also has "reverse" sequence (Retro-Inverso-Polypeptid).

Other examples of peptide derivatives are polypeptides modified at the side chain groups, the amino terminus or/and the carboxy terminus, for example modified at the amino group with e.g. a carboxylic acid or an alkyl group, or modified at the carboxylic acid group with an amino group or an ester group. The polypeptide and/or peptide derivative may also be present as a cyclic peptide.

Another object of the present invention is a nucleic acid encoding the above polypeptide. Examples of nucleic acids include DNA and RNA, in particular cDNA. For the recombinant production of the polypeptides, the nucleic acids can be cloned into customary eukaryotic or prokaryotic vectors and expressed in suitable host cells.

Another object of the present invention is a diagnostic agent comprising one or more of the above polypeptides or fragments thereof. The diagnostic agent may comprise the polypeptide or the fragment in free or carrier-bound form.

The fact that the polypeptide of the invention proves to be a highly specific and highly sensitive antigen for the detection of antibodies in body fluids of patients with rheumatic diseases, in particular inflammatory diseases with joints and the motor system, particularly preferably with rheumatoid arthritis, is highly surprising. Within the scope of the present invention, preferred body fluids are blood, serum and plasma, particularly preferred is serum.

The diagnostic agents of the present invention have a number of advantages. Because the polypeptide contains multiple antibody binding sites, it can effectively bind to monomer as well as multimeric antibodies. Another advantage of the mutated polypeptide is that it enables the provision of diagnostic agents that can identify patients with inflammatory and chronic diseases of the joints and motor system, in particular with rheumatoid arthritis, with a specificity of 99% and a sensitivity of 85%.

The prior art has not disclosed any diagnostic agent with considerable specificity or sensitivity which enables the use of citrulline-free proteins or peptides for the detection of rheumatic diseases, in particular rheumatoid Arthritis (P.J. Utz, Death, autoantigenin modifications, and tolerance; Arthritis Res., 2, (2000), 101-.

Another object of the present invention is a diagnostic kit for detecting rheumatic diseases, in particular rheumatoid arthritis, which comprises the above diagnostic agent. Furthermore, the diagnostic kit may comprise conventional components, such as buffers, solvents and/or labelling groups.

Suitable carriers are macromolecules such as DNA, RNA, pharmaceutically compatible polymers such as polyethylene, poly D, L-lactide-co-glycolide, synthetic biopolymers such as polylysine and dextran, and proteins such as serum albumin and hemocyanin. Preferably, dextran (Gregorius, K., Mouritsen, S. and Elsner, H.I., Hydrocoating: a new method for coating bioleules to solid phases, J.Immunol. methods 12(1995), 65-73) is used in the so-called "Water coating) -coating method".

Another object of the invention is a method for the in vitro detection of rheumatic diseases, in particular rheumatoid arthritis, in which method the concentration of autoantibodies in body fluids is determined. The methods allow for diagnosis, classification and/or assessment of severity of disease. The above diagnostic agent or the above diagnostic kit is used as a detection reagent.

In the method of the present invention, any method commonly used in the field of diagnosis may be used as the detection method, for example

(a) The method of the enzymology of the raw materials,

(b) methods based on luminescence, or

(c) A radiochemical method.

As a preferred detection method, radioimmunoassays, chemiluminescent immunoassays, immunoblotting assays or enzyme immunoassays such as ELISA can be considered in the method of the present invention.

In one embodiment of the method of the invention, the body fluid to be analyzed is added as a sample to the above-mentioned polypeptide bound to the carrier. After incubation of the sample, unbound components are washed away. The specific binding of the autoantibody to be detected to the polypeptide is detected by a secondary antibody having a labelling group.

Suitable secondary antibodies in the method of the invention are, for example, antibodies directed against human antibodies such as the Fc part of IgG, IgM, IgA or/and IgE, for example human IgG.

Suitable labelling groups in the method of the invention are, for example, enzymes such as peroxidase or alkaline phosphatase, radiolabels, or luminescent labelling groups such as acridinium compounds.

Alternatively, a competitive inhibition test using the polypeptides of the invention may also be performed, in which the binding of labeled Rheumatoid Arthritis (RA) -autoantibodies is inhibited in the presence of a sample, if RA-autoantibodies are also present in the sample.

The peptides of the invention may also be used as reagents for prognosis or/and for process control in the treatment of rheumatic diseases, in particular rheumatoid arthritis. Preferred prognostic agents here are peptides from the region of positions 30 to 65 or/and 55 to 70.

It is another object of the present invention to provide a medicament which selectively inhibits or prevents the formation of autoimmune complexes associated with rheumatic diseases, in particular inflammatory processes (particularly preferably in inflamed joints), but which does not lead to an overall blocking of antibody production.

According to the invention, this object is achieved by the use of a medicament which comprises the above-mentioned polypeptide or fragment and is suitable for use in human or veterinary medicine. The medicament may for example be used for the extracorporeal treatment of body fluids, such as blood or plasma, in order to capture autoantibodies contained therein using antibodies bound to a solid phase and reintroduce the treated body fluid into the patient.

The examples of the invention surprisingly show that the polypeptides of the invention are antigens which are primarily associated with the course of the disease, or even proteins which primarily initiate the disease. The course of the disease can be beneficially affected by selective removal of the antibody from the patient's body fluids.

Furthermore, the polypeptide or fragment thereof may also be administered directly in the form of a pharmaceutical composition, which may comprise pharmaceutically compatible carriers, solvents and/or adjuvants.

The pharmaceutical composition may be administered in the form of tablets, capsules, solutions, suspensions, aerosols, sprays (nasal or throat sprays), gels, plasters, etc.

The drug may be administered according to any known method, with oral administration and intravenous administration being particularly preferred.

The dosage may vary depending on the type and severity of the disease, and is generally in the range of 1 to 200 mg/day, preferably 10 to 200 mg/day.

In addition, RA patients can also be treated with the identified short, synthetic protein epitopes of the above polypeptides (particularly the C2-epitope) derived from the amino acid sequence of natural vimentin.

Analysis of B cell epitopes in RA patients yielded the surprising finding that: 91% of all patients interacted with a short, synthetic linear peptide epitope (C2-epitope) of the polypeptide of the invention.

Furthermore, it was found through mouse experiments that after such treatment, mice produced immune cells that successfully inhibited the division of antigen-specific immune cells.

The course and effectiveness of treatment of rheumatic diseases, particularly rheumatoid arthritis, can be controlled according to current diagnostic methods that rely solely on the laborious and time-consuming process of Disease Activity Score (DAS). In this method, the number of swollen joints, the number of painful joints, inflammation parameters (BSR or CRP) are calculated, and the status of the patient is recorded according to the visual analogue scoring method (visuelen Analog-Skala).

The 4 individual components are added up according to a formula. The resulting values provide relatively reliable and objective information about the actual activity and quality of treatment of the disease in the patient.

The polypeptides of the invention now enable the control of the course and effect of the treatment of rheumatic diseases, in particular rheumatoid arthritis.

For example, a rapid and effective treatment was observed in patients cross-reacting with the polypeptide at positions 30 to 65, which partially obtained a positive result after 2 years in the form of a complete cure.

Patients with low disease activity (average DAS score of 2.8) can be identified by ELISA based on polypeptides of the invention (average < 300U/ml). In relation to the effect of treatment with sulfasalazine or cortisone (DAS score below 1.5 after treatment), antibody titers were reduced to 1/6 of the starting value on average over the course of 1-2 years.

Patients with severe rheumatoid arthritis (average DAS score 4.9) had on average antibody titers of > 1000U/ml. Since the DAS score was not significantly altered by treatment with infliximab (Remicade) and/or methotrexate, the amount of antibodies to the polypeptides of the invention was reduced by approximately 30-50% in 50% of patients. In parallel with the altered antibody titers, the patients reported a significantly more positive overall assessment of the subjective disease status in the anamnesis records, i.e. the diagnostic agents of the invention are also suitable for quality control of the treatment of severe rheumatoid arthritis.

A significant advantage is that it is possible for the first time to provide reliable information qualitatively and quantitatively within minutes about the type, course and treatment of rheumatoid arthritis.

This surprising finding has led to the realization that the polypeptides of the invention can be used therapeutically, since, surprisingly, by their use it can be expected to reduce previously undesired drug effects while at the same time maintaining the efficacy of the drug.

In particular, the polypeptides of the invention surprisingly enable the provision of novel, previously unknown principles of action for the treatment of rheumatic diseases, in particular inflammation. The spectrum of action of the polypeptides of the invention differs from that of hitherto known inhibitors of inflammation, since selectively only antibody-dependent mediators (mediators) can be produced in a reduced number of ways. Another advantage of the polypeptides of the invention is that the side effect profile may be significantly smaller than that of the drugs known in the prior art, since no cross-reactivity with antibodies of healthy blood donors has been observed.

Thus, the above-described diagnostic and therapeutic agents of the present invention can be used for diagnosing, preventing or treating any symptoms based on the vimentin-dependent process.

Another object of the present invention are the putative proteins BF0786 and BF0713 of the periodontal disease pathogens Bacteroides forsythia (Bacteroides forsythus) and Prevotella intermedia (Prevotella intermedia), which have been found to be inhibitors of rheumatic diseases, in particular chronic inflammatory diseases of the joints and motor system.

These proteins and fragments thereof are therefore suitable for the production of diagnostic agents, diagnostic kits or medicaments for the detection, prevention and/or treatment of rheumatic diseases, in particular pain in joints and the motor system. For diagnostic and pharmaceutical applications, reference is made to the above detailed description of the vimentin analogs.

The protein BF0786 and its sequence have been described by Kuwahara, T.et al (Kuwahara, T.et al, Proc. Natl. Acad. Sci. U.S.A 101 (41)), 14919-14924 (2004)). The protein BF0713 and its sequence has been described by Cerdeno-Tarraga, A.M. et al (Cerdeno-Tarraga, A.M. et al, Science 307(5714), 1463-1465 (2005)). Medical applications in the diagnostic and therapeutic fields have not been disclosed to date.

It is another object of the present invention to find a substance which selectively blocks the formation of autoimmune complexes consisting of rheumatic-related autoantibodies with autoantigens, in particular vimentin, i.e. which only blocks the reaction of antibodies involved in pathogenesis, but does not generally affect antibody production and antibody reaction, thus enabling the sensitive and specific treatment or prevention of rheumatic-related pain diseases in the range of joints and the motor system.

It has now been found that ingredients of green tea can achieve this. Tariq, M. et al, "Prevention of collegen-induced arthritis in microorganism by apolyphenol separation from green tea" Proc. Natl. Acad. Sei. USA, Vol.96, p.4524-4529, (1999) discloses the epigallocatechin gallate (EGCG) component of green tea. This moiety binds to vimentin and blocks phosphorylation (S.Ermakova et al, The intermediate membrane protein differentiation a new target for The oligomeric atechophin gate; J.biol.chem.280(17), (2005), 16882-. The use of epigallocatechin gallate has shown that the formation of autoimmune complexes of antibodies to mutated vimentin can be blocked in a concentration-dependent manner.

In competition experiments it could be shown that the non-binding substance Epicatechin (EC) blocks antibody binding to a maximum of only 10%. Epigallocatechin gallate (EGCG) was used to show that up to 45% of antibody binding was blocked at concentrations of 10-100. mu.g/ml.

However, if green tea is extracted with a suitable organic or organic-aqueous solvent or solvent mixture, such as a mixture of dimethyl sulfoxide (DMSO), ethanol and water, an almost complete blocking of autoimmune complex formation is surprisingly obtained with a small amount of said extract. Furthermore, products obtained from such extracts, for example, by drying, lyophilization, fractionation, and the like, may also be used. The effect of the extract is contrary to the prior art, since specific blocking of antibody responses has not been disclosed so far for known single green tea substances and for natural substance mixtures. The extracts of the invention do not significantly alter antibody complex formation in the serum of patients with other autoimmune diseases.

These results are considered to be extremely surprising, since no independent binding of mutated vimentin has been disclosed in the prior art to date. Furthermore, the interaction of components of green tea with antibodies is not disclosed in the prior art.

Furthermore, it is known to date for EGCG to a) have an antioxidant effect, b) capture reactive intermediates of carcinogens, c) inhibit enzymes which activate carcinogens, and d) inhibit nitrosation and cell proliferation (in particular of tumor cells).

Finally, another object of the present invention is the use of the above-mentioned peptide, the above-mentioned peptide fragment, the above-mentioned reverse-reverse polypeptide or cyclic peptide for the discovery of blocking natural substances and/or chemical substances against antibody-driven inflammatory reactions.

Furthermore, based on the peptides of the present invention, bioassays can be developed, with which natural substance libraries, combinatorial libraries and chemical libraries can be analyzed for the presence of anti-inflammatory substances. By using disease-associated antibodies, substances that block the antigen of the invention directly or indirectly by blocking the antibody can be identified and purified from the above mixture. For these substances, delayed treatment of rheumatoid arthritis and complete cure at higher doses were observed in animal experiments.

Furthermore, the invention will be further elucidated by means of the following figures and examples.

Description of the figures

FIG. 1: the diagnostic specificity of detecting RA-autoantibodies using the mutated vimentin (●) was compared to the diagnostic specificity using citrullinated peptide (CCP) (. tangle-solidup.).

FIG. 2: the isolated CD 4-positive T cells inhibited the proliferative response of CD 4-negative cells following peptide immunotherapy.

FIG. 3: inhibition of the response of extracts from green tea, mutated vimentin, and combinations thereof to antibodies from RA serum.

FIG. 4: the extract from green tea is selective in blocking autoantibodies from RA patients.

FIG. 5: summary description of animal models of arthritis.

FIGS. 6 to 8: in vivo efficacy of extracts from green tea

Examples

Example 1: identification of mutant variants of human vimentin as RA-associated antigen

Cell extracts were prepared from U937 cells (human monocyte cell line). Citrullinated proteins were removed by pre-absorption using commercially available anti-citrullinated antibodies. Affinity chromatography using chicken anti-vimentin antibody was then performed, providing an eluate from which vimentin variants could be isolated that cross-react with antibodies from patients with RA. The resulting vimentin variants were further characterized by reverse phase chromatography, protease digestion, and amino acid sequencing. As a result thereof, the polypeptides described below having the amino acid sequences SEQ ID Nos. 2 to 9 can be identified.

Example 2: detection of RA-associated autoantibodies using mutated vimentin as detection antigen

Coli (e.coli) the recombinant mutant variant of the native vimentin from example 1 (SEQ ID No.9) was expressed and then purified by affinity chromatography using His-tag. With development buffer (Entfeltungbuffer) (50mM Tris, 2M guanidine hydrochloride, 5mM CaCl₂2mM DTT, 0.5mM EDTA, 5mM methylamine chloride, pH 7.4) A solution of 2mg/ml purified protein was pretreated at 4 ℃ for at least 14 hours and then diluted 1: 1000 in PBS. Mu.l of this solution were introduced into the wells of a microtiter plate (manufacturer: COSTAR) overnight at 4 ℃. Unbound polypeptides were removed by 3 washes (175. mu.l, buffered PBS/0.05% Tween). By using 150. mu.l of 3% bovine serum albumin in PBSThe wells were incubated with (BSA) solution to block possible non-specific cross-reactions. The closed, emptied plate was dried at 37 ℃ for 30 minutes and then stored at 4 ℃ under dry conditions.

The microtiter plates thus prepared were used for the quantitative detection of autoantibodies against mutated vimentin according to the principle of indirect enzyme immunoassay. For comparison, detection of autoantibodies against CCP (citrullinated peptide) was performed. For this, serum samples from healthy subjects and/or patients with different diseases were diluted 1: 100 with 1% BSA/PBS and incubated in wells for 30 min. Unbound serum antibodies were removed by repeated washing with PBS/0.05% Tween. Then, the enzyme-labeled detection antibody (in particular, peroxidase-conjugated anti-human IgG antibody, diluted 1: 10000) was incubated for 15 minutes.

After washing away excess detection antibody, 100. mu.l of substrate solution (TMB (3, 3 ', 5, 5' -tetramethylbenzidine) tablet dissolved in 10ml of 0.05M phosphate-citrate buffer and 8ml of hydrogen peroxide added immediately before use) was added and held for 15 minutes. The reaction was stopped by adding 100. mu.l of 1M HCl and the reaction product was yellow-coloured. The intensity of the yellow stain was determined photometrically at 450nm, where the absorbance is directly proportional to the concentration of autoantibodies sought.

The following measurements were obtained here:

serum numbering	Diagnosis of	OD 450nm of mutated vimentin	CCP[U/ml]
				1	Normal serum	0.127
2	Blood donor	0.089	6.5

3	Hepatitis (HAV)	0.107	12.5
				4	Borrelia Arthritis (Borrelian-Arthritis)	0.167	17.7
5	Rheumatoid arthritis	0.784	4.6
				6	Rheumatoid arthritis	0.984	361.4
7	Rheumatoid arthritis	2.456	1156.5
				8	Rheumatoid arthritis	1.709	8.6
9	Rheumatoid arthritis	1.342	1453.1

All patient sera diagnosed as "rheumatoid arthritis" showed significantly improved absorption values compared to normal sera and sera of patients with other diseases when measured in a Tecan "SPECTRA" photometer.

Example 3: development of diagnostic agents based on mutant variants of native vimentin

To develop a mutant vimentin-based diagnostic, different mutant polypeptide variants of native vimentin were cloned (see example 1) and expressed in e. To identify polypeptide variants with the highest sensitivity for detecting autoantibodies in RA, sera from a collection of Rheumatoid Arthritis (RA) patients (approximately 100) as defined by ACR criteria were used. To this end, similarly to example 2, the expressed and purified proteins were coated in microtiter plates and the cross-reactivity of autoantibodies in RA patients was analyzed in a conventional ELISA assay.

To obtain the greatest possible sensitivity, the mutated sequences obtained in the first round of screening were combined in a further reaction mixture. For the specificity of mutant polypeptide variants found by the study as diagnostic agents for RA, sera of 34 patients with other autoimmune diseases (in particular, SLE, sjogren's syndrome, IDDM) and 53 healthy people were used. The finally obtained mutant polypeptide variants were coated in microtiter plates analogously to example 2.

By using the mutant variants of example 2, surprisingly higher specificity (> 98%) and sensitivity than known in the prior art for citrullinated peptide (CCP) can be obtained in the analysis of autoantibodies in RA patients and patients with other autoimmune diseases (fig. 1).

Example 4: determination of self epitopes in mutated vimentin (autoipitope)

Anti-vimentin antibodies were tested in an ELISA for their ability to bind overlapping, biotinylated, synthetic 17-mer peptides of mutant vimentin (see example 2). The main question here is whether the antibodies of patients with RA are directed against the same B-cell epitope.

Surprisingly, in a cohort of patients with RA and different anti-vimentin antibody titers at position 102, 91% of the sera reacted with the linear peptide sequence from the amino-terminal region of vimentin. No cross-reactivity was observed for the carboxy-terminal region or the central alpha-helical rod-like domain. Sera of healthy subjects and patients with other autoimmune diseases (in particular, sjogren's syndrome, systemic lupus erythematosus or vasculitis) showed no response to the vimentin peptide sequence.

Particularly high reactivity is found in the region of amino acids 30 to 70, in particular in the region of amino acids 50 to 65.

Example 5: prognosis of RA patients

RA develops in very different ways and the decision on the treatment regimen is not final but has to be continuously controlled and adjusted when needed. Thus, 21 patients receiving drug therapy for RA were analyzed for response to the polypeptides of the invention. In a follow-up observation analysis (at least 7 times per patient over 1 to 2 years), cross-reactions to overlapping, biotinylated, synthetic peptides of the mutant vimentin were characterized in ELISA (see example 4). It can be ascertained here that, for example in the case of RA patients who cross-react with peptides in positions 30 to 65 of the vimentin sequence, a rapid and effective treatment is observed, which leads in part to a positive result (complete cure after 2 years). In contrast, in the case of RA patients cross-reactive with the peptides in positions 55 to 70, the treatment success could not be documented in the patient profile in any of the cases studied. Healthy patients showed no cross-reaction to any of the peptides analyzed.

Based on these data, the polypeptides of the invention can be used as diagnostic agents to control the course and outcome of treatment of rheumatoid diseases, in particular rheumatoid arthritis. That is, a quantitative ELISA using the peptide of the present invention can separate patients into drug "responders" and "non-responders" at the beginning of treatment.

Example 6: therapeutic efficacy of peptides

Specific messenger substances are responsible for inflammatory processes in the joint mucosa, cartilage and bone. The best known representative is Tumor Necrosis Factor (TNF). For many years, there have been transgenic mouse models for chronic polyarthritis that largely support anti-TNF therapy from the outset. These mice overexpress human TNF and are sufficient to cause the mice to develop severe, chronic, destructive polyarthritis.

In this context, the transgenic mice are treated with saline alone or injected with a solution of 1. mu.g of a peptide (50 to 65) of mutant vimentin (similar to PugaYung et al, Epitope-specific immunological analysis of protein immunization of protein in rhematoid arthritis, PNAS 2004101: 4228;. and Zwerina et al, Single and connective tissue of tumor necrosis factor, Interleukin-1, and RANKLpathways in tumor necrosis factor-induced injury: effector of immune necrosis, 2004 bone infection, and cardiac tissue engineering R. um. Jan.; 50-65). Then, swelling of the joints was measured, and joint mucosa and damage to bone and cartilage were examined at the fine tissue level. By injecting vimentin peptide, joint swelling was suppressed by 41%, and inflammation of joint mucosa was suppressed by 38%, and physiological saline did not show any effect. However, when combining multiple peptides of mutant vimentin, the inflammation can be almost completely controlled.

Regulatory T lymphocytes are considered to be promising mediators of peripheral tolerance. Therefore, in these animals studied, it was further investigated whether the limited allogeneic immune response induces the formation of regulatory cells in an antigen-specific manner. It was found here that the mice, after such a treatment, generate immune cells which successfully inhibit the division of antigen-specific immune cells, which are involved in having CD⁴⁺CD45RC^negPhenotypic intrahepatic T cells (figure 2).

Example 7: blocking autoantibody complex formation in vitro

To block the binding of antibodies to mutated vimentin, the following were used, either alone or in combination, at a final concentration of 1 μ g/ml:

1. epicatechin (EC)

2. Epigallocatechin gallate (EGCG)

3. DMSO extract from green tea

4. Mutant vimentin 1.4mg/ml

5. Mutated vimentin 0.7mg/ml

6. Citrullinated vimentin 1.5 mg/ml.

To prepare the extract from green tea, 5g of any commercially available tea was covered with 10ml of 80% DMSO and stirred overnight. The solution was centrifuged at 13000rpm for 10 minutes and defined as tea extract. In contrast to the above solid substances 1, 2 and 4 to 6, 10. mu.l/ml of the prepared tea extract was used. The antibody binding in the presence and absence of substances is analyzed in an ELISA, for which purpose microtitre plates are prepared analogously to example 2. Different sera from RA patients were used at dilutions ranging from 1: 100 to 1: 400. Blocking of antibody binding was induced by preincubation of 100 μ l of diluted serum with the indicated substances or tea solution for 10 minutes. The remaining cross-reactions were then analyzed by transferring 100 μ l of the serum-substance mixture to the wells of a given microtiter plate. Bound antibody was detected with HRP-conjugated anti-human IgG according to conventional ELISA embodiments. In fig. 3, the results obtained using patient serum number 400725 are illustrated.

The specificity of extracts from green tea for inhibition of autoantibodies (antibodies against mutant vimentin) from RA patients is shown in figure 4.

Example 8: blocking autoantibody complex formation in vivo

The oral absorption of the catechins of green tea is very low in the case of oral ingestion, so that only minimal serum concentrations are possible when normally drinking the tea (Zhu et al, Oralabsorption and bioavailability of tea catechins. plant medical 66(2000) 444-7; see also Schrader et al, Bioverfugukiverschendener Tee-Catechin im plant invon derDarreichungsform.Proc.Germ.Nutr.Soc.3(2001)36)。

Thus, the tea extract we prepared (see example 7) was freeze-dried and reconstituted with cocoa butter or salmon oil. After oral administration of an extract prepared from 5g of green tea (5g/kg body weight) to rats, blood was collected every half hour and analyzed using HPLC and GC/MC. In rats, at about 2 hours after ingestion, maximum plasma concentrations of 75 μ g/ml EC and EGCG (5-13% of the extract mass) and about 245 μ g/ml EGCG (to 50% of the extract content) can be measured. No toxic effects occurred in these animal experiments during the study period of 1 month.

If these results are transferred to humans, it can be concluded that the dosage form is used to obtain a plasma concentration of tea components (in particular catechins) that enables the formation of autoantibody complexes to be completely blocked.

Example 9: in vitro efficacy of green tea extract

Extraperitoneal administration of green tea extract at concentrations of 10 and 100mg per kg body weight, respectively, showed significant positive effects in an animal model of arthritis (figure 5: determination of paw swelling after adjuvant administration). Fig. 6 to 8 show the results.

Sequence listing

<110>Orgentec Diagnostika GmbH

<120> method for diagnosing rheumatic diseases

<130>34478PWO

<160>9

<170>PatentIn version 3.3

<210>1

<211>466

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<213> Intelligent people

<220>

<221> peptides

<222>(1)..(466)

<223> peptide sequence of native vimentin

<400>1

Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Gly

1 5 10 15

Gly Pro Gly Thr Ala Ser Arg Pro Ser Ser Ser Arg Ser Tyr Val Thr

20 25 30

Thr Ser Thr Arg Thr Tyr Ser Leu Gly Ser Ala Leu Arg Pro Ser Thr

35 40 45

Ser Arg Ser Leu Tyr Ala Ser Ser Pro Gly Gly Val Tyr Ala Thr Arg

50 55 60

Ser Ser Ala Val Arg Leu Arg Ser Ser Val Pro Gly Val Arg Leu Leu

65 70 75 80

Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe

85 90 95

Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp

100 105 110

Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn

115 120 125

Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser

130 135 140

Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln

145 150 155 160

Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp

165 170 175

Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu

180 185 190

Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln

195 200 205

Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val

210 215 220

Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu

225 230 235 240

Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile

245 250 255

Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val

260 265 270

Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu

275 280 285

Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg

290 295 300

Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg

305 310 315 320

Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr

325 330 335

Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala

340 345 350

Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu

355 360 365

Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln

370 375 380

Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr

385 390 395 400

Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro

405 410 415

Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro

420 425 430

Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu

435 440 445

Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp

450 455 460

Leu Glu

465

<210>2

<211>466

<212>PRT

<213> Intelligent people

<220>

<221> peptides

<222>(1)..(466)

<223> peptide sequence of mutant I

<400>2

Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Arg

1 5 10 15

Arg Pro Arg Thr Ala Ser Arg Pro Ser Ser Ser Arg Ser Tyr Val Thr

20 25 30

Thr Ser Thr Arg Thr Tyr Ser Leu Arg Ser Ala Leu Arg Pro Ser Thr

35 40 45

Ser Arg Ser Leu Tyr Ala Ser Ser Pro Arg Arg Val Tyr Ala Thr Arg

50 55 60

Ser Ser Ala Val Arg Leu Arg Ser Ser Val Pro Arg Val Arg Leu Leu

65 70 75 80

Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe

85 90 95

Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp

100 105 110

Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn

115 120 125

Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Arg Gln Arg Lys Ser

130 135 140

Arg Leu Arg Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln

145 150 155 160

Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp

165 170 175

Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu

180 185 190

Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln

195 200 205

Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val

210 215 220

Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu

225 230 235 240

Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile

245 250 255

Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val

260 265 270

Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu

275 280 285

Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg

290 295 300

Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg

305 310 315 320

Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr

325 330 335

Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala

340 345 350

Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Arg Arg Leu Gln Asp Glu

355 360 365

Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln

370 375 380

Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr

385 390 395 400

Arg Lys Leu Leu Glu Arg Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro

405 410 415

Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro

420 425 430

Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu

435 440 445

Thr Arg Asp Arg Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp

450 455 460

Leu Glu

465

<210>3

<211>466

<212>PRT

<213> Intelligent people

<220>

<221> peptides

<222>(1)..(466)

<223> peptide sequence of mutant II

<400>3

Met Ser Leu Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Gly

1 5 10 15

Arg Pro Gly Leu Ala Ser Arg Pro Ser Ser Ser Arg Ser Tyr Val Thr

20 25 30

Leu Ser Leu Arg Leu Tyr Ser Leu Arg Ser Ala Leu Arg Pro Ser Thr

35 40 45

Ser Arg Ser Leu Tyr Ala Ser Ser Pro Arg Gly Asp Tyr Ala Ser Gln

50 55 60

Asn Arg Trp Val Arg Leu Arg Ser Ser Val Pro Arg Val Arg Leu Leu

65 70 75 80

Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Leu Glu Phe

85 90 95

Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp

100 105 110

Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn

115 120 125

Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Arg Lys Ser

130 135 140

Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln

145 150 155 160

Val Asp Gln Leu Leu Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp

165 170 175

Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu

180 185 190

Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln

195 200 205

Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val

210 215 220

Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu

225 230 235 240

Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile

245 250 255

Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val

260 265 270

Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu

275 280 285

Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg

290 295 300

Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg

305 310 315 320

Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr

325 330 335

Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala

340 345 350

Val Glu Ala Ala Asn Tyr Gln Asp Leu Ile Gly Arg Leu Gln Asp Glu

355 360 365

Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln

370 375 380

Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Leu Tyr

385 390 395 400

Arg Lys Leu Leu Glu Arg Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro

405 410 415

Asn Phe Ser Ser Leu Asn Leu Arg Glu Leu Asn Leu Asp Ser Leu Pro

420 425 430

Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu

435 440 445

Thr Arg Asp Arg Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp

450 455 460

Leu Glu

465

<210>4

<211>466

<212>PRT

<213> Intelligent people

<220>

<221> peptides

<222>(1)..(466)

<223> peptide sequence of mutant III

<400>4

Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Gly Met Phe Gly

1 5 10 15

Arg Pro Gly Thr Ala Ser Gly Pro Ser Ser Ser Arg Ser Tyr Val Thr

20 25 30

Thr Ser Thr Arg Thr Tyr Ser Leu Arg Ser Ala Leu Gly Pro Ser Thr

35 40 45

Ser Arg Ser Leu Tyr Ala Ser Ser Pro Gly Arg Val Tyr Ala Thr Gly

50 55 60

Ser Ser Tyr Val Arg Leu Arg Ser Ser Val Pro Arg Val Gly Leu Leu

65 70 75 80

Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe

85 90 95

Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp

100 105 110

Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn

115 120 125

Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser

130 135 140

Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln

145 150 155 160

Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp

165 170 175

Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu

180 185 190

Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln

195 200 205

Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val

210 215 220

Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu

225 230 235 240

Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile

245 250 255

Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val

260 265 270

Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu

275 280 285

Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg

290 295 300

Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg

305 310 315 320

Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr

325 330 335

Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala

340 345 350

Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Gly Leu Gln Asp Glu

355 360 365

Ile Gln Asn Met Lys Glu Glu Met Ala Gly His Leu Arg Glu Tyr Gln

370 375 380

Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr

385 390 395 400

Gly Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro

405 410 415

Asn Phe Ser Ser Leu Asn Leu Gly Glu Thr Asn Leu Asp Ser Leu Pro

420 425 430

Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu

435 440 445

Thr Arg Asp Arg Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp

450 455 460

Leu Glu

465

<210>5

<211>466

<212>PRT

<213> Intelligent people

<220>

<221> peptides

<222>(1)..(466)

<223> peptide sequence of mutant IV

<400>5

Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Gly Met Phe Gly

1 5 10 15

Arg Pro Gly Thr Ala Ser Arg Pro Ser Ser Ser Arg Ser Tyr Val Thr

20 25 30

Thr Ser Thr Arg Thr Tyr Ser Leu Pro Ser Ala Leu Gly Pro Ser Thr

35 40 45

Ser Pro Ser Leu Tyr Ala Ser Ser Pro Gly Gly Arg Tyr Ala Thr Pro

50 55 60

Ser Ser Tyr Arg Gly Leu Arg Ser Ser Val Pro Arg Val Gly Leu Leu

65 70 75 80

Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe

85 90 95

Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp

100 105 110

Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn

115 120 125

Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser

130 135 140

Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln

145 150 155 160

Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp

165 170 175

Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu

180 185 190

Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln

195 200 205

Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val

210 215 220

Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu

225 230 235 240

Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile

245 250 255

Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val

260 265 270

Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu

275 280 285

Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg

290 295 300

Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg

305 310 315 320

Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr

325 330 335

Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala

340 345 350

Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu

355 360 365

Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln

370 375 380

Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr

385 390 395 400

Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro

405 410 415

Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro

420 425 430

Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu

435 440 445

Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp

450 455 460

Leu Glu

465

<210>6

<211>466

<212>PRT

<213> Intelligent people

<220>

<221> peptides

<222>(1)..(466)

<223> peptide sequence of mutant V

<220>

<221>MISC_FEATURE

<222>(1)..(466)

<223> Xaa ═ citrulline

<400>6

Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Arg

1 5 10 15

Arg Pro Arg Thr Pro Ser Arg Pro Ser Ser Ser Arg Ser Tyr Val Thr

20 25 30

Thr Ser Thr Arg Thr Tyr Ser Leu Arg Ser Pro Leu Arg Pro Ser Thr

35 40 45

Ser Arg Ser Leu Tyr Pro Ser Ser Pro Thr Arg Arg Tyr Pro Thr Xaa

50 55 60

Ser Ser Ala Val Arg Leu Arg Ser Ser Val Pro Arg Val Arg Leu Leu

65 70 75 80

Gln Asp Ser Val Asp Phe Ser Leu Pro Asp Ala Ile Asn Thr Glu Phe

85 90 95

Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp

100 105 110

Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn

115 120 125

Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Arg Gln Arg Lys Ser

130 135 140

Arg Leu Arg Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln

145 150 155 160

Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp

165 170 175

Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu

180 185 190

Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln

195 200 205

Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val

210 215 220

Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu

225 230 235 240

Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile

245 250 255

Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val

260 265 270

Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu

275 280 285

Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg

290 295 300

Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg

305 310 315 320

Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr

325 330 335

Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala

340 345 350

Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu

355 360 365

Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln

370 375 380

Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr

385 390 395 400

Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro

405 410 415

Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro

420 425 430

Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu

435 440 445

Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp

450 455 460

Leu Glu

465

<210>7

<211>466

<212>PRT

<213> Intelligent people

<220>

<221> peptides

<222>(1)..(466)

<223> peptide sequence of mutant VI

<220>

<221>MISC_FEATURE

<222>(1)..(466)

<223> Xaa ═ citrulline

<400>7

Met Ser Thr Xaa Ser Val Ser Ser Ser Ser Tyr Xaa Arg Met Phe Gly

1 5 10 15

Gly Pro Gly Thr Ala Ser Xaa Pro Ser Ser Ser Arg Ser Tyr Val Thr

20 25 30

Thr Ser Thr Xaa Thr Tyr Ser Leu Gly Ser Ala Leu Xaa Pro Ser Thr

35 40 45

Ser Arg Ser Leu Tyr Ala Ser Ser Pro Gly Arg Val Tyr Ala Thr Xaa

50 55 60

Ser Ser Tyr Val Arg Leu Xaa Ser Ser Val Pro Gly Val Arg Leu Leu

65 70 75 80

Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe

85 90 95

Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp

100 105 110

Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn

115 120 125

Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser

130 135 140

Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln

145 150 155 160

Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp

165 170 175

Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu

180 185 190

Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln

195 200 205

Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val

210 215 220

Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu

225 230 235 240

Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile

245 250 255

Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val

260 265 270

Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu

275 280 285

Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg

290 295 300

Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Xaa

305 310 315 320

Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr

325 330 335

Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala

340 345 350

Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Xaa Leu Gln Asp Glu

355 360 365

Ile Gln Asn Met Lys Glu Glu Met Ala Xaa His Leu Arg Glu Tyr Gln

370 375 380

Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr

385 390 395 400

Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro

405 410 415

Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro

420 425 430

Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu

435 440 445

Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp

450 455 460

Leu Glu

465

<210>8

<211>466

<212>PRT

<213> Intelligent people

<220>

<221> peptides

<222>(1)..(466)

<223> peptide sequence of mutant VII

<220>

<221>MISC_FEATURE

<222>(1)..(466)

<223> Xaa ═ citrulline

<400>8

Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Gly

1 5 10 15

Gly Pro Gly Thr Ala Ser Xaa Thr Tyr Ser Ser Xaa Gly Tyr Val Thr

20 25 30

Leu Ser Thr Xaa Thr Tyr Tyr Leu Gly Tyr Ala Leu Xaa Pro Ser Thr

35 40 45

Tyr Xaa Ser Leu Tyr Ala Tyr Ser Pro Gly Gly Val Tyr Ala Thr Xaa

50 55 60

Gly Ser Tyr Arg Arg Leu Xaa Gly Ser Val Pro Gly Val Arg Leu Leu

65 70 75 80

Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe

85 90 95

Lys Asn Thr Xaa Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp

100 105 110

Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn

115 120 125

Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser

130 135 140

Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln

145 150 155 160

Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp

165 170 175

Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu

180 185 190

Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln

195 200 205

Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val

210 215 220

Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu

225 230 235 240

Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile

245 250 255

Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val

260 265 270

Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu

275 280 285

Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg

290 295 300

Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg

305 310 315 320

Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr

325 330 335

Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala

340 345 350

Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu

355 360 365

Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln

370 375 380

Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr

385 390 395 400

Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro

405 410 415

Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro

420 425 430

Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu

435 440 445

Thr Xaa Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp

450 455 460

Leu Glu

465

<210>9

<211>466

<212>PRT

<213> Intelligent people

<220>

<221> peptides

<222>(1)..(466)

<223> peptide sequence of mutant VIII

<400>9

Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Arg

1 5 10 15

Gly Thr Gly Thr Ala Ser Arg Thr Ser Ser Ser Arg Ser Tyr Val Thr

20 25 30

Thr Ser Thr Arg Thr Tyr Ser Leu Arg Ser Ala Leu Arg Thr Ser Thr

35 40 45

Ser Arg Ser Leu Tyr Tyr Ser Ser Pro Gly Arg Val Tyr Ala Thr Arg

50 55 60

Ser Ser Tyr Val Arg Leu Arg Ser Ser Val Thr Arg Val Ser Leu Leu

65 70 75 80

Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe

85 90 95

Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp

100 105 110

Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn

115 120 125

Lys Ile Leu Leu Ala Glu Leu GIu Gln Leu Lys Gly Gln Gly Lys Ser

130 135 140

Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln

145 150 155 160

Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp

165 170 175

Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu

180 185 190

Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln

195 200 205

Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val

210 215 220

Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu

225 230 235 240

Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile

245 250 255

Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val

260 265 270

Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu

275 280 285

Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg

290 295 300

Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg

305 310 315 320

Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr

325 330 335

Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala

340 345 350

Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu

355 360 365

Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln

370 375 380

Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr

385 390 395 400

Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro

405 410 415

Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro

420 425 430

Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu

435 440 445

Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp

450 455 460

Leu Glu

465

Claims (33)

1. Polypeptide derived from a native vimentin having SEQ ID No.1, wherein the polypeptide comprises at least one additional arginine residue as compared to the native sequence.

2. The polypeptide of claim 1, characterized in that said polypeptide has an additional arginine residue at least one of positions 16, 17, 19, 41, 58, 59, 60, 68, 76, 140, 142, 147, 363, 406, or 452.

3. The polypeptide of claim 2, characterized in that said polypeptide has additional arginine residues in at least two of said positions.

4. The polypeptide of claim 1, characterized in that said polypeptide has an additional leucine residue at least one of positions 3, 20, 33, 36, 37, 94, 165, 361, 399 or 426 compared to the native sequence.

5. The polypeptide of claim 4, characterized in that said polypeptide has additional leucine residues in at least two of said positions.

6. The polypeptide of claim 1, characterized in that the polypeptide has an additional proline residue at least one of positions 21, 41, 43, 50, 54, 62, 64 or 89 compared to the native sequence.

7. The polypeptide of claim 6, characterized in that the polypeptide has additional proline residues in at least two of said positions.

8. The polypeptide of claim 1, characterized in that said polypeptide has an additional threonine residue at least one of positions 24, 35 or 99 compared to the native sequence.

9. The polypeptide of claim 8, characterized in that said polypeptide has additional threonine residues in at least two of said positions.

10. The polypeptide of claim 1, characterized in that the polypeptide has an additional tyrosine residue at least one of positions 25, 39, 42, 49, 55 or 67 as compared to the native sequence.

11. The polypeptide of claim 10, characterized in that said polypeptide has additional tyrosine residues in at least two of said positions.

12. Polypeptide according to claim 1, characterized in that at least one arginine residue is present as a citrulline residue.

13. The polypeptide of claim 12, characterized in that said polypeptide has a citrulline residue in at least one of positions 4, 12, 23, 28, 36, 45, 50, 64, 71, 100, 320, 364 or 378.

14. The polypeptide of claim 13, characterized in that said polypeptide has citrulline residues in at least two of said positions.

15. A fragment consisting of at least 6 amino acids derived from native vimentin having SEQ ID No.1, wherein said fragment comprises at least one region having at least one additional arginine residue and exhibits reactivity to rheumatoid related autoantibodies.

16. A peptide derivative of the polypeptide or fragment of any one of claims 1 to 15.

17. Peptide derivative according to claim 16, characterized in that it is selected from the group consisting of retro-or/and retro-polypeptides and cyclic peptides.

18. A diagnostic agent, characterized in that it comprises a polypeptide or peptide derivative as defined in any one of claims 1 to 17.

19. Diagnostic kit for the detection of rheumatic diseases, characterized in that it comprises a diagnostic agent as defined in claim 18.

20. The diagnostic kit of claim 19, characterized in that it is used for the detection of rheumatoid arthritis.

21. The diagnostic kit of claim 19 or 20, characterized in that said vector is DNA, RNA, a pharmaceutically compatible polymer, a synthetic biopolymer or a protein.

22. Method for the in vitro detection of rheumatic diseases, in which method the concentration of autoantibodies in body fluids is determined and optionally used for the diagnosis, classification and severity assessment of said diseases, characterized in that a diagnostic agent as defined in claim 18 or a diagnostic kit as defined in any of claims 19 and 20 is used.

23. The method of claim 22, wherein said method is used to detect rheumatoid arthritis.

24. A medicament, characterized in that it comprises a polypeptide or peptide derivative as defined in any one of claims 1 to 17.

25. Use of a polypeptide or peptide derivative as defined in any one of claims 1 to 17 for the preparation of a medicament for the prevention and/or treatment of rheumatic diseases.

26. Use according to claim 25, characterized in that the rheumatic disease is rheumatoid arthritis.

27. Use of the proteins BF0786 and/or BF0713 for the preparation of a medicament for the prophylaxis and/or treatment of rheumatic diseases, in particular rheumatoid arthritis.

28. Use of the proteins BF0786 or/and BF0713 as diagnostic reagents for the detection of rheumatic diseases, in particular rheumatoid arthritis.

29. Use of a polypeptide or peptide derivative as defined in any one of claims 1 to 17 for the discovery of blocking natural and/or chemical substances against inflammatory reactions driven by said antibody or/and said antigen.

30. Use of epigallocatechin gallate (EGCG) for the prevention or/and treatment of rheumatic diseases, in particular rheumatoid arthritis.

31. Use of green tea extract for preventing or/and treating rheumatic diseases, especially rheumatoid arthritis.

32. Use according to claim 31, characterized in that an organic extract or an organic-aqueous extract, or a product obtainable from such an extract, is used.

33. Use according to claim 31 or 32, characterized in that the extract contains epigallocatechin gallate (EGCG) as active ingredient.