Ra antigen peptides

FIELD: biotechnologies.

SUBSTANCE: claimed invention relates to novel class II antigen peptides of main histocompatibility copmplex, which are bound with "МНС" class II molecule.

EFFECT: claimed invention allows expanding arsenal of technical means used in early diagnostics of rheumatoid arthritis.

2 cl, 5 dwg, 4 tbl, 3 ex

 

The present invention relates to a new natural-processed by the RA antigenic peptides, which are hypothesized to be a marker of erosive and neurosurge RA. These antigenic peptides presented HLA-DR class II molecules of the main histocompatibility complex person. In addition, the present invention relates to antigenic polypeptides associated with class II molecules of the main histocompatibility complex, as well as antibodies that react with these antigenic peptides, nucleic acids coding for these antigenic peptides, the structures of nucleic acids and cells-hosts for the expression of these antigenic peptides. Antigenic peptides declared in accordance with the present invention, as well as polypeptides from which they are obtained, can be used as markers for the diagnosis of RA and as therapeutic agents in the composition of anti-RA vaccines.

Rheumatoid arthritis (RA). Rheumatoid Arthritis), usually denoted by the term chronic arthritis is a systemic autoimmune disease, which is one of the most severe forms of inflammation of the joints (Feldmann, M. et al., Cell 85 (1996) 307-310; Dedhia, H.V. & DiBartolomeo, A., Critical care clinics 18 (2002) 841-854). In typical cases of RA causes joint pain, leads to deformation and expressed regenashipton. The disease can manifest and not with articular syndrome, especially in patients positive for antibodies, referred to as the "rheumatoid factor (RF). Rheumatoid Factor) (Mageed, R.A., in: van Venrooij, W.J. & Maini, R.N., eds., Manual of biological markers of disease, Kluwer Academic Publishers (1996) 1-18). RA is more common in were Caucasian, with a predisposition to RA is affected by genetic factors and environmental factors. These factors have a significant influence on the occurrence and progression of this autoimmune disease. Approximately 4% of the General population have an increased genetic susceptibility to RA, of which 20% (approximately 1% of the total population) due to exposure to unknown non-hereditary factors for developing RA. In addition, it was shown that RA significantly more common in women; thus, the risk of disease in women is three times higher than in men, suggesting that sex hormones may also be involved in the pathogenesis.

In the initial stages of RA progresses slowly. The most common early symptoms are sweating palms, morning stiffness in the joints of the fingers and symmetrical joint inflammatory nature. In addition, you may encounter rheumatoid nodules, which is a sign of defeat cartilage tissue is th. A simplified diagram of the pathogenesis of RA can be represented as follows: the immune system produces antibodies to healthy tissue. These antibodies attack the articular cartilage that leads to inflammation of the joint and, at later stages, to its destruction. This destruction stimulates the immune system, which begins to produce more antibodies. In addition, the formation of cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1), which further amplify the inflammatory response (Houssiau, F.A., Clin Rheumatol 14 Suppi 2 (1995) 10-13). In response to infiltration of immune cells such as macrophages and T-cells, begins swelling of the synovial membrane. These cells are actively involved in the mechanism of cell death and maintain inflammation in the joint (Fox, D.A., Arthritis Rheum 40 (1997) 598-609; Choy, E.H. & Panayi, G.S., N Engi J Med 344 (2001) 907-916). All these processes form a vicious circle, including the formation of autoantibodies, inflammation and destruction of joint.

In typical cases of RA occurs chronically, 85-90% of all patients experience a slow progression of the disease. Malignant forms of the disease, leading to complete loss of function of joints up to disability, are found in 10-15% of cases. When such forms of RA patients noted a persistent inflammation of the joints and the presence of rheumatoid nodules. what these patients suffer from painful chronic pain, and persistent inflammation leads to severe rigidity of the joints of the fingers and irreversible deformation and dislocation of joints.

Diagnostics

There is increasing evidence that a therapeutic effect on the early stages of the disease can prevent the destruction of joints (Egmose, .et al., J Rheumatol 22 (1995) 2208-2213; Van der Heide, A. et al., Ann Intern Med 124 (1996) 699-707). Since treatment with disease modifying Antirheumatic drugs (DMARDs). Disease-Modifying Antirheumatic Drugs) was considered to be justified and managed to achieve a satisfactory ratio of risk/benefit and cost/effectiveness, became mandatory immediately after the onset of the disease differential diagnosis of RA and other forms of arthritis (Kirwan, J.R. & Quilty, B., Clin Exp Rheumatol 15 (1997) 15-25). The diagnosis is established on the basis of clear criteria identified from the medical history, clinical examination and laboratory data. The American Rheumatism Association has published a list of criteria that make it possible to collect objective evidence in favor of RA (Amett, F.C., et al., Arthritis Rheum 31 (1987) 315-324). However, to date there is no single test that is specific for RA. For a diagnosis of RA uses some biological and biochemical markers, such as C-reactive protein (CRP). C-reactive Protein), speed sedan what I erythrocytes (erythrocyte sedimentation rate), antinuclear antibodies (ANA). Antinuclear Antibody) and RF. However, these markers are not specific, since they can appear in other inflammatory or autoimmune diseases. In particular, RF is an autoantibody, which is present in serum is approximately 50% of patients with RA. Since it was proved that RF can be detected in serum and in other inflammatory diseases, such as Sjogren syndrome, endocarditis or chronic hepatitis, RF ceased to be used as a diagnostic marker for RA. The above biological and biochemical markers themselves have diagnostic value and, in addition, can be used to determine the degree of disease activity and prognosis, as well as for the treatment and classification of patients with RA (Nakamura, R., J Clin Lab Anal 14 (2000) 305-313).

Recently developed a set of criteria for the diagnosis of RA, which includes clinical and biochemical indicators in the early stages of the disease to conduct differential diagnostics between self-limiting RA, persistent erosive RA and persistent neurosignal RA (Visser, H. et al., Arthritis Rheum 46 (2002) 357-365). Self-limiting form of RA is characterized by a natural remission: when the patient is not observed signs of arthritis within a certain period of time. Rozumiyete established on the basis of the presence of erosive changes on x-rays of the hand and/or foot. In particular, the use of antibodies that recognize cyclic citrullinaemia peptides can be effective, and also suggests an important role citrullinaemia antigens in the pathogenesis of erosive RA (Schellekens, G.A. et al., J Clin Invest 101 (1998) 273-281; Vincent, .et al., J Rheumatol 25 (1998) 838-846). Early diagnosis of erosive RA allows you as soon as possible to start using DMARDs, which will facilitate the timely monitoring of pathological process and improve disease outcome (Symmons, D.P.M. et al., J Rheumatol 25 (1998) 1072-1077; Anderson, J.J. et al., Arthritis Rheum 43 (2000) 22-29). Similarly, early diagnosis self-limiting form of RA and neurosurge RA can prevent unnecessary drug therapy potentially toxic drugs (Fries, J.F. et al., Arthritis Rheum 36 (1993)297-306).

Therapy

The purpose of anti-inflammatory therapy is the relief of pain syndrome in order to improve the daily activity of patients. Currently fully cure RA is impossible, but with the help of modern methods of treatment can slow the progression of the disease or even to stop its development. Due to individual differences of each single patient requires its own individually customized therapy, and, as mentioned above, it is desirable to make the diagnosis as early as possible. Therapy of RA is complex is Noah and is lifelong use of drugs, as well as physiotherapy and radiation therapy. The main therapeutic agents for the treatment of RA are DMARDs (e.g. methotrexate, solifenacin, hydroxychloroquine, Leflunomide, azathioprine, steroids, non-steroidal anti-inflammatory drugs (NSAIDs) or monoclonal antibodies against Pro-inflammatory cytokines TNF-α, IL-1β or their receptors (http://rheuma-online.de). These medicinal agents have a common mechanism of action, which is to reduce the inflammatory response by suppressing the immune system. The main disadvantages are their low specificity with respect to RA, side effects and failure to act on the root cause of the development of RA.

Autoimmunization

Autoimmunization starts when initiated specific adaptive immune response directed against its own antigens (autoantigens) and manifested at the initial stage of the formation of Autonomous T or cells. The natural result of the adaptive immune response against the foreign antigen, is the elimination of the specified antigen from the body. In the case where the adaptive immune response develops its own antigen, the specified antigen in most cases not completely eliminated from the body and maintains the immune response. As a consequence, the effector m the mechanisms of immune protection lead to chronic inflammatory processes in tissues. Mechanisms of tissue damage in autoimmune diseases is not essentially different from the mechanisms that operate when the body's immune defense and in the development of hypersensitivity reactions. Although currently it is not very clear, what is the triggering factor for autoimmunization, recently summarized the factors that likely contribute to the development of autoimmune diseases and selection of targets for autoantigens (Marrack, P. et al., Nat Med 7 (2001) 899-905).

Autoimmune diseases are controlled by the properties of certain genes of each individual factor or factors in the external environment. The genes of the host determine the susceptibility to autoimmunization on at least three levels. First, some genes affect the overall reactivity of the immune system and, therefore, can predetermine the development of the individual, certain autoimmune diseases, or even the development of several diseases of various types. Second, it altered immunoreactivity leads to the formation of certain proteins that are regulated by genes that affect the recognition of antigenic peptides to T-cells. Thirdly, other genes determine the ability of target tissues to regulate the immune attack, in particular, affecting the activity of the effector cells of the immune system, which the INIC is irout immune aggression. The last two genes determine which antigens will be targets for autoantibodies and, therefore, what bodies will be subjected to attack and be attacked.

In addition, environmental signals influence the development of autoimmunization on the same three levels, affecting the overall reactivity of the immune system, antigen-specificity and the status of the potential target tissues. Finally, there is the cross-interaction of environmental factors and genetic factors.

The major histocompatibility complex (MHC). Main Histocompatibility Complex)

Population-based studies, genotyping and modern advances in molecular biology have shown that carriage of certain genes encoding proteins of the main histocompatibility complex (MHC), is a high risk of developing RA (Stastny, P., Tissue Antigens 4 (1974) 571-579; Wordsworth, P. et al., PNAS 86 (1989) 10049-10053; Wordsworth, P.&Bell, J., Springer Semin Immunopathol 14 (1992) 59-78). In particular, carriers of certain alleles of HLA class II molecules, such as HLA-DRB1*0101, *0401, *0404 *0405, some ethnic groups increases susceptibility to RA (Reveille, J., Curr Opin Rheumatol 10 (1998) 187-200). So, more than 90% RF-positive patients detected a carrier of one of these predisposing alleles. HLA molecules of the class II represent the MNF-surface proteins that bind antigenic peptide is IDA within cells and exhibit them on the surface of antigen-presenting cells to interact with receptors on CD4+T-lymphocytes, what triggers cellular immune response (Banchereau, J.& Stemman, R., Nature 392 (1998) 245-252). RA-the Association of certain HLA class II molecules and the presence of large numbers of activated CD4+T cells, apparently, is a circuit that triggers the development of the disease, which are associated with disease of HLA-DR molecules exhibit associated with disease (e.g., synovial) autoantigens and cause the stimulation and proliferation of synovial T cells, which further supports the inflammatory process (Striebich, ..et al., J Immunol 161 (1998) 4428-4436).

HLA-DR (in short: DR) proteins of class II MHC are heterodimer consisting of monomorphic α-chains and extremely polymorphic β-chains that bind protein antigens in peptidebased the groove. The specified groove typically has four main pocket for binding side chains in the relevant provisions 1, 4, 6 and 9 peptide (Stem, L.J. et al., Nature 368 (1994) 215-221). Allelic variants of HLA class II molecules have different ability to bind antigenic peptides. This explains the fact that individuals with different HLA alleles have a different set of antigenic peptides, which leads to differences in the intensity of the immune response (Messaoudi, I. et al., Science 298 (2002) 1797-1800).

Peptides that bind MHC molecules of class II, usually longer and more heterogeneous in size (11-25 am is nakilat), than peptides that bind MHC molecules of class I (8-10 amino acids). This difference is due to the fact that peptidase the groove of the class II proteins is open and, while peptides are held in the center of the groove, the ends may extend beyond it in different ways (Jones, DURING, Curr Opin Immunol 9 (1997) 75-79). In the end, the class II molecules usually bind a set of overlapping peptides, which have a common Central sequence, called "T-cell epitope", but vary in length.

More than ten years ago it was discovered that DRβ chain encoded by RA-associated DRB1 alleles, despite their polymorphism, all have a common segment is identical or almost identical amino acids at positions 67-74, known as "cross-epitope" (Gregersen, R.K. et al., Arthritis Rheum 30 (1987) 1205-1213). Until such time as it has been established that immunity to autoantigens is a Central element in the pathogenesis of RA, there was the assumption that cross-epitope may participate in the pathological binding of the corresponding DR molecules with at least two mechanisms: first, by selecting relevant autoantigenic peptides for display, and, secondly, by selection of suitable specific self-reactive T cells during ontogeny. Three-dimensional spatial structure of the DR molecules that the same confirms, that cross-epitope is located in Central α-helix, fenceroy peptideatlas groove on one side (Stem, L.J. et al., Nature 368 (1994) 215-221). Therefore, cross-epitope is strategically located so that it can cooperate with an associated peptide and T-cell receptor.

However, one of the unresolved issues of rheumatology is the question of what arthritogenic antigens and epitopes in the human body are the trigger and trigger the development of RA. Although currently been identified autoantibodies of various specificities in serum and synovial fluid of patients is still unclear whether antibodies, which are released in the destruction of cartilage, run the pathological process, or their release is simply a consequence of inflammation (Corrigall, V.M. & Panayi G.S., Crit Rev Immunol 22 (2002) 281-293). It is also very difficult to determine the pathogenetic mechanisms by which antigen is present in the body, including the joints, but the pathological process is directed primarily or exclusively on the joints.

Autoantigens

In the study of serum or, less frequently, T-cells isolated from patients with chronic RA, were able to identify a large number of possible RA of autoantigens. One of the clearly defined systemspecific antigens, which is, presumably, refers to DR molecules is collagen type II (CII), the main protein of articular cartilage. Antibodies to CII in high concentrations found in the serum and synovial fluid of patients with RA, although it is still not clear whether these anti-CII antibodies pathogenic when RA (Banerjee, S. et al., Clin Exp Rheumatol 6 (373-380). Snowden et al. showed that peripheral T blood cells isolated from patients with RA, begin to proliferate in the presence of CII and most intensively in patients with anti-CII-antibodies. However, this response was observed only in 50% of patients (Snowden, N. et al., Rheumatology 40 (1997) 1210-1218). The mice immunization with CII-induced arthritis in mice expressing alleles DRB 1*0401 and *0101 MHC class II (Rosloniec, E.F., et al., J Exp Med 185 (1997) 1113-1122; Rosloniec, E.F. et al., J Immunol 160 (1998) 2573-2578). Immunodominant epitope in both *0401 and *0101 alleles in transgenic mice was directed against the peptides with the remnants of CII 261-273 person (Fugger, L. et al., Ew J Immunol 26 (1996) 928-933). A similar epitope CII demonstrated the ability to stimulate T-cell response in patients with RA, particularly in the early stages of the disease. T-cells of the synovial fluid had the highest reactivity (Kirn, H.Y. et al., Arthritis Rheum 42 (1999) 2085-2093).

Although there have been found other cartilage proteins that can potentially be a RA antigens, OR-binding epitopes were about the detected only in cartilage glycoprotein 39 person (HCgp39). This protein is secreted by synovial cells and articular chondrocytes and its quantity in the plasma and synovial fluid is increased during inflammation (Vos, K. et al., Ann Rheum Dis 59 (2000) 544-548). As in the case of CII, the introduction of HCgp39 induces arthritis in mice. In patients with RA was determined by the immune response of T cells from the peripheral blood for the introduction of HCgp39 (Verheijden, G.F. et al., Arthritis Rheum 40 (1997) 1115-1125). The dominant epitope that recognize T-cells in patients carriers DR4, is located between residues 263-275 and identical to the immunodominant epitope detected by the DRB 1*0401 transgenic mice after immunization with native HCgp39 (Sora, A.R., et al., Arthritis Rheum 42 (1999) 1497-1507). Although the immune response to this protein and is not specific to this disease, it correlates with disease activity in patients with RA (Baeten, D. et al., Arthritis Rheum 43 (2000) 1233-1243). Antibodies to HCgp39, however, are determined in the serum of patients with inflammatory diseases such as inflammatory bowel disease and systemic lupus erythematosus (SLE), although in lower concentrations than in RA.

When trying to identify antigen-specific T cells in patients with RA, to determine synovial CD4+T cells reacting with CII or HCgp39 in DR4+patients used soluble DR4 peptide tetramer complexes (Kotzin, B.L. et al., PNAS 97 (2000) 291-296). CII-DR4 complex specific associated with CI-reactive T-cell hybridomas, but not defined fraction of synovial CD4+cells. Almost similar results were obtained with HCgp39-DR4 complex, which allowed us to make the assumption that the main oligoclonal populations of CD4+T cells present in the joints in RA, are not specific for dominant CII and HCgp39 dominants described above.

In General, despite the presence of some essential features of the Association CII and HCgp39 with the pathogenesis of RA, evidence that they are major antigens in RA, is not enough. Direct evidence that CII or HCgp39 proteins restrictionenzyme on MHC class II antigen-presenting cells and subsequent stimulation and activation of synovial CD4+T cells, is clearly not enough. In addition, a big problem when using animal models is that their susceptibility to RA are unknown until the end, because CII-induced arthritis in immunized rats or mice in many ways different from RA.

Natural-processed peptides associated with MHC class II

An alternative approach for the identification of RA-specific autoantibodies and T cells is the use of sequence analysis of natural-processed peptide antigens that are associated with molecules of MHC class II. Using monoclonal antibodies molecule MN is class II, determining a predisposition to RA may be selected from the appropriate cells. RA-associated peptide antigens can be suirvey acids from purified HLA class II molecules. A mixture of small proteins can be separated by HPLC, and protein sequences can be determined using sequencing by Admino or mass spectrometry. Because the methods protein purification and sequencing techniques have their limitations, so far only managed to extract protein sequences from MHC molecules isolated from cultured b-cell lines or a large volume of tissue, and to analyze only some proteins are present in large numbers (Kropsher et al., J. Exp.Med. 175 (1992) 1799-1803; Chicz, R. et al., J Exp Med 178 (1993) 27-47). Creating high-resolution microcapillary columns for HPLC and more sensitive mass spectrometers has allowed more effective analysis of MHC-related proteins (Dongre, A.R. et al., Eur J Immunol 31 (2001) 1485-1494; Engelhard, VH et al., Mol Immunol 39 (2002) 127-137).

In the present invention for research antigenic peptide of a set of HLA-DR4 molecules isolated from autologous dendritic cells (DCs). Dendritic Cells), which was pre-treated with serum or synovial fluid of patients with RA, used the method of isolation of modified peptides and their secv the scan. The main advantage of this modern technique is the use of human DCs, which are involved in RA-associated processing of antigens and their presentation, not models, transgenic animals or artificial b-cell lines.

DCs in large numbers are present in the synovial fluid and tissues in RA and originate from circulating immature precursors (Thomas, R. et al., J Immunol 152 (1994) 2613-2623). They are most active antigen-presenting cells that Express a large number of MHC molecules and a variety of accessory molecules (Mellman, I. et al., Trends Cell Biol 8 (1998) 231-237). Recent research in this area has shown that ex vivo differentiated human DCs and macrophages, which are phenotypically similar to antigen-presenting cells of the synovial fluid from the affected RA joints, have the ability to generate and presentation of immunodominant epitopes of SI and HCgp39 (Tsark, B.C. et al., J Immunol 169 (2002) 6625-6633). DC has the ability to preimenovati CD4+helper T cells and effectively activate cytotoxic CD8+T cells (Ridge, T. et al., Nature 393 (1998) 474-478). Therefore, the peptides bind to the MHC molecules of class II and prezentirane DCs play a major role in the pathogenesis of diseases in which the immune response involving T-cells.

In addition, the problem associated with the absence of what is it sufficient information, regarding RA antigenic peptides associated with MHC class II, can be solved by getting a new natural-processed by the RA antigenic peptides associated with MHC class II, and polypeptides, of which you can get these antigenic peptides, and their use as markers of RA.

The present invention relates to a new natural-processed antigenic peptides, which are hypothesized to be a marker of erosive and neurosurge RA. These antigenic peptides presented HLA-DR molecules MHC class II derived from dendritic cells that process the serum or synovial fluid of patients with a diagnosis of erosive or neurosignal RA. Antigenic peptides to MHC class II declared in accordance with the present invention include: (a) at least the amino acid sequence peptidase site selected from the group comprising SEQ ID NOs. 49-57 and SEQ ID NOs. 103-122, or (b) at least amino acid sequence peptidase site selected from the group comprising SEQ ID NOs. 49-57 and SEQ ID NOs. 103-122, with additional N-and C-terminal flanking sequences of the respective sequence selected from the group comprising SEQ ID NOs. 1-39 or SEQ ID NOs. 58-102; and the stated sequence derived from induced is-interferon lysosomal tearducts, integrin beta-2, phosphatidylinositol-4,5-phosphate-3-kinase; activator of plasminogen urokinase type section V-III heavy chain of immunoglobulin (Unb), protein DJ-1, apolipoprotein b-100, the 26S proteasome is not ATP-aznoe regulatory subunit 8, receptor interleukin 1, fibromyaliga, GM-CSF/IL-3/IL-5 receptor sorting of Nexia 3, the heavy chain H4 inhibitor, inter-α-trypsin, complement C4, complement C3 (α-chain), complement C3 (β-chain), protein 3 similar to the enriched glutamic acid-binding SH3-domain protein induced by interleukin-4 protein 1, hemopexin, Hsc70-interactive protein, constant chain (Ii)responsible for protein 2 receptor, retinoic acid, fibronectin, cathepsin b, tripeptidyl-peptidases II, legumain, receptor for platelet activating factor, poly-alpha-2,8-sialyltransferase and ras-related peptide Rab-11B. The present invention also relates to these antigenic peptides and proteins, from which they receive, and their use as markers of erosive and neurosurge RA. In addition, the present invention relates to specific antigenic peptides associated with MHC molecules of class II antibodies reacting with these antigenic peptides, nucleic acids coding for these antigenic peptides, and the structures of nucleic acids, cell host and SP is the event expression of these antigenic peptides. The present invention also relates to methods of separating and identifying RA antigenic peptides.

Figure 1: Schematic illustration of the analysis of tissue samples, mediated by dendritic cells (DC-mediated analysis). Dendritic cells (DCs), the most specialized antigen-presenting cells (APCs). Antigen-Presenting Cells), treated with medium containing the antigen (e.g., synovial fluid), under optimum conditions for the capture and processing of antigen. As a control using DCs, cultured under the same conditions but in the absence of antigens in synovial fluid. After maturation, DCs loaded with antigen molecules MHC class II clear and relevant antigenic peptides associated with MHC class II, distinguish and identify.

Figa: Chromatogram of the main peak (ION-TRAP MS) antigenic peptides associated with MHC class II, which were isolated from dendritic cells, pre-treated serum of the patient of RA. Peptides elute directly with RP-C18-HPLC column into the mass spectrometer ion trap for immediate MS/MS identification. The numbers correspond to the retention times (highest value) and molecular weight (lowest value) the most obvious protein peaks in the mixture at the appropriate time.

Figure 2 B: ION-TRAP MS spectrum of antigenic peptides during retention the project, average of 65.4 minutes Marked the peak of the advanced segments and coordinate with double ionic form of the peptide of the inhibitor interalpha-trypsin ITIH4 (see Table 3).

Figw: ION TRAP MS/MS spectrum of double-peptide ion at m/z constituting 977.1. Fragmented mass together with the mass of the parent ion analyze relatively non-selective database of the person using the SEQUEST algorithm. The found sequence MPKNVVFVIDKSGSMSGR (single-letter code) corresponds to the dominant epitope of ITIH4 (271-288) inhibitor, inter-alpha-trypsin. Of the given series of N-terminal and C-terminal Y-ions are marked.

Figure 3: Summary data for differential binding activity of the tested probable RA antigens on linking with the allele HLA-DRB1*0401. Prospective HLA-DRB1* 0401-binding site placed in the grey border. As a measure of affinity, determine the concentration of peptide required to reduce the binding of a specified quantity of biotinylated (307-319) peptide at 50% (so) when a competitor binding. Reciprocal value (1/so) correlates with the affinity of the peptide. As reporter molecules in the study used TO (307-319) peptide, the hemagglutinin of influenza virus (Rothbard, J.B. et al., Cell 52 (1988) 515-523).

Antigenic peptides declared in accordance with the present invention, are particularly the peptides, associates and presented by MHC molecules, and therefore they have the ability to activate or increase the sensitivity of T cells. Antigenic peptides presented by MHC molecules of class II, therefore, are antigenic peptides associated with MHC molecules of class II, or antigenic peptides to MHC class II; however, antigenic peptides presented by MHC molecules of class I are antigenic peptides associated with MHC molecules of class I, or antigenic peptides to MHC class I.

Peptides derived from proteins encoded in the genome of the organism or ARS, is designated as "automated". It is assumed that the main function of automatical submitted to the DCs in the peripheral organs of the lymphatic system, is to ensure tolerance of T cells to Automatica. Tolerance implies the failure of the immune response to the antigen; in that case, when the antigen is formed in the tissues of the body, the tolerance is known as autotolerance.

Antigens derived from his own body, called the "autoantigens". Adaptive immune response directed against autoantigens, called an autoimmune response. Similarly, adaptive immunity is specific for autoantigens, called autoimmunity. The term autoreactive the awn combines immune responses, directed against autoantigens. RA, presumably due to an autoimmune response, which involve self-reactive T-cells and/or self-reactive antibodies. To immunogenum peptides include, without limitation specified) antigenic peptides capable of inducing or stimulating the cellular or humoral immune response. These peptides can also react with antibodies.

Peptides derived from proteins encoded in the genome of bacteria, viruses, or other foreign pathogens, and different from automatico, called "alien antigens" or "alien" peptides. They have the ability to induce T-cell response directed against foreign proteins from which they were received.

RA antigenic peptides are automated that fulfill the role of autoantigens and, as a consequence of the disease, mistakenly run autoreactivity to their own body tissues.

The present invention relates to antigenic peptides MHC class II, including: a) at least the amino acid sequence peptidase site selected from the group comprising SEQ ID NOs. 49-57 or SEQ ID NOs. 103-122, or b) at least amino acid sequence peptidase site selected from the group comprising SEQ ID NOs. 49-57 or SEQ ID NOs. 103-122, with additional N - The C-terminal flanking sequences of the respective sequence, selected from the group comprising SEQ ID NOs. 1-39 or SEQ ID NOs. 58-102. Preferably antigenic peptides to MHC class II have a length of less than 26 amino acids, more preferably, their length ranges from 11 to 25 amino acids. Even more preferably, the length of the antigenic peptides, notified in accordance with the present invention, ranged from 11 to 19 amino acids. Most preferably, the composition of the antigenic peptide, notified in accordance with the present invention, included peptidase area, including four anchor amino acids.

The present invention relates to antigenic peptides MHC class II, including: a) at least the amino acid sequence peptidase plot SEQ ID NO. 49 or b) at least amino acid sequence peptidase plot SEQ ID NO. 49 with an additional C - and N-terminal flanking sequences of the respective sequence selected from the group comprising SEQ ID NOs. 1-3.

In addition, the present invention relates to antigenic peptides MHC class II, including: a) at least the amino acid sequence peptidase plot SEQ ID NO. 103 or b) at least amino acid sequence peptidase plot SEQ ID NO. 103 with an additional N - and C-terminal flanking the series is due to corresponding sequences of SEQ ID NOs. 58 and 59.

In addition, the present invention relates to antigenic peptides MHC class II, including: a) at least the amino acid sequence peptidase plot SEQ ID NO. 104 or b) at least amino acid sequence peptidase plot SEQ ID NO. 104 with additional N - and C-terminal flanking sequences corresponding to sequences SEQ ID NOs. 60.

In addition, the present invention relates to antigenic peptides MHC class II, including: a) at least the amino acid sequence peptidase plot SEQ ID NO. 105 or b) at least amino acid sequence peptidase plot SEQ ID NO. 105 with additional N - and C-terminal flanking sequences corresponding to sequences SEQ ID NOs. 61.

In addition, the present invention relates to antigenic peptides MHC class II, including: a) at least the amino acid sequence peptidase plot SEQ ID NO. 106 or b) at least amino acid sequence peptidase plot SEQ ID NO. 106 with additional N - and C-terminal flanking sequences corresponding to sequences SEQ ID NOs. 62.

In addition, the present invention relates to antigenic peptides MHC class II, including: a) graynamore amino acid sequence peptidase plot SEQ ID NO. 107 or (b) at least amino acid sequence peptidase plot SEQ ID NO. 107 with an additional N - and C-terminal flanking sequences corresponding to sequences SEQ ID NOs. 63.

In addition, the present invention relates to antigenic peptides MHC class II, including: a) at least the amino acid sequence peptidase plot SEQ ID NO. 50 or b) at least amino acid sequence peptidase plot SEQ ID NO. 50 with an additional N - and C-terminal flanking sequences corresponding to sequences SEQ ID NOs. 5.

In addition, the present invention relates to antigenic peptides MHC class II, including: a) at least the amino acid sequence peptidase plot SEQ ID NO. 108 or b) at least amino acid sequence peptidase plot SEQ ID NO. 108 with additional N - and C-terminal flanking sequences corresponding to sequences SEQ ID NOs. 64-67.

In addition, the present invention relates to antigenic peptides MHC class II, including: a) at least the amino acid sequence peptidase plot SEQ ID NO. 109 or b) at least amino acid sequence peptidase plot SEQ ID NO. 109 additional the N - and C-terminal flanking sequences corresponding to sequences SEQ ID NOs. 68.

In addition, the present invention relates to antigenic peptides MHC class II, including: a) at least the amino acid sequence peptidase plot SEQ ID NO. 110 or b) at least amino acid sequence peptidase plot SEQ ID NO. 110 with additional N - and C-terminal flanking sequences corresponding to sequences SEQ ID NOs. 69 and 70.

In addition, the present invention relates to antigenic peptides MHC class II, including: a) at least the amino acid sequence peptidase plot SEQ ID NO. 111 or b) at least amino acid sequence peptidase plot SEQ ID NO. 111 with additional N - and C-terminal flanking sequences corresponding to sequences SEQ ID NOs. 72.

In addition, the present invention relates to antigenic peptides MHC class II, including: a) at least the amino acid sequence peptidase plot SEQ ID NO. 112 or b) at least amino acid sequence peptidase plot SEQ ID NO. 112 with additional N - and C-terminal flanking sequences corresponding to sequences SEQ ID NOs. 73.

Antigenic peptides associated with MHC class II declared in accordance with the present invention is obtained from the induced γ-interferon lysosomal tearducts (SEQ ID NOs. 1-3), integrin beta-2 (SEQ ID NOs. 58-59), phosphatidylinositol-4,5-phosphate-3-kinase (SEQ ID NO:60); plasminogen activator urokinase type (SEQ ID NO:61), section V-III heavy chain immunoglobulin VH26) (SEQ ID NO:62), protein DJ-1 (SEQ ID NO:63), apolipoprotein b-100 (SEQ ID NOs. 4 and 5), 26S proteasome is not ATP-aznoe regulatory subunit 8 (SEQ ID NOs. 64-67), receptor interleukin 1 (SEQ ID NO:68), fibromyalia (SEQ ID NOs.: 69 and 70), GM-CSF/IL-3/IL-5 receptor (SEQ ID NOs. 71 and 72), sorting of nexin 3 (SEQ ID NO: 73), the heavy chain H4 inhibitor, inter-α-trypsin (SEQ ID NOs.: 6-12), complement C4 (SEQ ID NOs.: 13-18), complement C3 (α-chain) (SEQ ID NOs.: 19-23, 74 and 75), complement C3 (β-chain) (SEQ ID NOs. 76 and 77), protein 3 similar to the enriched glutamic acid-binding SH3-domain protein (SEQ ID NOs.: 24-27)induced by interleukin-4 protein 1 (SEQ ID NOs.: 28-30), hemopexin (SEQ ID NOs. 31-35 and 78), Hsc70-interactive protein (SEQ ID NOs. 36-39), constant chain (Ii) (SEQ ID NOs. 79-83)responsible for protein 2 receptor, retinoic acid (SEQ ID NOs. 84-86), fibronectin (SEQ ID NOs. 87-91), cathepsin B (SEQ ID NO: 92), tripeptidyl-peptidases II (SEQ ID NOs. 93 and 94), legumain (SEQ ID NO.: 95), receptor of platelet activating factor (SEQ ID NO: 96), poly-alpha-2,8-seatransport (SEQ ID NO:97) and ras-related peptide Rab-11B (SEQ ID NOs. 98 and 102).

Single peptidase groove of the MHC molecules of class II has a length of approximately 25, but unlike molecules MHC class I on both sides of the groove are the open (Stem LJ et al., Nature 1994; 368, 215-221). Thus, natural and processed antigenic peptides isolated from MHC molecules of class II human, have a minimum length, which is about 11 residues, and can reach a maximum length of 25 residues (Chicz RM, et al., J Exp Med 1993; 178, 27-47).

The stability of the MHC-peptide interaction provides more than a dozen hydrogen bonds involving backbone of the peptide, and komplementarnosti between specific pockets of the binding groove and appropriately spaced side chains of the amino acids of the peptide. Amino acid residues of the peptide corresponding to certain pockets, designated as the "anchor" of amino acids. With regard to most HLA-DR alleles specified anchor amino acids are localized in the respective positions P1, P4, P6 and P9. The combination of the amino acids in these 4 anchor positions provides highly stable binding with the corresponding HLA-DR allelic product and differs from allele to alalu. Peptidase area, as defined in the present description, is a sequence of nine amino acids, which includes four anchor amino acids. Peptidase plot antigenic peptide MHC class II declared in accordance with the present invention, represented by SEQ ID NO. 49 for peptides derived from induced by the interferon-γ lysosomal tearducts (SEQ ID NOs. l-3); SEQ ID NO. 103 for peptides derived from integrin beta-2 (SEQ ID NOs. 58 and 59); SEQ ID NO. 104 for peptides derived from phosphatidylinositol-4,5-phosphate-3-kinase (SEQ ID NO:60); SEQ ID NO. 105 for peptides derived from plasminogen activator urokinase type (SEQ ID NO: 61); SEQ ID NO. 106 for peptides derived from V-III plot heavy chain of immunoglobulin (VH26) (SEQ ID NO: 62); SEQ ID NO. 107 for peptides derived from the DJ-1 protein (SEQ ID NO:63); SEQ ID NO. 50 for peptides derived from apolipoprotein b-100 (SEQ ID NOs. 4 and 5); SEQ ID NO. 108 for peptides derived from the 26S proteasome is not ATP-aznoe regulatory subunit 8 (SEQ ID NOs. 64 and 67); SEQ ID NO. 109 for peptides derived from the receptor interleukin-1 (SEQ ID NO: 68); SEQ ID NO. 110 for peptides derived from the fibroids oculina (SEQ ID NOs.: 69 and 70); SEQ ID NO. 111 for peptides derived from GM-CSF/IL-3/IL-5 receptor (SEQ ID NOs. 71 and 72); SEQ ID NO. 112 for peptides derived from the sorting of nexin 3 (SEQ ID NO: 73); SEQ ID NO. 51 for peptides derived from H4 heavy chain inhibitor, inter-α-trypsin (SEQ ID NOs. 6-12); SEQ ID NO. 52 for peptides derived from complement C4 (SEQ ID NOs. 13-18); SEQ ID NO. 53 for peptides derived from complement C3 (α-chain) (SEQ ID NOs. 19-23, 74 and 75); SEQ ID NO.113 for peptides derived from complement C3 (β-chain) (SEQ ID NOs. 76 and 77); SEQ ID NO. 54 for peptides derived from protein 3 similar to the enriched glutamic acid-binding SH3-domain protein (SEQ ID NOs.: 24-27); SEQ ID NO. 55 for peptides derived from in ciruelos interleukin-4 protein 1 (SEQ ID NOs. 28-30); SEQ ID NO. 56 for peptides derived from hemopexin (SEQ ID NOs. 31-35 and 78); SEQ ID NO. 57 for peptides derived from Hsc70-interactive protein (SEQ ID NOs. 36-39); SEQ ID NO. 114 for peptides derived from the constant chain (Ii) (SEQ ID NOs. 79-83); SEQ ID NO. 115 for peptides derived from answering protein 2 receptor, retinoic acid (SEQ ID NOs. 84-86); SEQ ID NO. 116 for peptides derived from fibronectin (SEQ ID NOs. 87-91); SEQ ID NO. 117 for peptides derived from cathepsin B (SEQ ID NO: 92); SEQ ID NO. 118 for peptides obtained tripeptidyl-peptidases II (SEQ ID NOs. 93 and 94); SEQ ID NO. 119 for peptides derived from legumain (SEQ ID NO: 95); SEQ ID NO. 120 for peptides derived from the receptor of platelet activating factor (SEQ ID NO: 96); SEQ ID NO. 121 for peptides derived from poly-alpha-2,8-sialyltransferase (SEQ ID NO:97, and SEQ ID NO. 122 for peptides derived from ras-related peptide Rab-11B (SEQ ID NOs. 98 and 102).

Peptidase plot may also have at least one, at least two, at least three, at least four or at least five modifications of the amino acid sequence and still retain binding capacity of unmodified peptidebased plot. Preferably the modified peptidase area contains at least three of the four anchor amino acids unmodified peptidebased plot. Amino acid option is Katsia can be a conservative amino acid substitution, as is described below.

The additional energy of binding is provided by hydrogen bonds, in education involving residues located anterior to the P1 anchor amino acids and posterior to the P9 anchor amino acids. Accordingly, in most natural-processed peptides nonameric Central plot (P1-P9) on the C - and N-ends are flanked 3-4 residues. Therefore, most of the peptides are composed of residues 15-17. Longer peptides are outside of the groove, thus providing access to ectopeptidases that otscheplaut both ends.

So, antigenic peptides to MHC class II declared in accordance with the present invention include (a) at least the amino acid sequence peptidase site selected from the group comprising SEQ ID NOs. 49-57 and SEQ ID NOs. 103-122, or b) at least amino acid sequence peptidase site selected from the group comprising SEQ ID NOs. 49-57 and SEQ ID NOs. 103-122, with additional N - and C-terminal flanking sequences of the respective sequence selected from the group comprising SEQ ID NOs. 1-39 and SEQ ID NOs. 58-102, preferably with N - and C-terminal flanking amino acid sequences that provide additional energy of binding.

Preferably antigenic peptides to MHC class II, yavlenie in accordance with the present invention, have binding capacity, corresponding to that of the molecules of MHC class II and are between one-tenth and ten times the IC50the corresponding peptide selected from the group comprising: SEQ ID NOs. 1-39 and SEQ ID NOs. 58-102. The binding ability of the peptide is measured by determining the concentration required to reduce binding of the labeled reporter peptide by 50%. This value is called the IC50. Antigenic peptides to MHC class II declared in accordance with the present invention maintain their binding ability with respect to related molecules MHC class II up until the values of the IC50there are between one-tenth and ten times corresponding IC50 of a given peptide.

Because peptide trimming every time there is an individual way both before and after bonding with peptidases groove, the presence of several truncated variants with common nonamanis crustal section is a common feature of peptides associated with MHC class II. It was shown that the C - or N-terminal truncated variants of the epitope can induce divergent T-cell responses (Arnold et al., (2002) J. Immunol. 169, 739-749).

You can specify several parameters that can affect the relative redundancy of truncated variants of a certain epitope, e.g. the excess or integrity related antigen, antigen-associated proteins, excess proteases, the types of available proteases or availability of competing antigens and/or peptides. Since the availability of the antigen is the main parameter that can be correlated with the origin of the sample, the ratio of certain truncated variants of the epitope may be of diagnostic value.

Peptide declared in accordance with the present invention is a peptide or non-natural-processed equivalent (such as mutated peptide antigen), or has been allocated, has been separated or purified from components which surround it in natural conditions, for example in tissues, such as pancreatic tissue, liver, spleen, ovary, testis, muscle, joint tissue, neural tissue, gastrointestinal tissue, or in the natural body fluids, such as blood, serum, synovial fluid or urine. Typically, the peptide is considered to be "isolated"when the drug which includes the peptide declared in accordance with the present invention, contains at least 70% of the dry matter of the indicated peptide and less than 30% of the proteins and naturally-existing organic molecules with which it is associated in vivo. Preferably the preparation of the peptide, as claimed in the present invention contains at least 80%, more preferably at least 90%, even more preferably at least 99% of the dry matter of the indicated peptide. Because the peptide, obtained by chemical synthesis, by their nature, purified from components which surround it in natural conditions, this synthetic peptide is considered "selected".

The present invention also relates to analogs of antigenic peptides declared in accordance with the present invention. The term "analog" includes those peptides that possess a functional activity of these antigenic peptides, including binding capacity IC50and the ability to be recognized by antibodies and immune system cells. Analogues have almost the same value IC50as the corresponding peptides. The term "analog" also includes conservative substitutions or chemical derivatives of the peptides.

The term "analog" refers to any polypeptide having an amino acid sequence that is almost identical to the sequence presented here, in which one or more amino acid residue has been conservatively replaced by a residue functionally identical to the previous one; however, the specified polypeptide has the same functional activity as the peptides described herein. Examples of conservative substitutions are the Amena one nonpolar (hydrophobic) amino acids, such as phenylalanine, tyrosine, isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) amino acid to another, such as arginine for lysine, glutamine for asparagine, threonine to serine and Vice versa; replacement of one basic amino acids such as lysine, arginine or histidine for another, or the substitution of one acidic amino acids such as aspartic acid or glutamic acid for another.

The term "conservative substitution" also refers to the use of chemical derived amino acids instead of the usual amino acids. The term "chemical derivative" refers to a polypeptide having one or more amino acid modified with a chemical reaction of a functional side group. Examples of such modified molecules are, for example, molecules in which free amino groups have been modified with getting aminohydroxylation, p-toluene-sulfanilic groups, carbobenzoxy groups, t-butoxycarbonyl groups, Chloroacetic groups, acetyl groups or formyl groups. Free carboxyl groups can be modified to obtain salts, methyl or ethyl esters or other esters or hydrazides. The free hydroxyl group can be modified to obtain the O-acyl or O-alkyl derivatives. The imidazole nitrogen of the amino acid histidine mo is et to be modified to obtain N-im-benzylglycine. Also to chemical derivatives include such proteins or peptides, which include one or more derivative of any of the twenty existing in the nature of amino acids. For example: 4-hydroxyproline may be substituted for Proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine, ornithine or citrulline may be substituted for lysine.

Antigenic peptides to MHC class II declared in accordance with the present invention, and the proteins from which they are obtained, can be used as markers of RA and as therapeutic agents in the composition of anti-RA vaccines. The term "marker"as used here, refers to biomolecule, preferably a protein or polypeptide that is expressed in a group of patients with established disease, such as RA, and reaches its excess, which is significantly increased or decreased compared with the control group.

The token is declared in accordance with the present invention, can be used as a prognostic marker to predict susceptibility to disease, for example to predict the susceptibility to RA; as a diagnostic marker, allowing to determine the presence of disease, for example for the diagnosis of RA; as a differential diagnostic marker, on the basis of which vtorogo it is possible to carry out differential diagnostics of various forms of the disease, for example, various forms of RA; as a prognostic marker to predict disease outcome, for example to predict RA, and as a response marker to determine the effectiveness of treatment, for example, as a response marker in the treatment of RA.

According to another variant implementation of the present invention antigenic peptides to MHC class II include: (a) at least the amino acid sequence peptidase site selected from the group comprising SEQ ID NOs. 49-57 and SEQ ID NOs. 103-122, or b) at least amino acid sequence peptidase site selected from the group comprising SEQ ID NOs. 49-57 and SEQ ID NOs. 103-122, with additional N - and C-terminal flanking sequences of the respective sequence selected from the group comprising SEQ ID NOs. 1-39 and SEQ ID NOs. 58-102, and are used as markers of erosive and/or neurosignal RA.

According to another variant implementation of the present invention antigenic peptides to MHC class II include: (a) at least the amino acid sequence peptidase plot SEQ ID NO. 49 or b) at least amino acid sequence peptidase plot SEQ ID NOs. 49. with additional N - and C-terminal flanking sequences of the respective sequence selected from the group, Lucaya SEQ ID NOs. 1-3, and are used as markers of erosive and/or neurosignal RA.

In addition, the present invention relates to antigenic peptides MHC class II, which comprise: a) at least the amino acid sequence peptidase plot SEQ ID NO. 103 or b) at least amino acid sequence peptidase plot SEQ ID NO. 103 with an additional N - and C-terminal flanking sequences corresponding to sequences SEQ ID NOs. 58 and 59 and are used as markers of erosive and/or neurosignal RA.

In addition, the present invention relates to antigenic peptides MHC class II, which comprise: a) at least the amino acid sequence peptidase plot SEQ ID NO. 104 or b) at least amino acid sequence peptidase plot SEQ ID NO. 104 with additional N - and C-terminal flanking sequences corresponding to the sequence SEQ ID NO. 60 and are used as markers of erosive and/or neurosignal RA.

In addition, the present invention relates to antigenic peptides MHC class II, which comprise: a) at least the amino acid sequence peptidase plot SEQ ID NO. 105 or b) at least amino acid sequence peptidase plot SEQ ID NO. 105 D. the additional N - and C-terminal flanking sequences corresponding to the sequence SEQ ID NO. 61 and are used as markers of erosive and/or neurosignal RA.

In addition, the present invention relates to antigenic peptides MHC class II, which comprise: a) at least the amino acid sequence peptidase plot SEQ ID NO. 106 or b) at least amino acid sequence peptidase plot SEQ ID NO. 106 with additional N - and C-terminal flanking sequences corresponding to the sequence SEQ ID NO. 62 and are used as markers of erosive and/or neurosignal RA.

In addition, the present invention relates to antigenic peptides MHC class II, which comprise: a) at least the amino acid sequence peptidase plot SEQ ID NO. 107 or (b) at least amino acid sequence peptidase plot SEQ ID NO. 107 with an additional N - and C-terminal flanking sequences corresponding to the sequence SEQ ID NO. 63 and are used as markers of erosive and/or neurosignal RA.

In addition, the present invention relates to antigenic peptides MHC class II, which comprise: a) at least the amino acid sequence peptidase plot SEQ ID NO. 50 or b) at least amino acid sequence peptidase plot SEQ ID NO. 50 will complement lname N - and C-terminal flanking sequences corresponding to the sequence SEQ ID NO. 5 and are used as markers of erosive and/or neurosignal RA.

In addition, the present invention relates to antigenic peptides MHC class II, which comprise: a) at least the amino acid sequence peptidase plot SEQ ID NO. 108 or b) at least amino acid sequence peptidase plot SEQ ID NO. 108 with additional N - and C-terminal flanking sequences corresponding to sequences SEQ ID NOs. 64-67 and are used as markers of erosive and/or neurosignal RA.

In addition, the present invention relates to antigenic peptides MHC class II, which comprise: a) at least the amino acid sequence peptidase plot SEQ ID NO. 109 or b) at least amino acid sequence peptidase plot SEQ ID NO. 109 with additional N - and C-terminal flanking sequences corresponding to the sequence SEQ ID NO. 68 and are used as markers of erosive and/or neurosignal RA.

In addition, the present invention relates to antigenic peptides MHC class II, which comprise: a) at least the amino acid sequence peptidase plot SEQ ID NO. 110 or b) at least amino acid sequence peptidase plot SEQ ID NO. 110 updat the additional N - and C-terminal flanking sequences corresponding to sequences SEQ ID NOs. 69 and 70 and are used as markers of erosive and/or neurosignal RA.

In addition, the present invention relates to antigenic peptides MHC class II, which comprise: a) at least the amino acid sequence peptidase plot SEQ ID NO. 111 or b) at least amino acid sequence peptidase plot SEQ ED NO. 111 c additional N - and C-terminal flanking sequences corresponding to sequences SEQ ID NOs. 72 and 59 and are used as markers of erosive and/or neurosignal RA.

In addition, the present invention relates to antigenic peptides MHC class II, which comprise: a) at least the amino acid sequence peptidase plot SEQ ID NO. 112 or b) at least amino acid sequence peptidase plot SEQ ID NO. 112 with additional N - and C-terminal flanking sequences corresponding to the sequence SEQ ID NO. 73 and are used as markers of erosive and/or neurosignal RA.

The present invention, as described above, also relates to antigenic peptides MHC class II associated with molecules of MHC class II.

Multimer (for example, dimers, trimers, tetramer, pentamers, hexamers or oligomers) molecules MHC class II containing covalently or ecovalence include the major peptides, declared in accordance with the present invention, when conjugated with a detectable label (e.g. a fluorescent molecule, a radioactive label or an enzyme that catalyzes a reaction resulting in the formation of the product that absorbs or emits light with a certain wavelength) can be used to identify the subject (e.g., in humans) the number of T-cells, bearing on its surface receptors specific and, therefore, connects the above-mentioned complexes. The relatively high content of such T cells may be a diagnostic sign of the disease or to indicate that T cells are involved in building immunity to the disease. In addition, continuous monitoring of the relative amount of multimer-binding T cells may be useful for monitoring the disease course or treatment efficiency. Such methods of analysis were developed using tetramers of MHC molecules of class I, including peptides derived from the virus HIV-1 or influenza-15 (Altaian et al., (1996), Science 274:94-96; Ogg et al. (1998) Science 279:2103-21061), so it is expected that the corresponding polymers MHC class II will also be useful. These complexes can be obtained by chemical cross-linking of purified molecules, MHC class II, about edinennyh in the presence of the desired peptide, or by modifying the already known recombinant methods used to obtain molecules MHC class II containing one specific peptide (Kazono et al. (1994) Nature 369:151-154; Gauthier et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95:11828-118331). The monomer molecules MHC class II these polymers can be a native molecules formed from full-sized alpha - and beta-chains. Alternatively, they may represent a molecule composed of the extracellular domains of alpha - and beta-chains or domains of alpha - and beta-chains, which form the "walls" and "bottom" peptideatlas the trough.

The present invention also relates to an antibody, fragments or derivatives, which are directed against the above antigenic peptides to MHC class II or react with them. General method for producing antibodies are well known and described, for example, Kohler and Milstein, 1975, Nature 256, 494 or J.G.R. Hurrel, Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press Inc., Boco Raron, FL (1982). Antibodies may be polyclonal or preferably monoclonal or represent only fragments of antibodies, such as F(ab')2, Fab, Fv or scFv. Antibodies declared in accordance with the present invention, can also be humanitarianism (Merluzzi S. et al., (2000) Adv. Clin. Path, 4(2):77-85), or a human antibodies (Aujame L. et al., Hum. Antibodies, (1997), 8(4):155-168).

This is completed with the invention also relates to a nucleic acid molecule, encoding antigenic peptides to MHC class II declared in accordance with the present invention comprising: a) at least the amino acid sequence peptidase site selected from the group comprising SEQ ID NOs. 49-57 and SEQ ID NOs. 103-122, or b) at least amino acid sequence peptidase site selected from the group comprising SEQ ID NOs. 49-57 and SEQ ID NOs. 103-122, with additional N - and C-terminal flanking sequences of the respective sequence selected from the group comprising SEQ ID NOs. 1-39 and SEQ ID NOs. 58-102. Preferably the nucleic acid molecule is a DNA molecule.

In addition, the present invention relates to a nucleic acid molecule that encodes antigenic peptides to MHC class II declared in accordance with the present invention and related molecules MHC class II.

The present invention also relates to recombinant constructs a nucleic acid comprising the nucleic acid molecule as described above is functionally associated with the expression vector. The expression vector, suitable for use in accordance with the present invention, contains at least one controlling the expression element is functionally linked with a nucleotide sequence encoding an antigenic peptide or anti the config peptide, associated with the molecule MHC class II. Recombinant design expression can be a DNA structure.

The elements controlling the expression is inserted into a vector for the control and regulation of expression of the nucleotide sequence that encodes an antigenic peptide, notified in accordance with the present invention. Examples of the elements controlling the expression, are, without limitation specified, the lac system, the operator and the promoter of phage lambda, yeast promoters and promoters derived from virus polyoma, adenovirus, retrovirus, or SV40. Additional preferred or required operational elements include, without limitation specified, a leader sequence, termination codons, polyadenylation signals, and any other sequences necessary or preferred for adequate transcription and subsequent translation of the nucleotide sequence in the system of the host. The person skilled in the art it is obvious that the right combination is required or preferred elements controlling the expression will depend on the selected system of the host. It is also clear that the expression vector should include additional elements that are required for transfer and subsequent replication of the expression vector containing the nucleotide serial is lnost, the system owner. Examples of such elements include, without limitation specified, originy replication and breeding markers. The person skilled in the art also understand that these vectors can be easily constructed using standard techniques (DNA Isolation and Sequencing", Bruce A. Roe, Judy S. Crabtree and Akbar S. Khan, Published by John Wiley&Sons, 1996); in addition, they are commercially available.

The present invention also relates to the body-the owner or the cell host, which embed recombinant construct nucleic acid containing the nucleic acid molecule as described above is functionally associated with the expression vector. The cells of the host, transformed with nucleic acid structures, declared in accordance with the present invention include eukaryotic cells such as animal cells, plant, insect and yeast cells, and prokaryotic cells such as E. coli. The ways in which the design of nucleic acid containing the nucleotide sequence, can be built into a cell include, without limitation specified, microinjection, electroporation, transduction, or transfection using DEAE-dextran, lipofection, the use of calcium phosphate and other methods well known to experts in the art (Sambrook et al. (1989) in "Molecular Cloning. A Lboratory Manual", Cold Spring Harbor Press, Plainview, New York).

According to a preferred variant implementation of the present invention are applied eukaryotic expression vectors that function in eukaryotic cells. Examples of such vectors include, without limitation specified, retroviral vectors, the expression vector based on the vaccinia virus, adenovirus vectors, vectors based on herpes simplex virus, vectors based avian pox, plasmid or baculovirus transfer vectors. Preferred eukaryotic cell lines include, without limitation specified, COS cells, Cho cells, HeLa cells, NIH/3T3 cells, 293 cells (ATCS# CRL15731), T2 cells, dendritic cells, monocytes or b cells transformed by Epstein-Barr-15.

Antigenic peptide declared in accordance with the present invention can be obtained, for example, by extraction from a natural source (for example, elution of molecules MHC class II); by expression of a recombinant nucleic acid that encodes a peptide; or by chemical synthesis. The peptide, which is produced by the system of cells, different from the source from which it occurs in nature, is "dedicated"because he will be separated from the components that surround it in natural conditions. Recombinant peptide, expressio the text of the body-master, can be obtained in the form of the crude lysate or can be purified using standard methods used for purification of proteins and is well known to specialists in this field, which include differential precipitation, chromatography with the exception of size, ion-exchange chromatography, isoelectric focusing, electrophoresis gel, affine and immunoaffinity chromatography and others. The degree of purification or selection can be measured using any suitable method, such as mass spectrometry or HPLC analysis. The peptides can be obtained by synthetic means, using techniques described Memfield, (1986) Science 232: 341-347, and Barany and Merrifield, The Peptides, Gross and Meienher, eds (N.Y., Academic Press). Chemical synthesis can be carried out in solution or on solid phase or by using an automated synthesizing device (Stewart and Young, Solid Peptide Synthesis, 2nded., Rockford 111., Pierce Chemical Co. (1984)).

Thus, the present invention also relates to a method for producing antigenic peptides to MHC class II, including: a) at least the amino acid sequence peptidase site selected from the group comprising SEQ ID NOs. 49-57 and SEQ ID NOs. 103-122, or b) at least amino acid sequence peptidase site selected from the group comprising SEQ ID NOs. 49-57 and SEQ ID NOs. 103-122, with additional and N - and C-terminal flanking sequences of the respective sequence, selected from the group comprising SEQ ID NOs. 1-39 and SEQ ID NOs. 58-102. This method includes the step of culturing host cells containing the construct recombinant nucleic acid molecule, as described above, in conditions that ensure the expression of the indicated peptide, and a stage of isolation of the peptide from the cell or cellular environment.

The present invention also relates to a method of separating and identifying RA antigenic peptides associated with MHC class II, femtomolar quantities, comprising: a) obtaining an immature dendritic cells in an amount to provide from 0.1 to 5 μg molecules MHC class II; b) ensuring contact of the cells with serum or synovial fluid and indexing maturation of dendritic cells by adding TNF-alpha; C) isolation of complexes of antigenic peptide-molecule MHC class II from the cells using methods that include the solubilization of cells and isolation of complexes of molecules MHC class II antigenic peptides by thus or immunoaffinity chromatography; d) laundering of isolated complexes of molecules MHC class II antigenic peptides water in vitro ultrafiltration; d) elution associated antigenic peptides with MHC molecules of class II at a temperature of 37°C diluted triperoxonane acid and (e) separation, identification and identificationdata peptides using liquid chromatography and mass spectrometry. In addition, the step (e) liquid chromatography includes a first stage linear elution with material in the opposite phase by volume, sufficient for elution of most impurities, prior to elution of the peptide. Moreover, the method may also include the step (g) analysis of the identified peptides with known databases and special programs designed for comparative analysis of data obtained from numerous well-known databases.

The amount of tissue or body fluid needed to obtain, for example, 100 ng of molecules MHC class II, depending on the number of cells expressing MHC class II, and the ratio of the expression of MHC molecules class II: for example, 100 ng of MHC class II equivalent to about 2·105Mature DCs or 5·106monocytes peripheral blood or about 5·107mononuclear cells of peripheral blood (RVMS), which can be selected from about 50 ml of blood.

For the purification of complexes of molecules MHC class II antigenic peptides from cells or tissue membrane of cells or tissues must be solubilisate. Lysis of cells can be carried out using methods well known to specialists in this field, for example, using cycles of freezing-thawing and using detergents, as well as combinations of these methods. Pre is a respectful way of lysis is solubilization with detergents, preferably TX-100, NP40, n-octylglucoside, Zwittergent, Lubrol, CHAPS, more preferably TX-100 or Zwittergent 3-12. The decay products of the cells and cell nuclei must be removed from the cell lysate containing dissolved complexes of the receptor-peptide, using centrifugation. Thus, complexes of molecules MHC class II antigenic peptides isolated from the cells using detergent solubilization.

In addition, complexes of molecules MHC class II antigenic peptides purified from the cell lysate using the thus or immunoaffinity chromatography. For thus and immunoaffinity chromatography using antibodies specific for molecules MHC class II and suitable for use according to the specified methods. It is preferable to use monoclonal antibodies covalently or ecovalence (e.g., Protein A) associated with the beads, for example sivaratnam or agarose beads. The panel of antibodies to HLA used in accordance with the specified methods, includes: anti-HLA-DR antibodies: L243, TU36, DA6.147, preferably L243; anti-HLA-DQ antibodies: SPVL3, TU22, TU169, preferably TU22 and TU169; anti-HLA-DP antibody B7/21, and anti-HLA-a, b, C antibodies W6/32 and W.

Monoclonal antibodies specific to different molecules MHC class II can be obtained industrial (e.g., Pharmingen Dianova) or purified from the supernatant of the respective cells hybridoma using Protein a or Protein G affinity chromatography. Purified monoclonal antibodies can be combined using various methods, well known to experts in the field, preferably by covalent binding of amino groups of the antibody to CNBr-activated separate.

Immunovaccine MHC molecules may be carried out by incubation of the beads coated with the antibody from the cell lysate with rotation for several hours or using chromatography by passing the cell lysate through microcolony. Laundering beads can be carried out in Eppendorf tubes or microbalance. Efficiency thus can be estimated using SDS-PAGE and Western blotting using antibodies that recognize denatured MHC molecules (anti-HLA-DR; 1V5; anti-HLA class I; HC10 or NSA).

Isolated complexes of molecules MHC class II antigenic peptides before elution washed with water or kaboolian buffer to remove any residual detergent. As slabosolenaja buffer you can use Tris, phosphate or acetate buffer with a concentration of from 0.5 to 10 mm, preferably in a concentration of 0.5 mm. According to a more preferred variant implementation of the present invention, the complexes of molecules MHC class II antigenic peptides washed ultrapure water (programmable scale), which traditionally is about used for HPLC analysis preferably ultrapure water (programmable scale) from MERCK. Stage laundering can be carried out by means of ultrafiltration. The ultrafiltration can be performed in vitro ultrafiltration with a cut-off at mol. the mass of 30 KD, 20 KD, 10 KD or 5 KD, preferably 30 KD and displacement of the tubes from 0.5 to 1.0 ml tubes "Ultrafree"; Millipore). Laundering in test tubes for ultrafiltration can be carried out from 4 to 12 times, preferably from 6 to 10 times in volume, 10-20 times greater than the amount of beads bearing complexes of the receptor-peptide, preferably in the amount of 15 times the volume of the beads. Erwerbende peptides can be separated from the remaining molecules MHC class II using the same tubes for ultrafiltration. Erwerbende peptides can then be dried.

When the elution of peptides from MHC molecules of class II get a complex mixture of natural-processed peptides from the source potential of the antigen and of the polypeptides of the intracellular and extracellular origin. Only after elution can be divided peptides and to analyze their sequences.

Antigenic peptides declared in accordance with the present invention, can be suirvey using various methods, well known to experts in this field, preferably with the use of dilute acid, such as diluted the CSOs acetonitrile (Jardetsky TS et al., Nature 1991 353, 326-329), diluted acetic acid, followed by heating (Radensky AY et al., Nature 1991, 353, 622-626; Chicz RM, et al., Nature 1992, 358, 764-768) or divorced triperoxonane acid at 37°C (Cortese N. et al., J Exp Med 1992, 175, 1799-1803). More preferably the peptides elute at 37°C diluted triperoxonane acid.

Selected antigenic peptides then separate, detect and identify. Detection confirms that the amino acid sequence of individual peptides in a mixture selected antigenic peptides can be installed using methods suitable for the identification and sequencing of peptides in femtomolar quantities. Identification allows you to determine which proteins or polypeptides were derived antigenic peptides, and what sequence of these proteins and polypeptides are in their composition.

At the first stage of a complex mixture buervenich peptides may be separated by any known possible chromatographic methods, for example by means of chromatography with reversed phase, anion exchange chromatography, cation exchange chromatography, or a combination of these methods. Preferably the separation is carried out using chromatography with a C18-reverse phase or using back-phase/cation-exchange two-dimensional HPLC, denoted as MudPit (Washbum MP et al., Nat BiotechnoL, (2001) 19, 242-247).

The separation of the OS is p using HPLC using microcapillary tubes made of quartz glass, connecting to the generator elektrorazpredelenie mass spectrometer or with a device for microfractionation that allocates a fraction on the tablet for MALDI analysis.

Liquid chromatography involves the fractionation of the peptide with strong ion-exchange material and a hydrophobic material with a reverse phase. For elution of peptides with ion exchange material and the material of the reverse phase consistently use a variety of schemes, including elution with salt and with the use of organic solvents, such as acetonitrile. The elution of material from the reverse phase is carried out in several stages linear gradients of different lengths and tilt. Contamination of the sample that is subjected to fractionation, can represent any contamination which competes with the determination of protein peaks in the mass spectrometer. Thus, in order to prevent joint elution, before elution of peptides must be eluted impurities in sufficient volume of solvent. Depending on which column is used for liquid chromatography, the amount of solvent sufficient for elution of impurities before elution of peptides can 100-200 times the volume of the column.

Suitable are various methods of mass spectrometry (MS), preferably MALDI-post source decay (PSD) MS or tandem mass spectrum of the test with spray ionization (ESI-MS, from the English., Electrospray lonization Tandem Mass Spectrometry), most preferably ESI-MS with ion trap.

Sequences of individual peptides can be identified using techniques well known to specialists in this field. Preferably the sequence analysis is performed using the fragmentation of peptides and subsequent analysis of the fragments on the computer using algorithms such as MASCOT or SEQUEST. Both of these computer algorithm for cross-correlation analysis both theoretically and experimentally (using tandem mass spectrometry) data obtained using a database of protein and nucleotide sequences.

Isolated and identified antigenic peptides declared in accordance with the present invention can be confirmed using MHC-binding site, MHC-binding ability and/or T-cell recognition.

MHC-binding site

Peptides associated with particular MHC molecules (allelic variants), have common structural features, denoted as binding sites required for formation of stable complexes with MHC molecules. Peptide ligands, erwerbende with molecules MHC class I are relatively short and are composed of from 8 to 11 amino acids. In addition, 2 or 3 Bo the new chain peptide are related to the binding. The position of the corresponding side chains of amino acid allelic variants of HLA varies, usually two so-called "anchor" amino acid residue located at positions 2 and 9. With regard to a specific anchor position, only one and two amino acids in the norm can function as an anchor amino acids such as leucine or valine V in position 2 in the case of HLA-A2.

As for molecules, MHC class II, the length of the peptides varies from 11 to 25 amino acids, as longer peptides communicate better, both end peptidebased groove are open. The majority of HLA class II molecules have up to four anchor amino acid residue in the corresponding positions P1, P4, P6 and P9, located in nenamerno crust area. Specified core plot, however, is located at a different distance from the N-terminal part of the peptide. In most cases korovou section is preceded by 2-4 N-terminal residue. Therefore, in most HLA class II molecules P1 anchor residues are located at positions 3, 4 or 5. Peptides, erwerbende with molecules HLA-DR class II share a large hydrophobic P1 anchor amino acid residue represented by tyrosine, phenylalanine, tryptophan, methionine, leucine, isoleucine or valine.

A well-defined type of anchor amino acid residues and the position form peptidase plot, currently known for the most frequently occurring allelic products of HLA class II. A computer program that allows you to confirm the sequence of the peptides, called "Tepitope and provided by the company Vaccinome.

MHC-binding capacity

The ability of the peptides identified using the method stated in accordance with the present invention, to associate the appropriate molecules MHC class II can be tested using techniques well known to specialists in this field, for example by separating the molecules of MHC class II and synthetic peptides with amino acid sequences identical to those identified using the method stated in accordance with the present invention (Kropstnner H et al., J. Exp.Med. 1992; 175, 1799-1803; Vogt AB, et al., J Immunol. 1994; 153, 1665-1673; Sloan VS et al., Nature 1995; 375, 802-806). Alternatively, for verification of the identified epitope can be applied cell research linking using cell lines expressing MHC class II, and the biotinylated peptides (Amdt SO et al., EMBO J. 2000; 19, 1241-1251).

In both cases, the corresponding binding capacity of the peptide is measured by determining the concentration required to reduce binding of the labeled reporter peptide by 50%. The specified value indicate the as IC 50. When the binding of the peptides with significant affinity to the corresponding molecules MHC class II IC50 values do not exceed the value of the IC50for certain control peptides more than 10 times.

Similar studies linking can be carried out in order to test the ability of the peptides to contact alternative MHC-class II molecules, i.e. molecules of MHC class II, other than those with which elute these peptides using the method stated in accordance with the present invention. Diagnostic methods of using these peptides or therapeutic methods declared in accordance with the present invention, using both peptides, and peptides derived from it, can be applied to the subjects expressing these alternative molecules MHC class II.

Recognition by T-cells

The verification procedure epitopes may include the study of the ability of the peptides identified using the method stated in accordance with the present invention, to activate populations of CD4+T cells. Synthesize peptides having amino acid sequences that are identical to those identified in accordance with the present invention, or the corresponding cow sequence, p is obtained from the "female" group of peptides, identified according to the present invention. Then study the ability of synthetic peptides to activate CD4+T cells, obtained from (a) entities expressing interest MHC molecules of class II and having at least one symptom of the disease; and (b) control subjects expressing interest MHC molecules of class II, have no symptoms. An additional control group may include people with symptoms, but not expressing interest MHC molecules of class II.

In some diseases (for example, the pathogenesis of which involved an autoimmune component) measured the reactivity of CD4+T cells in the tested subjects [in the control group described in paragraph (b)] proves the assumption that the relevant peptide is an epitope that activates CD4+T cells, which, in turn, initiate, contribute to and exacerbate the relevant diseases. Other diseases (such as cancer or infectious diseases without autoimmune component) is similar to the nature of the reactivity (the same as in the previous example) indicates that the relevant peptide is an epitope that activates CD4+T cells that can provide immunity to the specified C is the disease or at least, to reduce clinical manifestations.

The response of CD4+T cells can be measured using various in vitro methods, well known to specialists in this field. For example, it is possible to cultivate a whole mononuclear cells from peripheral blood (RVMS) both in the presence and in the absence of interest synthetic peptide, and then to measure their proliferation, for example, by incorporation into DNA [3H]-thymidine. What proliferating T cells are CD4+T-cells can be confirmed by the Department of CD4+T cells from RUMS before the study, and by adding inhibitory antibodies that bind to CD4+molecule of T cells and thus inhibit the proliferation of the latter. In both situations, the proliferative response is inhibited only if CD4+T-cells are proliferating cells. Alternatively, CD4+T cells can be purified from RVMS, and their proliferative response to the presence of the peptides studied in the presence of APC expressing the appropriate MHC molecules of class II. These ARS can be b-lymphocytes, monocytes, macrophages, or dendritic cells, or whole RVMS. As ARS can also be used an immortalized cell line derived from b-lymphocytes, monocytes, macrophages or dendritic cells. ARS m is able to Express the endogenous MHC molecules of class II, of interest, or to Express transfetsirovannyh polynucleotide encoding these molecules. In all cases, before the study of ARS can be deprived of the ability to proliferation by processing, for example, ionizing radiation or mitomycin-C.

An alternative to study cell proliferation may be research based on measuring the production of cytokines by CD4+T-cells using methods well known to specialists in this field. The cytokines include, without limitation specified, interleukin-2 (IL-2), gamma interferon (EFN-γ), interleukin-4 (IL-4), TNF-alpha, interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12) or TGF-beta. For ways to measure their products include, without limitation specified, ELISA and bosslaguna in which to study the reactivity (e.g., proliferation) of cells that responds to the products of the respective cytokine in the presence of the test specimen.

Alternatively, it is possible to directly visualize the cytokine production of CD4+lymphocytes using intracellular immunofluorescent staining and flow cytometry.

In addition, antigenic peptides to MHC class II declared in accordance with the present invention can be used for the diagnosis of RA. Thus, another embodiment of the present invention which is the use of antigenic peptides declared in accordance with the present invention, as markers of RA.

Preferably as markers of RA use of antigenic peptides to MHC class II, including (a) at least the amino acid sequence peptidase site selected from the group comprising SEQ ID NOs. 49-57 and SEQ ID NOs. 103-122, or (b) at least amino acid sequence peptidase site selected from the group comprising SEQ ID NOs. 49-57 and SEQ ID NOs. 103-122 with additional N - and C-terminal flanking sequences of the respective sequence selected from the group comprising SEQ ID NOs. 1-39 or SEQ ID NOs. 58-102.

According to another variant implementation of the present invention antigenic peptides declared in accordance with the present invention, can be used as markers of response to assess the effectiveness of the treatment regimen. Essentially, you can determine the initial level of antigenic peptides, and then to enter a specific medicinal agent and consistently monitor the level of antigenic peptides, however, changing the specified level will allow to evaluate the effectiveness of therapy.

In addition, antigenic peptides detected only at certain stages or phases of the disease, preferably RA, can be used as markers of a certain hundred and the AI diseases. Essentially, regularly measure the level of antigenic peptides associated with a certain disease stage, which allows to obtain information related to disease stage and progression of the disease.

The present invention also relates to the use of polypeptides, which are RA antigenic peptides as markers for diagnosis and monitoring of disease, preferably RA, and, in particular, erosive form of RA in contrast neurosignal form of RA. By reason of the use of the corresponding proteins is the fact that DCs are present in most tissues, where they carry out the seizure of exogenous antigens using specific receptors and through specialized intracellular mechanisms (for example, macropinocytosis) subsequent representation processioning antigen as peptide on the MHC molecules of class II. Previous research showed that the frequency of detectable peptide epitope in the case of MHC molecules, class II, for example RA antigenic peptides, in most cases reflects the excess protein from which specified a specific peptide.

Thus, not only the RA antigenic peptides and corresponding proteins can serve as markers of RA.

Thus, according to another variant, the wasp is estline of the present invention as markers of RA using the polypeptides, selected from the group including induced by interferon-γ lysosomal tearducts (SEQ ID NO. 40), integrin beta-2 (SEQ ID NO.123), phosphatidylinositol-4,5-phosphate-3-kinase (SEQ ID NO:124), plasminogen activator urokinase type (SEQ ID NO: 125), V-III sites of the heavy chain of immunoglobulin (VH26) (SEQ ID NO: 126), DJ-1 protein (SEQ ID NO:127), apolipoprotein B-100 (SEQ ID NO: 41), 26S proteasome is not ATP-asnow regulatory subunit 8 (SEQ ID NO. 128), the receptor for interleukin-1 (SEQ ID NO: 129), fibromatosis (SEQ ID NOs.: 130), GM-CSF/IL-3/IL-5 receptor (SEQ ID NOs. 131), sorting nexin 3 (SEQ ID NO: 132), H4 heavy chain inhibitor, inter-α-trypsin (SEQ ID NO. 42), complement C4 (SEQ ID NO: 43), complement C3 (SEQ ID NO: 44), protein 3 similar to the enriched glutamic acid-binding SH3-domain protein (SEQ ID NO: 45), induced by interleukin-4 protein 1 (SEQ ID NO: 46), hemopexin (SEQ ID NO. 47), Hsc70-interactive protein (SEQ ID NO: 48), constant chain (Ii) (SEQ ID NO: 133), responsible for protein 2 receptor retinoic acid (SEQ ID NO: 134), fibronectin (SEQ ID NO: 135), cathepsin (SEQ ID NO: 136), tripeptidyl-peptidase II (SEQ ID NOs. 137), legumain (SEQ ID NO: 138), the receptor of platelet activating factor (SEQ ID NO: 139), poly-alpha-2,8-sialyltransferase (SEQ ID NO:140), ras-related peptide Rab-11 (SEQ ID NO: 141). Preferably the polypeptide is used as a marker of erosive RA. Also is preferred to use the polypeptide as a marker neurosignal RA. Osobennostyami is the use of induced interleukin-4 protein as a marker for RA. To date, the polypeptide presented in figure 1, was unknown as a marker of RA, but it is considered one of the most important candidates for this role.

The diagnosis of RA can be diagnosed on the basis of studying the expression and/or composition of the polypeptide or peptide marker of RA that can be done through various methods, including enzyme linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation and immunofluorescence. In the tested sample obtained from the patient, determine the presence of altered expression and/or structure of the polypeptide or peptide, notified in accordance with the present invention. Altered expression of the polypeptide or peptide may represent, for example, quantitatively altered expression (i.e. change the number of the produced polypeptide); the change in the structure of the polypeptide represents a qualitative change in the expression of the polypeptide (for example, expression of a mutant polypeptide or peptide, resulting from alternative splicing).

Both of these changes (quantitative and qualitative) can also occur. The term "change" in the expression or structure of the polypeptide, as used here, refers to the changes in the expression or structure of the polypeptide or peptide in the test sample compared to expression of isostructural polypeptide or peptide in the sample. Control sample is a sample that corresponds to the test sample (for example, it is from the same type of cells), but isolated from the organism of the individual, not suffering from RA. Changes in the expression or structure of the peptide or polypeptide in the test sample when compared to the control sample is indicative of presence of RA or predisposition to RA. For an analysis of these changes in the expression or structure of a peptide or polypeptide that is declared in accordance with the present invention may be used various methods, including spectroscopy, colorimetry, electrophoresis, isoelectric focusing and immunological analysis (see for example, David et al., Pat. No. 4,376, 110), such as the Western blot turns (see also Current Protocols in Molecular Biology, in particular, Chapter 10). For example, according to one of embodiments of the present invention, it is possible to use an antibody able to bind with the polypeptide (for example, as described above), preferably the polypeptide labeled with the designated label. Antibodies may be polyclonal or preferably monoclonal. You can use the intact antibody or its fragment [e.g., Fab or F(ab')2). The term "labeled", used in relation to the sample or antibody, means the direct introduction of the label in the sample or the antibodies is about by joining (that is, the physical binding) detected connection to the sample or antibody, as well as the indirect introduction of the label in the sample or antibody through reaction with another reagent that can directly be determined. Examples of indirect injection marks are the definition of the first antibody with a second antibody labeled with a fluorescent label, or end-labeling DNA sample with Biotin such that it can be determined using streptavidin labeled with a fluorescent label.

To measure the level or amount of the peptide or polypeptide in the test sample, and comparing the obtained results with a control sample can be applied Western blotting using antibodies that specifically binds to a peptide or polypeptide that is declared in accordance with the present invention, as described above. Preferably the level of a peptide or polypeptide in the test sample is measured using a homogeneous or heterogeneous immunoassay. If the level or amount of the polypeptide in the test sample is higher or lower than the level or amount of the polypeptide in the control sample, and this difference is significant, this indicates a modified expression of the polypeptide, and the presence of RA or the predisposition towards it.

Thus, the present invention also relates to a diagnostic composition comprising the antibody, R is airwomen with antigenic peptide-MHC class II, declared in accordance with the present invention.

According to another variant implementation of the present invention antigenic peptides declared in accordance with the present invention, or proteins, from which they receive can be used for the prevention and treatment of diseases, preferably RA.

One aspect of the present invention is a therapeutic aspect, when one or more of the identified antigenic peptide is used to vaccinate patients against RA. During the course of vaccination antigenic peptide to induce antigen-specific T-cell tolerance in a patient, which will directly contribute to the regression of the disease or slow the development of disease.

A promising approach to induce specific immune tolerance, which may find application in future clinical studies, is the use of DNA vaccines, contributing to the development of tolerance. It was shown that DNA vaccines, contributing to the development of tolerance and coding individual autoantigens, reduce T-cell proliferative response (Ruiz, P. et al., J Immunol 162 (1999) 3336-3341), while DNA vaccines, contributing to the development of tolerance and delivering autoantigen in conjunction with IL-4 also induce protective Tn2 response (Garren, H. et al., Imunity 15 (2001) 15-22). Examples polinucleotide specific tolerized therapies that are under development are methods based on the use of protein antigens, natural-processed peptides, altered peptide ligands, other biomolecules, such as DNA or proteins, or peptides with post-translational modifications, as well as antigens, administered orally and cause the so-called "oral tolerance" (see review by Robinson, W.H. et al., Clin Immunol 103 (2002) 7-12). One of the possible side effects of such therapy, contributing to the development of tolerance, are autoimmune reactions.

For this purpose, you can enter the patient corresponding RA antigenic peptides in sufficient quantity to bind MHC molecules and cause peripheral tolerance of T cells.

Alternatively, antigenic peptides, notified in accordance with the present invention, can be used to produce vaccines based on DCs. In this case, autologous DCs derived from monocytes of a patient can be treated with relevant peptides or recombinant protein comprising the peptide sequence.

Thus, the present invention relates to pharmaceutical compositions containing antigenic peptides to MHC class II, including (a) to what ina least the amino acid sequence peptidase plot selected from the group comprising SEQ ID NOs. 49-57 and SEQ ID NOs. 103-122, or (b) at least amino acid sequence peptidase site selected from the group comprising SEQ ID NOs.

49-57 and SEQ ID NOs. 103-122 with additional N - and C-terminal flanking sequences of the respective sequence selected from the group comprising SEQ ID NOs. 1-39 or SEQ ID NOs. 58-102; antibody reacting with the specified antigenic peptide or polypeptide selected from the group comprising SEQ ID NOs. 40-48 and SEQ ID NOs. 123-141, and, optionally, a pharmaceutically acceptable excipient, solvent or carrier. Antigenic peptide must be present in sufficient quantity to cause the tolerance of specific cells. Specified number depends on the used peptide, the method of administration, the severity of the disease to be cured, as well as the General condition of the patient and is usually in the range from 1 to 50 mg/ml, for example, for peptides associated with dendritic cells.

Acceptable excipient, diluent or carrier may be a phosphate buffer saline solution for in vitro and physiological saline for use in vivo.

The term "vaccine"as used here, refers to both active immunization, i.e. for the introduction of the peptides in vivo to stimulate the immune tolerance which the spine in vivo directly from the patient, and passive immunization, i.e. using peptides to stimulate tolerance of CD4+T-lymphocytes in vitro or to stimulate autologous or allogeneic dendritic cells, which are then re-administered to the patient.

The present invention also relates to antigenic peptides, antibodies, nucleic acids, cells, hosts, methods, compositions and their application as described above, in particular with regard to the presented examples.

A more complete understanding of the present invention can be obtained from the following specific examples, which are presented here only to illustrate the invention and do not limit its essence, if not otherwise specified, taking into account the following figures.

Examples

The examples below are illustrated by the figures described above, and based on the methodology summarized in figure 1 and disclosed in detail below. If not stated otherwise, all examples used industrial reagent according to the manufacturer's instructions.

The method of the invention

Dendritic cells and their cultivation

The study carried out with the use of dendritic cells that were differentiated from monocytes, as described below. Monocytes obtained from peripheral blood. The shelter is taken from healthy donors, with the following haplotypes: (1) HLA-DRB1*0401, *03011, (2) HLA-DRB1*0401, *0304, (3) HLA-DRB1*0401, *0301, (4) HLA-DRB1*0401, *0701, HLA-DRB1*0401, *0407.

All cells were cultured in RPMI medium 1640 (RPMI)enriched with 1 mm pyruvate, 2 mm glutamine and 10% V / V heat inactivated fetal calf serum (Gibco BRL, Rockville, MD).

The selection of mononuclear peripheral blood cells (PBMCs)

Peripheral blood obtained from the blood Bank of Mannheim (Germany) as a standard compositions presented leukocyte film from healthy donors. To prevent coagulation using heparin (200 IU/ml of blood, Liquemine, Roche). Mononuclear cells from peripheral blood (PBMCs) was isolated using centrifugation in LSM® (1,077-1,080 g/ml; ICN, Aurora, OH) at 800g (room temperature) for 30 minutes. PBMCs collected at the interphase and washed twice PRMI containing 20 mm Hepes (500g for 15 min, 300g for 5 minutes). To remove red blood cells PBMCs treated with ALT buffer (140 mm ammonium chloride, 20 mm Tris, pH 7.2) for 3 minutes at 37°C. PBMCs washed twice RPMI containing 20 mm Hepes (200g for 5 minutes).

Obtaining dendritic cells from monocytes in peripheral blood

Monocytes isolated from PBMCs using positive sorting using anti-CDH magnetic beads (Miltenyi Biotech, Auburn, CA) according to manufacturer's instructions. Monocytes were cultured in RPMI enriched with 1% nonessential aminoxy the lots (Gibco, BRL, Rockville, MD), 50 ng/ml recombinant human granulocyte-macrophage colony-forming human factor (GM-CSF). Granulocyte Macrophage-Colony Stimulating Factor, S.A.

1,1·107U/mg) (Leucomax; Novartis, Basel, Switzerland) and 3 ng/ml recombinant IL-4 (S.A. 2,9·104U/g) (R&D Systems, Minneapolis, MN). To obtain immature dendritic cells monocytes were seeded in the amount of 0.3·106/ml in 6-hole plates and cultured for 5 days.

The quality of the immature dendritic cells derived from monocytes, is regularly assessed using flow cytometry; it is considered to be acceptable in the case when the cells have the following phenotype: CD1a (high), CD3 (negatives), CD14 (low), CD19 (negatives), CD56 (negatives), CD80 (low), CD83 (negatives), CD86 (low) and HLA-DR (high). In contrast, Mature dendritic cells (see below) have the following phenotype: CD1a (low), CD80 (high), CD83 (high), CD86 (high) and HLA-DR (high). Monoclonal antibodies to CD1a, CD3, CD14, CD19, CD56, CD80, CD83, as well as relevant izotopicheskie controls acquire the company Pharmingen (San Diego, CA).

Treatment of dendritic cells with serum or synovial fluid

Serum or synovial fluid within 30 minutes is irradiated with 137Cs (70 TBq). Makeup for dendritic cells serum or antigen selected from Zinovii, 6·106immature dendritic cells treated or 1 ml serum or 0.6 of synovial liquids is I. At the same time induce the maturation of dendritic cells by adding 10 ng/ml of recombinant tumor necrosis factor alpha man (TNFα; S.A. 1,1·105IU/µg). As control 6·106immature dendritic cells incubated only with TNFα.

After 24 hours of culturing dendritic cells are harvested by centrifugation at 300g for 10 minutes. Cells washed PBS and transferred into Eppendorf tubes. After centrifugation at 400g for 3 min, the supernatant is completely removed and the cells frozen at -70°C.

Obtaining beads with anti-HLA class II

Anti-HLA-DR monoclonal antibody (mAb) L243 (ATS, Manassas, VA) obtained by culturing the corresponding cell line hybridoma mouse. MAb L243 purified using Protein And sepharose (Pharmacia, Uppsala, Sweden) and put on CNBr-activated sepharose beads (Pharmacia) to a final concentration of 2.5 mg/ml according to the manufacturer's instructions. L243 beads stored in PBS, containing 0.1% Zwittergent 3-12 (Calbiochem, La Jolla, CA).

Purification of HLA-DR peptide complexes in the nanoscale

Precipitation frozen dendritic cells resuspended in a ten-fold volume of ice-lyse buffer [1%Triron X-100, 20 mm Tris, pH 7.8, 5 mm MgCl2containing protease inhibitors, such as hemostatic, pepstatin, PMSF and leupeptin (Roche, Mannheim, Germany)] and are lysed in a horizontal shaker at 1000 rpm, 40C for 1 is. Cell lysate cleared from cell debris and nuclei by centrifugation at l0000g, 4°C for 10 min, the Lysate is incubated together with L243 beads (5-10 µl L243 the beads in 100 ál of cell lysate) in a horizontal shaker at 1000 rpm, 4°C for 2 hours Immunoprecipitation HLA-DR complexes associated with L243 beads, precipitated by centrifugation at l000g, 4°C for 1 min and washed four times with 500 µl of 0.1% Zwittergent 3-12 (Calbiochem) in PBS.

The cleaning efficiency of HLA-DR-peptide complexes is controlled by analyzing the corresponding cell lysates before and after and thus aliquots of beads using Western blotting using anti-HLA-DRα-specific mAb 1B5 (Adams, I.E. et al., Immunology 50 (1983) 613-624).

Elution of HLA-DR-associated peptides

HLA-DR-peptide complexes associated with L243 beads, resuspended in 100 μl of N2On (HPLC-gradient; Merck, Darmstadt, Germany), transferred into tubes for ultrafiltration, Ultrafree MC, with cut-off at the rate of 30 KD (Millipore, Bedford, MA) and washed 10 times with 100 μl of N2On (HPLC-gradient) by centrifugation for 1-2 minutes at l0000g at room temperature. For elution of related peptides add 60 μl of 0.1%triperoxonane acid (Fluka, Buchs, Switzerland) in N2On (HPLC-gradient) and incubated for 30 minutes at 37°C. Erwerbende peptides collected in a new Eppendorf tubes by centrifugation in si is the subject Ultrafree at 10000g for 3 min at room temperature and immediately lyophilizer in a vacuum centrifuge Speed-Vac™.

Fractionation using two-dimensional liquid chromatography (LC. from the English. Liquid Chromatography) nanobotic

For sequencing of complex mixtures of peptides with high throughput using multi-dimensional technology to identify peptides (MudPIT). Multidimensional Protein Identification Technology) (Washbum, M.P. et al., Nat Biotechnol 19 (2001), 242-247), which is based on the fractionation using liquid chromatography with subsequent mass spectrometric analysis of sequences.

For this purpose lyophilized peptides, erwerbende with HLA molecules, resuspended in buffer containing 5% (vol./about.) acetonitrile (ACN)and 0.5% (vol./about.) acetic acid, 0,012% (vol./about.) heptacosanoic acid (HFBA) and 1% (vol./about.) formic acid. The mixture of peptides fractionary on the micro capillary column of fused silica glass (inner diameter 100 mm · 375 μm), obtained using a laser puller Model P-2000 (Sutler Instrument Co., Novato, CA). Microcolony fill 3 μm/C18 material with a reverse phase (C18-ACE 3 μm [ProntoSIL 120 will receive 3 entries-C18 ACE-EPS, Leonberg, Germany]), then 3 cm 5 μm cation exchange material (Partisphere SCX; Whatman, Clifon, USA).

Perform fully automatic gradient separation of peptides by LC Packings UltiMate HPLC (LC Packings, San Francisco, USA), including 8 steps using the following buffer: 5% ACN/ 0,012% HFBA/ a 0.5% acetic acid (buffer A), 80% ACN/ 0,012% HFBA/ 0,5% acetic is islote (buffer); 250 mm ammonium acetate/ 5% ACN/ 0,012% HFBA/ a 0.5% acetic acid (buffer C) and 1.5 mm ammonium acetate/ 5% ACN/ 0,012% HFBA/ a 0.5% acetic acid (buffer D). The first phase, the duration of which is 116 minutes, includes: 75 minute gradient of buffer b from 0% to 40%, a 10-minute gradient of buffer from 40% to 80%, 6 minutes of incubation in 80%buffer b and 10 minutes of equilibration 100%buffer A. the Following 5 phases (each lasting 164 minutes) characterised by the following scheme: 5 min 100%buffer A, 5-minute gradient buffer With 0% to x%, 5 min 100%buffer A, A 30 minute gradient of buffer b from 0% to 10%, the 55-minute gradient of buffer In 10% to 35%, a 20 minute gradient of buffer from 35% to 50%, the 10-minute gradient of buffer In 50% to 80%, 6 minutes of incubation in 80%buffer b and 10 minutes of equilibration 100%buffer A. the Percentage of buffer C (x) for stages 2-6 are: 20, 40, 60, 80 and 90%. 30 minute gradient of buffer b from 0%to 10%, which is the first stage of the linear elution with material reverse phase, is necessary in order to adequately separate the elution of peptides and elution main contaminant (m/z=945), which leads to loss of most hydrophilic peptide peaks. Step 7 involves the following phases: 5 min 100%buffer A, 20 min 100%buffer C, a 5-minute gradient of buffer b from 0% to 10%, a 35-minute gradient of buffer In 10% to 35%, 50-minute gradient of buffer from 35% to 50%, 10 minutes is the gradient of buffer In 50% to 80%, 5 minutes of incubation in 80%buffer b and 10 minutes of equilibration 100%buffer A. the Stage 8 is identical to step 7, except that instead of buffer use the buffer D.

Mass spectrometry MS/MS with ion trap

HPLC column directly attached to the mass-spectrometer ion trap Finnigan LCQ Deca XP Plus (Thermo Finnigan, San Jose, USA)equipped with a nano-LC source of ionizing elektrorazpredelenie. Mass spectrometry MS/MS is carried out according to the manufacturer's instructions. Peptides identified using the SEQUEST program (U.S. patent 6 693 017 and 5 538 897).

Mass spectrometry MALDI-TEESE

Peptides marked on the tablet AnchorChip, crystallized together with the matrix [5 mg/ml; α-cyano-hydroxy-cinnamic acid (Merck, Darmstadt, Germany), 50%acetonitrile, with 0.1%triperoxonane acid). For the quantitative analysis of the full set of peptides samples will be analyzed by the mass spectrometer wall-mounted™ MALDI-THIES (Broker, Bremen, Germany) according to manufacturer's instructions.

Identification of sequences using SEQUEST and differentiated data analysis

Data MS/MS fragmentation analyzed using SEQUEST (Thermo Finnigan, San Jose, USA). From the internal database of proteins, based on public databases Swiss-Prot, TrEMBL, SEQUEST selects for each spectrum, all peptide sequences that correspond to the molecular mass of the parent which it measures the degree of correspondence between the experimental spectrum and theoretical, in silico generated spectrum. The list includes only candidate sequence that gives the highest score.

Peptide sequences obtained using SEQUEST analysis, as well as related information about the exact weight, the account settings and the origin of the peptides retain at appropriate appropriate database and subjected to additional processing. In order to secure the preservation of the only significant sequences with satisfactory SEQUEST score to impose certain restrictions. The two most important limitations include: (i) keep only those sequences that have a correlation coefficient (CC). Correlation Coefficient exceeding a certain value, and (ii) of the remaining sequences, save those that have a predefined Delta-correlation coefficient (CC). For both these criteria, the selected minimum values are based on empirical knowledge about how to interpret the results of SEQUEST.

The data set is determined as the sum of data from a specific range of the spectrum. Technical solution database and computer program allows the query as one set of data, and to compare multiple databases. This decision database and computer programs allow osushestvljali samples which provides no program SEQUEST. For example, you can query one data set provide information about the distribution account among the saved spectrum sequences, the presence of additional variants of the sequences along the length or total suppositionally, and also about how protein derived peptide sequence. Because the presence of truncated variants of one epitope is a General property of peptides associated with MHC molecules of class II, the presence of variants in length in the data set provides additional strong evidence that in this spectrum there is the epitope.

The most important feature of the analysis of multiple databases is that it makes it possible to identify a common subset of sequences that satisfies the given criteria. These criteria should be based on the similarity of the sequences; for example, among all sequences that are in the database, selects only those sequences that have at least one General suppositionally. The specified comparative analysis of different databases provides a differentiated approach (RA samples vs. control samples) and, thus, allows to optimize the search for potential pepti the different markers of RA.

Indicators account pairwise similarity of sequences is calculated using a standard computer program that implements the standard algorithm for string comparison. Subsequently, these figures account is used for combining related sequences (sequences that share a common suppositionally) in a completely separated clusters that are carried out with additionally developed a computer program, which is based on the well-known algorithm (hierarchical cluster analysis, UPGMA).

The obtained clusters (e.g. clusters of truncated variants of the peptides is then used to identify related sequences in different databases.

In addition, the program for data analysis allows you to quickly and accurately carry out the following:

from the output, obtained using SEQUEST, select those sequences that satisfy reliable empirical criteria;

- save the data in a database specially developed manually for research.

- get information on all sequences stored in each database. This information is valuable because it helps determine the importance of the individual sequences in a given database and, consequently, in numerous the x databases;

to ensure a differentiated approach, namely to study the actual sequence of one sample compared to the other, through the comparative analysis of multiple databases.

Purification of HLA-DR molecules

HLA-DR molecules purified from 1010EBV-transformed b-cell lines or T2-transfectants using affinity chromatography using an anti-DR monoclonal antibody L243, as described above (Kropshofer H. et al., PNAS 92 (1995) 8313-8317).

The analysis of binding peptides in vitro

ON(307-319), PKYVKQNTLKLAT, is an immunodominant epitope of the hemagglutinin of influenza virus, which is well associated with HLA-DR molecules and used as a reporter peptide in the analysis of binding peptides in vitro (Rothbard, J.B. et al., Cell (1988) 52:515-523).

Peeled dissolved in the detergent HLA-DR4 molecules (200 nm) are incubated together with biotinylated (307-319) peptide (200 nm) and graded by the number of competing peptide (100 nm - 10 μm) for 24 hours at 37°C in binding buffer (50 nm sodium phosphate, 50 nm sodium citrate, pH of 4.8, 0.1% Zwittergent 3-12) in a total volume of 50 µl. Competing peptides originate from possible RA antigens identified in this study as well as the use of synthetic peptides firm Medprobe (Lund, Sweden).

Then 3·10 ál of 10-fold diluted in PBS containing 0.05%tween-20 and 1%BSA, and cu is irout for 2 h in microtiter tablet (Nalge Nunc), which pre-applied anti-DR monoclonal antibody L243 (during the night). Immediately after receiving the samples by incubation with 0.1 μg/ml EU-labeled streptavidin (Wallay Oy, Turku, Finland) for 45 minutes according to the manufacturer's instructions. After an intense laundering of 0.05%Tween-20 in PBS measure the fluorescence of europium with fluorimetry with a temporal resolution (VICTOR 1420, Wallac/Perkin Elmer Life Sciences) for calculating the binding of biotinylated(307-319) peptide with HLA-DR molecules (Aradt, S.O. et al., EMBO J. 19 (2000) 1241-1251).

Example 1

In this example, to identify new markers HLA-DR-associated peptides isolated from serum and synovial fluid of patients with neurosignal RA, use the technique shown in figure 1.

6·106immature dendritic cells treated with 1 ml of serum (5 samples) or 0.6 ml of synovial fluid (2 sample) patients with neurosignal RA and cultured for 24 hours in the presence of 10 ng/ml TNFα. As a control were cultured 6·106dendritic cells in the presence of TNFα (10 ng/ml) and 1 ml of PBS, but without the addition of serum. In an additional experiment 6·106dendritic cells treated with 1 ml of serum from two healthy individuals and cultured for 24 hours in the presence of TNFα (10 ng/ml).

Dendritic cells are lysed with detergent TX-100 and HLA-DR molecules emit using mAb L23. HLA-DR-associated peptides elute with 0.1%TFA and analyzed by high throughput 2D-LC-MS/MS methods. Identification of peptides is performed using the SEQUEST algorithm. Peptide sequences obtained using the SEQUEST analysis, and additional information about the exact weight, the account settings and the origin of the peptides retain at appropriate appropriate database and subjected to further processing.

Peptide sequences obtained from untreated DCs (control 1) and from DCs treated with serum of healthy people (control 2), compared with peptide sequences derived from DCs treated with serum of patients with neurosignal RA. Among the RA-specific sequences for further analysis selected only those peptides that are re-identified at least three out of five samples neurosignal RA.

Each serum sample identify approximately 600±150 individual peptide sequences (correlation coefficient SS>3.0 and Δ>0.15). In samples of synovial fluid in the number of individual peptide sequences significantly less (400±30). Approximately 80-85% of the peptides detected in RA samples, you can also select and control samples, indicating high reproducibility of the method. In most of the cases there are several options one epitope of different lengths, that is a characteristic feature of antigens associated with MHC class II, and confirms the reliability of the results (Jones, DURING, Curr Opin Immunol 9 (1997) 75-79). Another confirmation of the quality of the received data is the fact that some of the identified peptides or proteins have already been described as associated with molecules of MHC class II epitopes derived from ubiquitous proteins, such as Hsp70, enolase, Annex II, cathepsin With or collagen II and MHC molecules (HLA-A, -B, -C, -E, -G, and 2-microglobulin) and CLIP (Chicz, R. et al., J Exp Med 178 (1993) 27-47; Sinigaglia, F. & Hammer, J., Curr Opin Immunol 6 (1994) 52-56; Amold-Schild, D. et al., J Immunol 162 (1999) 3757-3760; Vogt, A.B. & Kropsher, H., Trends Biochem Sci 4 (1999) 150-154) is also often defined.

RA-specific peptide sequence is additionally confirmed by binding to the allele DRB1*0401, determining predisposition to RA, using the TEPITOPE program (Hammer, J. et al., Adv Immunol 66 (1997) 67-100). This program allows you to conduct a quantitative and qualitative determination of T-cell epitopes.

The data obtained in the course of the study, refer only to the epitope, which is found exclusively in samples from patients with neurosignal RA (with one exception) (table 1).

Induced by interferon-gamma lysosomal tearducts

Very interesting epitope identified in three of the seven serum samples and sinovia the Aulnay with neurosignal RA, derived from induced gamma interferon lysosomal tearducts (GILT): 16-dimensional GILT (192-207) with the amino acid sequence SEQ ID NO: 3 (table 1). Additional options specified epitope (by length) in the other three samples confirm the relevance of the specified epitope (table 1): 14-dimensional GILT (192-205; SEQ ID NO: 1) and 17-dimensional GILT (192-208; SEQ ID NO:2).

When analyzing the shortest variant, GILT (192-205), it was found that the epitope contains a suitable binding site allele DRB1*0401, determining predisposition to RA, and 196 M performs the function P1 anchors, M - P4 anchors, and 201 A - P6 anchors. According TEPITOPE counting the epitope is a measure of the binding (the threshold value)of 1%, which is similar to the indicator of the binding epitope of the hemagglutinin of influenza virus (307-319), representing a strong DRB1*040-linking agent (table 1) (Rothbard, J.B. et al., Cell 52 (1988) 515-523).

GILT constitutively expressed antigen-presenting cells such as dendritic cells, macrophages and b cells, and ensures maximizing subjected to endocytosis of antigens in MHC class II-containing compartments (MEAs) by enzymatic cleavage of disulfide bonds (Phan, U.T. et al., J Biol Chem 275 (2000) 25907-25914). It was shown that in b cells is a direct binding with GILT HLA-DR molecules (Arunachalam, V. et al., J Immunol 160 (1998) 5797-5806). Also what about the installed the relatively long second epitope GILT associated with HLA-DR3 molecules: 22-dimensional GILT (38-59), having the amino acid sequence SPLQALDFFGNGPPVNYKTGNL (Chicz, R. et al., J Exp Med 178 (1993) 27-47).

In addition to GILT (192-207) in some RA samples was identified a different epitope of the same protein, it was detected in control samples: GILT (210-227) with the amino acid sequence QPPHEYVPWVTVNGKPLE. The specified epitope was identified together with three options (length): 16-dimensional GILT (210-225), 17-dimensional GILT (210-226) and 19-dimensional GILT (210-228).

As the name suggests enzyme, expression of GILT may be the induction of proinflammatory cytokine interferon-gamma (IFN-γ) in the cells of various types, including macrophages, endothelial cells and fibroblasts (Luster, A.D. et al., J Biol Chem 263 (1988) 12036-12043). Because ifn-γ is detected in the inflamed joints of patients with RA, can be observed overexpression of GILT in sinovia and serum and, as a consequence, its capture DCs as an exogenous antigen. GILT (192-207) can be obtained on the basis of exogenous GILT. Another epitope GILT, which is also detected in the control samples can be obtained from the endogenous GILT expressed by DCs. Alternatively, both options, namely GILT (192-207) and GILT (210-227), can be obtained on the basis of endogenous GILT in that case, if the processing GILT and view what is happening from GILT epitope DCs were mean is correctly changed as a result of contact with RA-associated material.

The identified epitope induced by interferon-gamma lysosomal tearducts GILT (192-205), which was described in detail above, additionally analyzed in the study binding in vitro using synthetic GILT (192-205) and purified HLA-DR molecules (Figure 3). According to the calculation using the TEPITOPE peptide binds to HLA-DR4 with high affinity in comparison with viral (307-319) peptide.

Integrin beta-2

Detailed analysis revealed the presence of the epitope, which was identified in two of the seven serum samples and sinovia patient with neurosignal RA and which is obtained from the beta-subunit of integrin (ITB2): 17-dimensional ITB2 (315-331; SEQ ID NO: 58) with the amino acid sequence NIQPIFAVTSRMVKTYE (table 1). Was discovered one option length: 19-dimensional ITB2 (313-331; SEQ ID NO: 59), which confirms the reliability of the identified epitope (table 1).

Peptide sequence includes moderate HLA-DRB1*0401 binding site with 3161 and 3191, performing the function of the anchor amino acids P1 and P4, respectively, a measure of binding, calculated using TEPITOPE, 2%).

Integrins are a family of cell surface receptors that play an important role in embryogenesis, wound healing, immune response and cell adhesion. ITB2 is a heterodimeric receptor for intracellular and vascular Molek the adhesion (ICAMs and VCAMs) and is expressed exclusively on the surface of leukocytes. ICAMs and VCAMs belong to the immunoglobulin superfamily, which plays a major role in humoral and cellular immune response. Many cytokines, such as interferon-γ, IL-1 and TNFα, the regulation of which is increased in inflammatory diseases such as RA, induce the expression of ICAMs on the surface of endothelial cells. It was shown that the expression of ICAM-1 and VCAM-1 in synovial tissue of patients with RA is higher than in patients with osteoarthritis (Furuzawa-Carballeda, J. et al., Scand J Immunol 50 (1999) 215-222).

Linking TV and other integrins with ICAMs allows leukocytes to infilterate the target tissue, for example the site of inflammation. In order to carry out its function as a non-adhesive manner or to circulate in the tissues, white blood cells constitutively Express TV and other integrins with low ability to bind ligand. Interaction ITB2/ICAM-1 is a potential target for the effects of inflammation, autoimmune diseases and cancer (Yusuf-Makagiansar, H. et al., Med Res Rev 22 (2002) 146-167), which is a strong validation of the fact that the identified ITB2 epitope is a potential marker of RA.

Phosphatidylinositol 4.5-phosphate-3-kinase

Another epitope with moderate HLA-DRB1*0401 binding site was identified in two serum samples of patients with neurosignal RA: 17-dimensional PI3K (792-808; SEQ ID NO: 60) with the amino acid sequence of the NKVFGEDSVGVIFKNGD, selected from phosphatidylinositol-3-kinase (PI3K) (table 1). In the specified peptide 794V may function as a hydrophobic anchor amino acids PI, 797E - negatively charged anchor amino acids P4, a 799S - normal anchor amino acids DR4-P6 (a measure of binding calculated using TEPITOPE, 2%).

PI3K is widely expressed by cells of many types and phosphorylate lipids, mainly phosphatidylinositol-4,5-phosphate. This enzyme acts as a key transducer signal for receptor survival factors, including growth factors, cytokines and integrins (review Toker, A. & Cantley, L., Nature 387 (1997) 673-676). In addition, PI3K plays an important role in the transmission path of the signal Toll receptors (TLRs), which recognize a wide range of microbial products, summarized in the term pathogen-associated molecular products (PAMPs) (Fukao, T. & Koyasu, S., Trends Immunol 24 (2003) 358-363). Stimulation of TLRs with PAMPs, such as lipopolysaccharide (LPS) (endotoxin), triggers the synthesis of various cytokines, including IL-2, which is a key cytokine in TLR-mediated Thi responses. It has been shown that PI3K is an endogenous suppressor of TLR-mediated synthesis of IL-2 and restricts excessive movement Thi. Thus, an assumption was made that PI3K is a negative regulator of the natural immune response that prevents developed the e prolonged active immunity, which may be harmful to the host body. The information that autoimmune responses in RA based on the Th1 cytokines, allows binding of PI3K with the pathogenesis of RA. Although specific pathogen directly associated with the development of RA, hitherto unknown, there is reason to assume that RA develops in two stages, with the primary response, induced by foreign antigens, consistently goes into a self-sustaining autoimmune response (Klinman, D., Arthritis Rheum 48 (2003)590-593).

Plasminogen activator urokinase type

Another epitope defined only in samples from patients with neurosignal RA, was isolated from plasminogen activator urokinase type (uPA): 16-dimensional uPA (328-343; SEQ ID NO: 61) with the amino acid sequence YPEQLKMTVVKLISHR (table 1). Based on the definition of the HLA-DRB1*0401 binding, the sequence contains moderate binding site: 332L, T and 337V represent the estimated anchor amino acids P1, P4 and P6, respectively (figure bind obtained using TEPITOPE, 2%).

Plasminogen activators (PAs) are highly specific serine protease responsible for the conversion of plasmin in the plasminogen. Two types of PAs, urokinase type (uPA) and tissue-type (tPA), have been identified in mammals. The activity of PAs is controlled by partnerincrime (PAIs). The plasmin is involved in inflammatory reactions involving inducing cytokines, such as TGFβ, as well as in the degradation of cartilage and fibrin. The signs of constant coagulation in the joint affected by RA, together with increased synthesis of uPA and its activity in articular tissues allowed us to make the assumption that the system RA/plasmin associated with clinical severity of arthritis (review Busso, N. & Hamilton, J.A. Arthtritis Rheum 46 (2002) 2268-2279). On the basis of in vitro studies, it was found that in the inflamed joints of patients with RA there are several types of cells, in particular monocytes and fibroblasts, which can Express the PAs and PAIs, which, consequently, contributes to the improvement of these indicators in the in vitro samples. The activity of PAs (i.e. uPA) is stimulated by cytokines, such as IL-1 and TNFα, the regulation of which in the serum and synovial fluid of patients with RA increased. The above data prove that the uPA (328-343) can be estimated peptide marker of RA.

V-II (VH26) plot heavy chain immunoglobulin

One of the identified epitopes showing a very strong binding site of HLA-DRB1*0401, epitope is selected from a gene segment VH26 heavy chain immunoglobulin: 16-dimensional VH26 (95-110; SEQ ID NO: 62) with the amino acid sequence KNTLYLQMNSLRAEDT (table 1). the very high rate of binding (1%), obtained using TEPITOPE, confirmed by the fact that the role of a powerful anchor P1 performs 99Y, moderate anchors P4 - M and function of a typical HLA-DR4 P6 anchors performs 104S.

Immunoglobulins (Ig) is responsible for binding to the antigen and stimulate additional immune response, for example, by linking with the isotype-specific Fc receptors. Interestingly, the gene segment VH26 man, apparently encodes vysokoavidnyh synovial rheumatoid factor and, thus, influences the progression of RA (Wong, A. et al., Autoimmunity 20 (1995) 191-199). The fact that the identified epitope VH26 (95-110) was detected in serum in two seropositive patients with RA (table 1), correlates with the above observation.

DJ-1 protein

In two samples neurosignal RA using long-term studies were identified epitope, which is separated from the protein, called DJ-1: 16-dimensional DJ-1 (135-150; SEQ ID NO: 63) with the amino acid sequence NGGHYTYSENRVEKDG (table 1). According TEPITOPE counting the peptide contains a relatively weak HLA-DRB1*0401 - binding site with 139Y in the role of the P1 anchor 124 S role in P4 anchors and 144N in the role of possible P6 anchors (figure binding: 8%).

DJ-1 belongs to the family of ThiJ/PfpI protein whose members are in the process of evolution has been transferred from Archaea to Eukarya. ThiJ/PfpI proteins have a common ThiJ domain, that its structure can be carried is N. the domain glutamylcysteine type I (Lee, S.J. et al., J Biol Chem 25 (2003) Epub ahead of print).

DJ-1, which is expressed mainly by the tissues of the testes and to a lesser extent the tissues of other organs, was first identified as a new potential carcinogenic product that transforms NIH3T3 cells of mice together with ras. Meanwhile, were discovered additional physiological functions of DJ-1, including the existence of a strict correlation with Parkinson's disease and fertilization of sperm. It is assumed that DJ-1 has many functions - authors of the present invention were first presents data on the relationship of DJ-1 with RA.

Example 2

This example uses the same methodology as that described in Example 1. To identify potential markers that are specific against erosive RA, use a serum (six samples) and synovial fluid (two samples) from patients diagnosed with erosive RA.

Peptide sequences detected in samples from patients with erosive RA, compared with sequences identified in untreated DCs (control 1) and DCs treated with serum of healthy people (control 2). Among the RA-specific sequences for further analysis selected only those that are re-identified in at least three of the six samples erosive RA.

In this study, was found od the h epitope, which is identified with one exception, only in samples erosive RA.

Apolipoprotein B-100

The epitope which is mainly detected in the serum of patients with erosive RA (in four samples RA of eight) was isolated from apolipoprotein b-100: 16-dimensional Ares (227-2892) with the amino acid sequence SEQ ID NO: 4 (table 2). In addition, identify the option specified epitope in length (table 2): 17-dimensional Ares (2877-2893; SEQ ID NO:5). It can be assumed that the following DRB1*0401-binding site: 288L as P1 anchors, 288D as P4 anchors and 2886N as P6 anchors (figure bind 3%).

In early studies, EBV-B cells was detected communication epitope Ares (2885-2900), which overlaps with the epitope described herein with HLA-DR4 (Chicz, R. et al., J Exp Med 178 (1993) 27-47).

Apolipoprotein B-100 is part of lipoproteins very low density lipoproteins (VLDL) and low density lipoprotein (LDL) and performs the role of signal detection for cellular binding and capture of LDL particles Ares/E receptor (Yang, C.Y. et al., Nature 323 (1986) 738-742). Interestingly, the increased ratio of LDL cholesterol to HDL cholesterol observed in patients with recently diagnosed RA (Park, Y.B. et al., J Rheumatol 26 (1999) 1701-1704). Disturbed lipid profile with active RA can be improved through the use of DMARDs without the use of antihyperlipidemic medicinalfunds (Park, Y.B. et al., Am J Med 113 (2002) 188-193). As they began to register with increased mortality from cardiovascular disease among patients with chronic inflammatory diseases such as RA (Symmons, D.P. et al., J Rheumatol 25 (1998) 1072-1077), an assumption was made that the local inflammatory process in RA can lead to disruption of blood lipid spectrum and, therefore, increase the risk of atherosclerosis. The question of how the components of lipid metabolism are the cause of the pathogenesis of RA, or they only affect what is happening in RA, the immune response, remains open. However, the fact that patients with RA is marked modified lipid profile, allows you to confirm the assumption that the epitope Ares can be a potential serum marker of RA.

In control samples from two healthy individuals was identified version of Ares (2877-2892) in length, but not Ares (2877-2893). Because Apolipoprotein In part 1% of all plasma proteins, the presence of Ares epitopes in the control samples is not surprising. The obtained results allow to assume that the only option Ares in length (2877-2893; SEQ ID NO: 5) is specific for erosive RA.

The 26S proteasome is not ATP-asna regulatory subunit 8

The epitope, which is also often defined in most of the samples the t patients with erosive RA, both in serum and in synovial fluid, separated from the 26S proteasome is not ATP-aznoe regulatory subunit 8 (PSMD8): 15-dimensional PSMD8 (218-232; SEQ ID NO: 64) with the amino acid sequence GPNNYYSFASQQQKP (table 2). Were also identified three additional option length: 16-dimensional PSMD8 (218-233; SEQ ID NO: 65), 17-dimensional PSMD8 (218-234; SEQ ID NO: 66) and 18-dimensional PSMD8 (218-234; SEQ ID NO: 67) (table 2). The presence of variants in length confirms the reliability of the identified epitope derived from molecules of MHC class II antigenic peptide. The peptide contains a plot of moderate binding DRB1*0401 (TEPITOPE score linking: 3%). Binding to HLA-DR4 can be confirmed by studies linking in vitro using synthetic PSMD8 (218-233) peptide and purified HLA-DR4 molecules (Figure 3). According to the value of the IC50(with respect to the reporter peptide (308-319), PSMD8 (218-233; SEQ ID NO:65) is associated with HLA-DR4 with moderate affinity, which confirms the data obtained using the TEPITOPE. 15-dimensional PSMD8 (218-232; SEQ ID NO: 64) was identified in one of the untreated control samples.

About 1% of the cytoplasmic protein pool is represented by the proteasome, which is involved in ATP-dependent degradation of ubiquitous proteins. In addition, the proteasome is responsible for the processing of certain transcription factors (e.g. nuclear factor-KB), pin is the role of the cell cycle and the generation of antigens restrictively on MHC class I Regulatory subunit of the proteasome is required to ensure the selectivity degradation of peptides. It is known that not ATP-asna regulatory subunit 8, in particular, necessary for activation of the protein 28 that controls cell division (Cdc28), and is an essential regulator of the cell cycle in yeast.

It is important that a significant increase in levels of circulating proteasomes (cProteasomes) detected in sera obtained from patients with various systemic autoimmune diseases, including RA (Egerer, K. et al., J Rheumatol 29 (2002) 2045-2052). Apparently, there is a close relationship between levels of circulating proteasomes and activity of the disease and the concentration of C-reactive protein in patients with severe RA. Discusses the fact that circulating proteasome are the triggering factors of subsequent immune responses, which indicates that the managed antigens mechanism. The concentration of released proteasome antigen, apparently, reflects the degree of cell damage in autoimmune diseases. On the basis of their discoveries Egerer et al. concluded that circulating proteasome may represent a new marker of disease severity in autoimmune processes. Because the epitope PSMD8 (218-232; SEQ ID NO: 64) was identified in the serum samples of patients erosive RA, research conducted by the authors of the present invention, confirm this conclusion.

The receptor for interleukin-1

Another potential marker of erosive RA is the receptor for interlekin-1 (IL-1R), which was identified in two serum samples of patients with erosive RA:IL-1R (79-94; SEQ ID NO: 68) with the amino acid sequence EKLWFVPAKVEDSGHY (table 2). IL-1R (79-94; SEQ ID NO: 68) 83F in the role of PI anchors, 86A in the role of P4 anchors and 88V role in P6 anchor comprises a strong binding site of the allele DRB1*0401, determining predisposition to RA (TEPITOPE score linking: 1%). In the study of binding in vitro, it was shown that a synthetic peptide binds with molecules of HLA-DR4 with high affinity (Figure 3), similar TO that in (309-319) peptide.

Interleukin-1 (IL-1) is a proinflammatory cytokine that is involved in a variety of infectious immune responses and immune response in RA and other diseases (Dinarello, C., Blood 87 (1996) 2095-2147). It binds with its specific receptor, which transmits a signal that triggers cell proliferation or stimulate protein synthesis. Increased production of IL-1 is observed in patients with RA and plays a key role in the clinical manifestation of this disease (Dayer, J.M. Rheumatology 42 (2003) ii3-ii19). IL-1 is considered as the main mediator in RA, which operates through the act is vazio macrophages and T and b lymphocytes.

In addition, IL-1 is involved in inflammatory reactions inducyruya the expression of cell adhesion molecules and other cytokines and chemokines. IL-1 is also one of the main factors involved in bone and cartilage destruction in RA, because it stimulates the formation of matrix metalloproteinases. Therefore, IL-1/IL-IR is the first target for anti-inflammatory therapy. The use of recombinant receptor antagonist IL-1 person (IL-1RA) has been approved for the treatment of patients with RA.

Fibronodular

In three samples from patients with erosive RA was detected epitope selected from the secreted matrix protein fibromyalia (FM): 13-dimensional FM (178-190; SEQ ID NO: 70) with the amino acid sequence LRELHLDHNQISR (table 2). In addition, it was identified by the 14-dimensional FM (177-190; SEQ ID NO: 69), who also once was detected in untreated control sample. The epitope has a strong DRB1*0401-binding site with 181L in the role of the P1 anchor, 184D in the role of P4 anchors and 186N role in P6 anchors (TEPITOPE score linking: 1%).

Fibronodular belongs to the family of small-enriched leucine proteoglycans (SLRPs), which are associated with TGFβ and collagen and other molecules of the extracellular matrix. In vitro studies have shown that SLRPs regulate fibrillogenesis of collagen, a process necessary for the development, tissue repair and metastasized who I am. In order to better understand the functions of SLRPs in vivo, get mice deficient in SLRPs; it was shown that these mice develop a wide range of diseases (such as osteoporosis and osteoarthritis), most of which are directly due to impaired collagen fibrillogenesis (Ameye, L. & Young, M.F., Glycobiology 12 (2002) 107R-116R). Since the processes of formation and degradation of collagen significantly activated in inflamed joints in RA, can be observed and increase the level fibromyalia. This assumption is also confirmed by the detection of epitope FM in RA samples of synovial fluid (in three of four samples of synovial fluid).

The receptor for GM-CSF/IL-3/IL-5

In five of the eight samples from patients with erosive RA can identify the epitope of the β-chain of multiple receptor cytokines (CYRB): 15-dimensional CYRB (359-373; SEQ ID NO: 71) with the amino acid sequence ETMKMRYEHIDHTFE and its variant in length, 17-dimensional CYRB (359-375; SEQ ID NO: 72) (table 2). In the study of binding in vitro, it was shown that a synthetic peptide CYRB (359-375) binds with molecules of HLA-DR4 with moderate affinity (Figure 3). This corresponds to data obtained using TEPITOPE, which testify to the presence in the composition of the epitope moderately linking DRB1*0401 plot (TEPITOPE score linking: 3%). The epitope is clearly in excess was present in samples of RA and thus was detected in only one serum sample of a healthy person.

The receptor for GM-CSF/IL-3/IL-5 is a membrane protein type I, which is differentially expressed cell system disorders (review Geijsen, N. et al., Cytokine Growth Factor Rev 12 (2001) 19-25). Its ligand, GM-CSF, secreted CD4+cells and is an important stimulus for the formation of dendritic cells from precursor cells in the bone marrow. The important role of dendritic cells in the initiation and development of immune response allows to make the assumption that they also play an important role in the development of autoimmune inflammatory diseases such as RA, conveying autoantigen to the draining lymph node, where DCs are found with "untrained" T-cells and primesouth them. It was shown that the activity of GM-CSF is associated with proinflammatory effects in RA, which confirms the assumption that the identified epitope of the receptor CYRB (359-373; SEQ ID NO: 71) is a potential marker for the diagnosis of RA.

Sorting nexin 3

Another epitope, which is probably indicative of RA, isolated from the protein, referred to as sorting nexin 3 (SNX3): 16-dimensional SNX3 (142-157; SEQ ID NO: 73) with the amino acid sequence HMFLQDEIIDKSYTPS (table 2). Amino acids 144F, 147D and 1491 specified epitope can perform the functions P1, P4 and P6 anchor amino acids, respectively, in peptidebased the groove allele DRB1*0401, defining Pedras loannot to RA (TEPITOPE score linking: 2%).

Sorting nexin is a diverse group of cellular proteins transfer that share the phospholipid-binding site (review Worby, S.A. & Dixon, J. C., Nat Rev Mol Cell Biol 3 (2002) 919-31). The ability of these proteins to bind specific phospholipids, as well as their tendency to form protein complexes indicates their involvement in the regulation of migration through the membrane and sorting of proteins. Sorting nexin 3, in particular, is present in the cytosol and the endosomes and, apparently, takes part in the regulation of migration through the membrane from early endosomes to return to the endosomes. Does the sorting nexin 3 a role in the pathogenesis of RA, for example, influencing the route of antigen presentation, are currently unknown. Identification of the epitope SNX3 (142-157; SEQ ID NO:73), this study suggests there is a relationship between sorting by nexiam and autoimmunization.

Example 3

All peptide sequences identified in examples 1 and 2 in samples from patients with neurosignal and erosive RA, in the present example is used to search for common markers significant for both types of RA. RA-specific sequence again compared with the peptide sequences of the control samples (untreated DCs and DCs treated with serum of two healthy people) and to further the Academy of Sciences of the Lisa selected only those peptides, who repeatedly detected in at least three of the fifteen samples of RA (erosive and neurosurge RA).

Inhibitor, inter-alpha-trypsin

In ten of eleven serum samples (erosive and neurosignal RA) is detected epitope allocated from the heavy chain H4 inhibitor, inter-alpha-trypsin: ITIH4 (271-287) with the amino acid sequence SEQ ID NO: 8 (table 3). In addition to the specified long option ITIH4 epitope, it is possible to identify six variants of the same ITIH4 epitope length: 19-dimensional ITIH4 (271-289; SEQ ID NO: b), 18-dimensional ITIH4 (271-288; SEQ ID NO: 7), 16-dimensional ITIH4 (274-289; SEQ ID NO: 12), 15-dimensional ITIH4 (273-287; SEQ ID NO: 10), 15-dimensional ITIH4 (274-288; SEQ ID NO: 11) and 14-dimensional ITIH4 (274-287; SEQ ID NO: 9).

When analyzing the short version ITIH4 (274-287) it was found that the epitope is composed of a strong binding site of the allele DRB1*0401, determining predisposition to RA: 277F performs the function P1 anchors, 280D - P4 anchors and 282S - P6 anchors (figure binding: 1%).

ITIH4 belongs to the family of inter-alpha (IαI) inhibitors, which are a group of inhibitors of serine proteases that are associated with hyaluronic acid (and, apparently, involved in acute phase reactions (Salier, J.P. et al., Biochemical Journal, 315 (1996) 1-9).

Is a polysaccharide found in all body tissues, particularly in loose connective tissue, for example in sousta is Noah fluid (Evered, D. & Whelan, J., eds., The Biology of Hyaluronan, John Wiley & Sons (1989)). ON has an important structural function in cartilage and other tissues, where it stabilizes the extracellular matrix, forming aggregates with proteoglycans. It was also found that perform important biological functions, which include the regulation of cell activity by binding to proteins on the cell surface, such as CD44 and ICAM-1 (Knudson, S. C. & Knudson, W., FASEB J 7 (1993) 1233-1241; Hall, C.L. et al., J Cell Biol 126 (1994) 575-588). RA is accompanied by a significant increase in the overall level in synovial fluid and in the serum; this is based on the assumption that circulating ON comes from the affected rheumatoid arthritis joints (Engstrom-Laurent, A. et al., Scand J Clin Lab Invest 45 (1985) 497-504).

Systems and some members of the family IαI in a significant amount determined in synovial fluid of patients with RA (Jessen, I.E. et al., Biological Chemistry Hoppe-Seyler 375 (1994) 521-526). The role of IαI complex in inflammatory reactions may consist in the modification of the interaction of the CD44-HA that mediates activation and invasion of leukocytes (Isacke, S.M. & Yarwood, H., hit Biochem Cell Biol 34 (2002) 718-721). In addition, the synovial fluid of patients with RA contains a large amount of TSG-6, an anti-inflammatory glycoprotein belonging to the family of hyaladherin that relate to ON-binding proteins (Winiewski, H.G. et al., J Immunol 151 (1993) 6593-6601). It was shown that the complex TSF-6 and family members IαI inhibits the activity of plasmin, a Central molecule involved in the activation of enzymes associated with inflammation (Wisniewski, H.G. et A1., J Immunol 156 (1996) 1609-1615). Regulation of activity of plasmin some plasma acute phase proteins, namely TSG-6 and protein from the family of IαI, can play an important role in the pathogenesis of RA and to lead to higher levels, TSG-6 and protein from the family of IαI in the synovial fluid of inflamed joints.

These observations, as well as identification of multiple variants of the same epitope on the length and the presence of a strong HLA-DR4-binding site is convincing evidence in favor of that present in the serum samples epitope ITIH4 is a marker of RA.

Complement C4

In eight of the eleven samples of RA sera (erosive and neurosignal RA) was identified by another dominant epitope allocated from the complement C4: 15-dimensional C4 (1697-1711) with the amino acid sequence SEQ ID NO: 13 (table 3). You can also find five options specified epitope length (table 3): a 12-dimensional C4 (1697-1708; SEQ ID NO: 18), 13-dimensional C4 (1698-1710; SEQ ID NO:17), 14-dimensional C4 (1697-1710; SEQ ID NO: 15). 16-dimensional C4 (1697-1712; SEQ ID NO: 14) and 18-dimensional C4 (1697-1714; SEQ ID NO: 16). Moreover, the epitope has a very strong DRB1*0401-binding site: 1700Y performs the function of the 1 anchor, 170D - P2 anchors and 170N - P6 anchors (figure binding: 1%).

C4, approximately 0.5% of the protein mass of the plasma plays a major role in the activation of the Central cascade reactions of the complement system. The protein is synthesized as single-chain precursor and before the secretion of the enzyme cleaved with the formation of a trimer of identical α-, β - and γ-chains. The identified epitope C4 (1697-1711) is located at the far C-terminal site of the γ-chain. α-chain C4 advanced proteoliticeski cleaved activated C1 with the formation Sa anaphylaxia, which is a mediator of local inflammatory reactions (Moon, K.E. et A1., J Biol Chem 256 (1981) 8685-8692).

In General, complementry cascade is involved in the induction and progression of inflammatory reactions and is a major defense system against various pathogenic agents, including bacteria, viruses and other antigens (Morgan, B.P., Methods Mol Biol 150 (2000) 1-13). Inadequate activation, however, can lead to tissue damage and the manifestation of the disease (Speth, .et al., Wien Klin Wochenshr 111 (1999) 378-391).

It was repeatedly reported that activation of the complement system has a role in the pathogenesis of RA; the basis for such claims is the detection of elevated levels of metabolites comlement, including C4 and Sa, plasma, synovial fluid and synovial TC is neither patients with RA (Neumann, E. et al., Arthtritis Rheum 46 (2002) 934-945). In addition, collagen-induced arthritis (CIA) in mice is characterized by the presence of the products of the activation of complement (Linton, S.M. & Morgan, B.P., Mol. Immunol 36 (1999) 905-914). The development of CIA can be prevented by using anti-C5-monoclonal antibodies (Wang, Y. et al., PNAS 92 (1995) 8955-8959) or with soluble CR1, inhibitor system comlement entered using gene therapy (Dreja, H. et al., Arthritis Rheum 43 (2000) 1698-1709). Activation of factors comlement joints induce may present different immune complexes; an assumption was made that the internal stimulation of the immune system by infectious agents and cytokines can influence the initiation of RA (Friese, M.A. et al., Clin Exp Immunol 121 (2000) 406-414).

Two of the six epitopes C4, 15-gauge and 18-gauge, were also identified in the two control samples (table 3), suggesting that only some of the options specified epitope C4 in length are RA-specific, namely antigenic peptides with SEQ ID NOs: 14, 15, 17 and 18.

Complement C3

Another epitope, which was detected in samples of erosive and neurosurge RA, separated from complement C3 (alpha chain): 14-dimensional C3 (1431-1444) with the amino acid sequence SEQ ID NO: 21 (table 3). Also in the serum were identified six variants of the same epitope length (table 3): 13-dimensional C3 (1431-1443; SEQ ID NO: 23), 14-dimensional C3 (1429-144; SEQ ID NO: 74), 15-dimensional C3 (1431-1445; SEQ ID NO: 22), 15-dimensional C3 (1429-1443; SEQ ID NO: 20), 17-dimensional C3 (1427-1443; SEQ ID NO: 75) and 19-dimensional C3 (1426-1444; SEQ ID NO: 19). When analyzing the short version, C3 (1431-1443), was installed next DRB1*0401-binding site: 1434Y in the role of the P1 anchor, 1437D in the role of P4 anchors and A role in P6 anchors.

In samples of erosive and neurosurge RA was also found additional epitope allocated from complement C3 (α-chain): 19-dimensional C3 (157-175) with the amino acid sequence SEQ ID NO: 76 (table 3). One additional option along the length of the same epitope was identified in the serum (table 3): 20-dimensional C3 (157-176; SEQ ID NO: 77).

Complement C3 of approximately 1-2% of the total protein mass in plasma, plays a major role in the activation system complement and belongs to the family of acute phase proteins. Its processing using C3 convertase with the formation of C3a anaphylaxia and C3b is the Central element of both classical and alternative pathway activation of complement (Barrington, R. et al., Immunol Rev 180 (2001) 5-15). After activation, C3b can covalently to contact (via jet tiefer) with carbohydrates on the surface of cells or immune aggregates (Isaac, L. & Isenman, D.E., J Biol Chem 267 (1992) 10062-10069). The identified epitope C3 (1431-1444) is located at the C-terminal stretch of C3b.

As already discussed in relation to the epitope of complement C4 (1697-1711), appears the sun is more evidence, what system components comlement play an important role in the pathogenesis of RA. The results of this study, in which serum of patients with RA were identified two major epitope selected from the complement C3 and C4, underscore the close link between activation of the system complement and pathogenesis of RA. This coincidence is a strong argument in favor of the present C3/C4 epitopes are potential serum markers of RA.

Protein 3 similar to the enriched glutamic acid-binding SH3-domain protein

Another apropos, which is frequently detected in the serum of patients with RA (in six of the eleven samples erosive and neurosurge RA), epitope is allocated from protein 3 similar to the enriched glutamic acid-binding SH3-domain protein (SH3BGRL3): SH3BGRL3 (15-26) with the amino acid sequence SEQ ID NO: 25 (table 3). It was also identified three options specified epitope length (table 3): 14-dimensional SH3BGRL3 (13-26; SEQ ID NO: 26), 14-dimensional SH3BGRL3 (15-28; SEQ ID NO: 27) and 16-dimensional SH3BGRL3 (13-28; SEQ ID NO: 24). DRB1*0401-binding site is represented: 171 the role of the P1 anchor, 20Q in the role of P4 anchors and 22S in the role of P6 anchors (figure bind 4%).

SH3BGRL3 is a protein with a mol. mass CD, which belongs to the family of SH3BGR. The intended function of the protein is unknown, but noted its rolein as a modulator of biological activity glutaredoxin (Mazzocco, M. et al., Biochem Biophys Res Commun 285 (2001) 540-545). Thus, SH3BGRL3 not described in relation to RA.

It is interesting to note that the identified second epitope of the same protein, which visocosity in all samples of RA and control samples: 16-dimensional SH3BGRL3 (29-44) with the amino acid sequence DGKRIQYQLVDISQDN. There were numerous ranging in length variants of this epitope in many samples. When studying the shortest variant, SH3BGRL3 (31-42), it was found that the epitope contains almost similar with DRB1*0401 anchor residues compared to SH3BGRL3 (15-26): 331 serves as a P1 anchor, 36Q - as P4 anchors and 38V - as P6 anchors (by binding - 2). This similarity is confirmed by a comparative account of the binding.

The second epitope SH3BGRL3 confirms the reliability of the epitope SH3BGRL3 (15-26), since both peptides are derived from the same protein, but only one of them, namely the epitope SH3BGRL3 (15-26), apparently, is generated RA-specific way. A similar observation has already been described for GILT in Example 1.

Among the four different length options SH3BGRL3 most dlinnorazmernyh option, SH3BGRL3 (13-28), also identified in the sample of healthy individuals (table 3). However, this option is of a certain length were found only once, indicating a significant enrichment of apito the om SH3BGRL3 in RA.

Induced by interleukin-4 (IL-4) protein 1

In all investigated synovial fluids (erosive and neurosignal RA) and in eight of the 11 sera (erosive and neurosignal RA) identified only highly dominant epitope derived from homologous human induced IL-4 protein 1(Fig1): Fig1 (293-309) with the amino acid sequence SEQ ID NO: 28 (table 3): 16-dimensional Fig1 (293-308; SEQ ID NO: 30) 19-dimensional Fig1 (293-311; SEQ ID NO: 29). Moreover, amino acid sequence detects typical DRB1*0401 binding site: 299V serves as a P1 anchor E as P4 anchors and 304S as P6 anchors (by the binding of 1%).

Two different length variants of the same epitope, Fig1 (293-308) and Fig1 (293-309) were identified in one unprocessed sample and one control sample from a healthy person (table 3). However, the presence of epitope Fig1 almost all samples of RA, but not in all control samples strictly indicates the enrichment of RA.

Gene fig1 person first identified in stimulated IL-4 IN cell cultures (Chu, C.C. & Paul, W.E., PNAS 94 (1997)) 2507-2512). This gene is localized on chromosome 19q13.3-19q13.4, on the site, for which the previously shown a role in the development of sensitivity to autoimmune diseases, including systemic lupus erythematosus, arthritis, multiple sclerosis and the Sulin-dependent diabetes mellitus (Becker K.G. et al., PNAS 95(1998) 9979-9984). Because the expression of the indicated gene is significantly restricted immune tissues and its regulation depends on IL-4, a key modulator of the immune response, fig1, therefore, is the most likely candidate for the role of the gene that determines susceptibility to autoimmune diseases (Chavan, S.S. et al., Biochim Biophys Acta 1576 (2002) 70-80). Limited by HLA-DR4 presentation of the epitope Fig1 shows, first, that the protein Fig1 is produced and possibly involved in the development of RA. Polypeptide Fig1 was not known as a marker of RA up to the present time and it is believed that he is one of the most important candidates for the specified role.

Hemopexin

Another candidate for the role of marker RA, frequently detected in the serum samples (10 out of 11 samples) and samples of synovial fluid (two of the 4 samples) (erosive and neurosignal RA), is derived hemopexin (HPX): HPX (351-367) with the amino acid sequence SEQ ID NO: 32 (table 3). Found some different length options, which confirm the importance of the specified epitope (table 3): 13-dimensional HPX (351-363; SEQ ID NO: 33), 14-dimensional HPX (350-363; SEQ ID NO: 34), 15-dimensional HPX (351-365; SEQ ID NO: 35), 18-dimensional HPX (351-368; SEQ ID NO: 31) and 18-dimensional HPX (350-367; SEQ ID NO: 78). Moreover, the epitope contains very strong binding DRB1*0401 plot: 3551 serves as a P1 anchor, 358D - as P4 anchors and 360V as P6 anchors (by the binding of 1%).

Two different length variants of the same epitope, namely HPX (351-367; SEQ ID NO: 32) and HPX (351-365; SEQ ID NO: 35), can also be identified in the control samples of healthy persons (table 3). This suggests that only the options of a certain length specific for RA, namely antigenic peptides SEQ ID NOs. 31, 33, 34 and 78.

HPX is a glycoprotein in the blood plasma with mol. mass of 60 KD with high binding affinity to GEMU (Muller-Eberhard, U., Methods Enzymol 163 (1988) 536-565). It is mainly expressed in the liver and belongs to the acute phase proteins, the synthesis of which is induced in inflammatory processes. Rheumatoid arthritis is a chronic inflammatory autoimmune inflammatory disease characterized by elevated levels of acute phase proteins, including C-reactive protein and serum amyloid A (Nakamura, R., J Clin Lab Anal 14 (2000) 305-313). HPX is responsible for the cytokines IL-1 and IL-6, which are regulated elevated in patients with RA (Feldmann, M. &Maini, R.N., Rheumatology 38, Suppi 2 (1999) 3-7).

HPX is the main carrier for the transport of heme in plasma, and its principal role is to prevent indirect gem oxidative stress and loss associated with gem iron (Tolosano, E. & Altruda, F., DNA Cell Biol 21 (2002) 297-306). It can protect cells from oxidative stress and dotirovanie expression of intracellular antioxidants, such as oxygenase heme, metallothionein and ferritin. Metallothionein and cytosolic proteins expressed primarily in synovial fibroblasts (Backman, J. T. et al., Virchows Arch 433 (1998) 153-160). There is considerable experimental evidence in favor of the existence of oxidative stress in synovial tissues of patients with RA (see review Schett, G. et al., Arthritis Res 3 (2000) 80-86). Moreover, it was reported that HPX induces proliferation of T-lymphocytes person (Smith, A. et al., Exp Cell Res 232 (1997) 246-254). These data support the likelihood that HPX belongs to proteins, which are regulated elevated in serum and synovial fluid of patients with RA, testifying in favor of HPX (351-367) is a candidate for the role of RA-specific marker.

Hsc70-interactive protein

The epitope which is mainly identified in serum samples (4 out of 11 samples) (erosive and neurosignal RA) and which is also associated with response to stress, is of Hsc70-interactive protein Hip: Hip (83-98) with the amino acid sequence SEQ ID NO: 38 (table 3).

Identified two different length options specified epitope (table 3): 18-dimensional Hip (83-100; SEQ ID NO: 36) and 15-dimensional Hip (84-98; SEQ ID NO: 39). Identified additional option in one sample of synovial fluid (table 3): 15-dimensional Hip (85-99; SEQ ID NO: 37). When the teachings of the shortest variant Hip (84-98) was established, the epitope contains binding DRB1*0401 plot, giving by the binding of 8%: 891 serves as a P1 anchor, 92D as P4 anchors and 94D - as P6 anchors.

In the cytosol of eukaryotic cells proteins Hip and holes associated with Hsc70 to participate in the regulation chaperone activity of Hsc70 (Frydman, J. & Hohfeld, J., Trends Biochem Sci 22 (1997) 87-92). Protein Hip mol. mass of 42 KD is associated with the ATPase domain of Hsc70. Postulated that the Hip may increase the half-life of the complex chaperon the substrate, creating a molecular basis for effective interaction of Hsc70 with adjustable step-down image of hypernovae system. It is shown that Hsc70 and Hsc90 interact during Assembly of the protein in vitro (Jakob, U. & Buchner, J., Trends Biochem Sci 19 (1994) 205-211; Freeman, B.C. & Morimoto, R.I., EMBO J 15 (1996) 2969-2979) and play a role in the denaturation temperature (Schneider, .et al., PNAS 93 (1996) 14536-14541). The fact that Hsc70 and Hsc90 associated with adaptation to stress, ambiguous between Hip with responses to stress, including the induction of heat shock proteins in synovial tissue of patients with RA (see review Schett, G. et al., Arthritis Res 3 (2001) 80-86).

Binding properties ITIH4. C4. C3. SH3BGRL3. Fig1. HPX and Hip

To further study the binding properties described above antigenic peptides ITIH4, C4, C3, SH3BGRL3, Fig1, HPX and Hip with molecules HLA-DR carry out research associate in vitro (Figure 3): in accordance with their values IC50 against the reporter peptide (293-309) synthetic peptides ITIH4 (274-287), SH3BGRL3 (13-26) and Fig1(293-309) associated with HLA-DR with high affinity (Figure 3). Moderate binding to HLA-DR is determined for synthetic peptides C4 (1696-1709) and HPX (351-365). Synthetic peptide C3 (1431-1444) binds weakly to HLA-DR, which is consistent with the TEPITOPE score (9%). Do not define the binding for peptide Hip (84-98) (data not shown).

Invariant chain (Ii)

Among all candidates for marker peptides, which, apparently, can be indicators of erosive and neurosurge RA, there epitope, which comes from associated with HLA-DR invariant chain (Ii): 15-dimensional Ii (110-124; SEQ ID NO: 83) with the amino acid sequence ATPLLMQALPMGALP (table 3). Moreover, identified four different length options specified epitope: 16-dimensional Ii (109-124; SEQ ID NO: 81), 17-d-Ii (109-125; SEQ ID NO: 80), 18-gauge Ii (109-126; SEQ ID NO: 79) and 21-d-Ii (109-129; SEQ ID NO: 82) (table 3). The presence of different length variants corresponds to the identified peptide sequence detecting section moderate binding DRB1*0401 (account binding TEPITOPE 5%), and 114L serves as a P1 anchor, 117A - as P4 anchors and R as P6 anchors. The epitope was identified in two samples of erosive and three samples neurosignal RA. Two different length options Ii was detected in the untreated sample and two samples from healthy and is dividuum.

The peptide-loaded MHC molecules of class II controlled by two auxiliary molecules of the path class II, namely the invariant chain Ii and HLA-DM (see review Bakke, O. & Nordeng T.W., Rev Immunol 172 (1999) 171-187 and Kropshofer, H. et al., Immunol Today 18 (1997) 77-82). Trimers Ii contact maturing molecules MHC class II in the rough endoplasmic reticulum (ER) and block the peptide-binding groove, stabilizing molecule MHC class II and preventing ligand binding, available in AYR. Complexes of molecules of class II/Ii" are transported via the Golgi to the endosome, where Ii is cleaved with the formation of the nesting set associated with molecules of the class II peptide Ii (CLIP). The release CLIP allows endosomal peptides to contact with molecules of MHC class II, which are the receptors of T cells from the cell surface. The identified epitope Ii (110-124) overlaps with the C-end section of the CLIP. It is interesting to note that described in the literature reduced trunk CLIP interaction with RA-associated alleles HLA-DR (Patil, N.S. et al., J Immunol 167 (2001) 7157-7168), indicates that this interaction may contribute to the pathophysiology of autoimmunity in RA.

Responding to retinoic acid receptor protein 2

In four of the eleven serum samples (erosive and neurosignal RA) was identified epitope originating in responding to retinoic acid receptor protein 2 (RARRES2):22-dimensional RARRES2 (40-61; SEQ ID NO: 86) with the amino acid sequence HPPVQWAFQETSVESAVDTPFP (table 3). In the C-terminal site of the epitope can be identified by two different length options: 23-dimensional RARRES2 (40-62; SEQ ID NO: 84) and 24-dimensional RARRES2 (40-63; SEQ ID NO: 85) (table 3). 45W serves as PI anchors, 48Q - as P4 anchors and 50T - as P6 anchors; an epitope found the plot a moderate binding DRB1*0401 (account binding TEPITOPE is 3%).

RARRES2 is a small protein with a mol. mass 18, 6 KD, which is mainly expressed in the endothelium and the epidermis. Expression, apparently, is gormonzawisimah and identified responses to retinoic acid in the skin and some Osteuropa hormones originating in bone marrow stromal cells (Nagpal, S. et al., J Invest Dermatol 109 (1997) 91-95 and Adams, A.E. et al., J Cell Biochem 74 (1999) 587-595). Function RARRES2 virtually unknown, and its Association with RA, for example, due to the weakening of osteoclastogenesis is assumed.

Fibronectin

Other HLA-DR4-associated peptide that selectively detected in three of four samples of synovial fluid (erosive and neurosignal RA), is derived from fibronectin (Fn), a major glycoprotein of blood plasma and extracellular matrix: a 15-gauge Fn (1881-1895; SEQ ID NO: 90) with the amino acid sequence IYLYTLNDNARSSPV (table 3). Epitope detects very good linking the DRB1*0401, and 1885Y serves as a hydrophobic P1 anchors, 188N as P4 anchors and 1890N as P6 anchors (by binding TEPITOPE is 1%). Additionally identified four different length options: 16-dimensional Fn (1881-1896; SEQ ID NO:91), 16-gauge Fn (1880-1895; SEQ ID NO:88), 17-d Fn (1881-1897; SEQ ID NO:89) and 17-gauge Fn (1880-1896; SEQ ID NO:87), which is strictly confirms the significance of the identified Fn-epitope (table 3).

Fibronectin plays an important role in cell adhesion, cell motility and opsonization. It binds collagen and fibrin and mediates the adhesion of fibroblasts to collagen fibrils. Fibronectin is expressed strictly in lining the synovial cavity layer in patients with RA and osteoarthritis and correlated with hyperplasia of diseased joints. Identification of the epitope Fn (1881-1895) only in samples of synovial origin is consistent with these data and suggests a possible role vysokodoznogo fibronectin in the development of autoimmunity.

Cathepsin

In three of the fifteen samples of RA (erosive and neurosignal RA) is detected epitope derived from cathepsin B (CatB): 15-dimensional CatB (227-241; SEQ ID NO: 92) with the amino acid sequence YNSYSVSNSEKDIMA (table 3). Epitope detects moderate binding to DRB1*0401, and 230Y serves as a hydrophobic P1 anchor serine residues at positions 233 and 235 as possible P4 and P6 anchors (account is wyzwania TEPITOPE is 6%).

Cathepsin represents a thiol of peptidase and, apparently, involved in intracellular degradation and turnover of proteins, such as collagen. It is localized in the lysosomes of cells of different types, including leukocytes. It is shown that cathepsin In contributes to the destruction of cartilage in osteoarthritis and pathological proteolysis when RA and cancer (Cunnane, G. et al., Arthritis Rheum 44 (2001) 1744-1753). The enzymatic activity of cathepsin b and other lysosomal peptidases correlates with the progression of RA, which confirms the importance of epitope CatB (227-241) as a possible marker peptide RA (Sohar, N. et al., Biol Chem 383 (2002) 865-869).

Tripeptidyl-peptidase II

The other originating from peptidases epitope was detected in three of the fifteen samples of RA (erosive and neurosignal RA), which comes from tripeptidyl-peptidases II (TRR): 15-dimensional TRR (970-984; SEQ ID NO: 93) with the amino acid sequence AGSLTLSKTELGKKA (table 3). Additionally reveals a great length option TRR (970-985; SEQ ID NO: 94). Peptidases epitope contains typical binding DRB1*0401 plot, and 973L, 976S and T serve as P1, P4 and P6 anchors respectively (account binding TEPITOPE is 3%).

Similar to cathepsin In Tripeptidyl-peptidase II is involved in the breakdown of lysosomal proteins. Identification of the epitope TRR (970-984) in the context of RA indicates, first of ocher is d', that TR may be involved in reducing protein degradation in inflammatory processes and RA.

Legumain

Legumain (LGMN) completes the set of three peptidases that are discovered in this study. The identified epitope LGMN (99-112; SEQ ID NO: 95) with the amino acid sequence VPKDYTGEDVTPQN (table 3) reveals a typical plot of binding to the allele HLA-DRB1*0401, and 103Y serves as a P1 anchor E as P4 anchor, a 108V - as P6 anchors (by binding TEPITOPE is 1%).

Legumain detected in endosomal and lysosomal fractions of antigen-presenting cells such as dendritic cells (Schwarz, G. et al., Biol Chem 383 (2002) 1813-1816). It is designed solely for the hydrolysis asparaginase links. It is shown that it plays an important role in the processing of bacterial antigens for MHC molecules class II (Manoury, B. et al., Nature 396 (1998) 695-699). Remains unknown, involved whether legumain in the development of autoimmune diseases such as RA, but the fact that three of lysosomal peptidases with GILT and 268-proteasome identified in the course of this study suggests that the mechanisms of cleavage of proteins for antigen processing can be significantly modified by RA.

The receptor of platelet activating factor

In three of the eleven serum samples from patients with RA (e is osuny and neurosignal RA) was identified epitope, originating from receptor of platelet activating factor (PAFR): 13-dimensional PAFR (264-276; SEQ ID NO: 96) with the amino acid sequence DSKFHQAINDAHQ (table 3). The epitope according to the account linking TEPITOPE (3%) found the plot a moderate binding DRB1*0401, and 267F serves as a P1 anchor A as P4 anchors and 272N as P6 anchors (table 3).

The platelet activating factor (PAF) is a proinflammatory lipid mediator that is associated with coupled with G-protein seventh transmembrane receptor on the surface of several cell types. By binding to the PAF receptor transduces pleiotropic functions, including motility of the cells, smooth muscle contraction, as well as the synthesis and release of cytokines (see review Honda, Z. et al., J Biochem 131 (2002) 773-779). Pharmacological studies and obtaining PAFR (-/-) mice suggests that the functions of PAF different and are involved in allergic reactions, inflammation, neuronal activity, reproduction and development of atherosclerosis. It is interesting to note that PAF found in elevated concentrations in the synovial fluid of patients with RA and shows its role in the induction of neoangiogenesis, which is often observed in rheumatoid synovitis (Lupia, E. et al., Eur J Immunol 26 (1996) 1690-1694). This further testifies to the fact that the epitope PAFR (264-276) is associated the data with RA marker peptide.

Poly-alpha-2.8-sialyltransferase

Another epitope, which was detected in three of the eleven serum samples (erosive and neurosignal RA), comes from polysialyltransferase (PST): 15-dimensional PST (333-347; SEQ ID NO: 97) with the amino acid sequence MPLEFKTLNVLHNRG (table 3). It contains moderately linking DRB1*0401 area (by binding TEPITOPE 2%), and 337F, 340L and 342 V serve as PI, P4 and P6 anchors, respectively.

Polivanova acid is a carbohydrate consisting of a linear homopolymer alpha-2,8-linked sialic acid residues. Glycan mainly associated with adhesion molecule neurons (N-CAM) and is involved in many processes of morphogenesis of neurons by modulating adhesion properties of N-CAM. Membrane protein polysialyltransferase catalyzes the polycondensation of residues of sialic acid and vysokoaktivnye in the brain, lungs and kidneys of the fetus, heart, spleen and thymus of the adult and less efficiently expressed in peripheral blood leukocytes (Nakayama, J. et al., Proc Nati Acad Sci (1995) 7031-7035). Elevated levels PST detected in the serum of patients with metastatic tumors, and increased activity of this enzyme is associated with rheumatoid arthritis (Berge, P.O. et al., Klin Wochenschr 60 (1982) 445-449). Identification of the epitope PST (333-347) in samples from patients with RA shows in section the pout all, that this enzyme may be involved in the development of RA.

Ras-related protein Rab-11B

Last epitope, which was detected in serum samples from patients with RA, comes from Ras-related protein Rab-11B:13-dimensional Rab-11B (51-63; SEQ ID NO: 102) with the amino acid sequence RSIQVDGKTIKAQ (table 3). The peptide sequence was confirmed by sensitivity of the binding of RA allele DRB1*0401 using TEPITOPE software product (Hammer, J. et al., Adv Immunol 66 (1997) 67-100). This epitope and four additional different length variants, namely 14-dimensional Rab-11 (50-63; SEQ ID NO:100), 15-dimensional Rab-11B (49-63; SEQ ID NO:98), 17-dimensional Rab-11B (49-65; SEQ ID NO:101) and an 18-dimensional Rab-11 (49-66; SEQ ID NO:99), detected in three samples from patients with RA (erosive and neurosignal RA) (table 3). 15-dimensional Rab-11B (49-63) was identified in one of the twelve control samples.

The Rab proteins are small GTP, which play an important role in membrane transfer as endoclitics and assoclation ways. Rab-11B plays a significant role in the transport of internalized transferrin from recyclingof compartment to the plasma membrane. Moreover, Rab-11B identified in rat osteoclasts and may play an additional role in bone resorption. Epitope Rab-11B (51-63) indicates that a Rab-11 involved in the development of RA.

img src="https://img.russianpatents.com/890/8908484-s.jpg" height="120" width="172" />

1. Antigenic peptides to MHC class II, which are markers of rheumatoid arthritis and predisposition thereto and comprising: a) at least the amino acid sequence peptidase plot SEQ ID NO. 49 or b) at least amino acid sequence peptidase plot SEQ ID NO. 49 with additional N - and C-terminal flanking sequences corresponding to the sequence that is selected from the group comprising SEQ ID NOs. 1-3.

2. Antigenic peptides to MHC class II according to claim 1, associated with the molecule MHC class II.



 

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FIELD: medicine.

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4 cl, 1 tbl, 1 dwg, 4 ex

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FIELD: biology, biotechnologies.

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FIELD: chemistry, biotechnology.

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1 dwg, 1 tbl

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2 tbl, 5 dwg, 2 ex

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2 tbl, 5 dwg, 2 ex

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19 cl, 13 dwg, 6 ex

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19 cl, 13 dwg, 6 ex

FIELD: organic chemistry, medicine, pharmacy.

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EFFECT: improved preparing methods, improved and valuable medicinal properties of substances and compositions.

48 cl, 3 tbl, 112 dwg, 5 ex

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