BR102013017639B1 - MIMETIC PEPTIDES TO AUTOANTIGENS AND THEIR USE IN IMMUNODIAGNOSIS OF JUVENILE IDIOPATHIC ARTHRITIS - Google Patents

Abstract

autoantigen mimetic peptides and their use in immunodiagnosis of juvenile idiopathic arthritis juvenile idiopathic arthritis (aij) is characterized by persistent arthritis of unknown cause that begins before 16 years of age. Currently, the diagnosis of aij is based on clinical history and detection of rheumatoid factor, which has low specificity, sensitivity and its prognostic value is controversial. Due to the need for new diagnostic methods, the present invention developed, from the dba1/j mouse line (a murine model of human arthritis), peptides that mimic autoantigens present in aij. these peptides were selected from libraries that display random peptides on the surface of phages, which bound to antibodies purified from the serum of mice with arthritis. the fourteen peptides obtained during the selection were able to discriminate patients with aij from healthy children and can potentially be used to build new platforms for the early detection of aij.

Description

[0001] The present invention relates to recombinant peptides that bind to antibodies present in the serum of patients with juvenile idiopathic arthritis. The selected peptides were specifically immunoreactive with the serum of children with juvenile idiopathic arthritis, efficiently discriminating healthy from patients and can be potentially used in immunodiagnostics, fluorescent or radioactive particle carriers for imaging diagnosis, drug carriers in the treatment and in vaccine compositions for the control of juvenile idiopathic arthritis.

[0002] Juvenile Idiopathic Arthritis is defined as a disease occurring before 16 years of age, primarily characterized by the presence of persistent arthritis in one or more joints, for at least 6 weeks, after excluding other causes. It is a chronic inflammatory disease that, if not treated early, can cause permanent damage (Ravelli A, Martini A. Juvenile idiopathic arthritis. The Lancet 369:767-778, 2007).

[0003] Juvenile Idiopathic Arthritis comprises a heterogeneous group of chronic arthropathies that affect approximately 1-3/1000 children (Murray KTSD, Glass DN. Pathogenesis of juvenile chronic arthritis: genetic and environmental factors. Archives of Disease in childhood 77:530- 534, 1997). In the first world, it is classified as the most common rheumatic disease in childhood and one of the most frequent chronic diseases in this youth group. The prevalence is 0.07 - 4.01 /1000 children and an overall incidence of 0.83 - 86.0/100,000 children per year, with a marked difference in incidence between different countries, also justified by the difficulty of diagnosis and by the different existing classifications (Oen K. Comparative epidemiology of rheumatic diseases in children. Current Opinion in Rheumatology 12:410-414, 2000. Manners PJ, Bower C. Worldwide prevalence of juvenile arthritis why does it vary so much? The Journal of Rheumatology 29:1520-1530 , 2002). In Brazil there is no epidemiological study, but Juvenile Idiopathic Arthritis is not a rare disease, it is the second rheumatic disease with the highest incidence in childhood, surpassed only by rheumatic fever (Alver M. et al., Prevalence of Rheumatic Fever in Children from Children from a Public High School in Belo Horizonte. Arquivos Brasileiros de Cardiologia 65:331-334, 1995. Terreri MT. et al., Resourse utilization and cost of rheumatic fever. Journal of Rheumatology 28:1394-1397, 2001).

[0004] Early diagnosis is exclusionary and includes seven different forms. However, it can be facilitated by recognizing the three major forms of presentation: oligoarticular (50%-60% of cases), polyarticular (30%-35% of cases) and systemic (10%-20% of cases). Soon after its onset, the course of JIA is uncertain, but this evolution becomes reasonably predictable after defining the patient's definitive clinical pattern (Cassidy JT. Juvenile Rheumatoid Arthritis. in Kelley's Textbook of Rheumatology, Company: Philadelphia p:1297- 1313, 2001. Cassidy JT. Juvenile Rheumatoid Arthritis, in Kelley's Texbook of Rheumatotology, Company: Philadelphia p:1579-1596, 2005).

[0005] There is agreement that Juvenile Idiopathic Arthritis is a disease of complex and multifactorial pathogenesis, resulting from the interaction of environmental and genetic factors. Among the numerous risk factors suggested for the onset of the disease, positive family history stands out (Clemens LE. et al., Sibling pairs affected by chronic arthritis of childhood: evidence for a genetic predisposition. The Journal of Rheumatology 12:108 -113, 1985. Prahalad S. Genetic analysis of juvenile rheumatoid arthritis: approaches to complex traits. Current Problems in Pediatric and Adolescent Health Care 36:83-90, 2006). Juvenile Idiopathic Arthritis has often been described as a complex genetic trait, where several genes interact to result in a specific phenotype (Glass DN, Giannini EH. Juvenile rheumatoid arthritis as a complex genetic trait. Arthritis & Rheumatism 42:2261-2268, 1999). In monozygotic twins, the concordance rate for JIA is around 25 to 40% when one of the twins is affected by the disease, and the prevalence of JIA among carrier siblings is 15 to 30 times higher than in the general population (Prahalad S, Glass DN. A Comprehensive Review of the Genetics of Juvenile Idiopathic Arthritis Pediatric Rheumatology Online Journal 6:11, 2008). Juvenile Idiopathic Arthritis subtypes share genetic and phenotypic characteristics with other autoimmune diseases, which are believed to be the result of the interaction of genetic and environmental factors. The identification of genetic factors associated with susceptibility or protection to autoimmune diseases is important for several reasons, such as identifying variants that have the potential to significantly improve our understanding of the pathogenesis of the disease, which in turn will lead to better diagnosis and classification of phenotypes. Furthermore, the discovery of new pathways involved in the pathogenesis of the disease may lead to new treatment modalities ( Angeles-Han S, Prahalad S. The Genetics of Juvenile Idiopathic Arthritis: What Is New in 2010? Current Rheumatology Reports 12:87-93 , 2010).

[0006] Recently, several studies have shown that clinically distinct autoimmune diseases have ordinary action of genetic susceptibility factors. For example, polymorphisms in the genes that encode HLA molecules are associated with several autoimmune diseases. Associations with HLA variants have been validated and confirmed in different populations of autoimmune diseases, such as Diabetes Mellitus Type I, Rheumatoid Arthritis, as well as JIA. However, HLA variants only explain part of the genetic susceptibility to many autoimmune diseases, suggesting that variations outside the HLA region might also contribute to disease susceptibility. For example, known variants in the major histocompatibility complex (MHC) have been reported to account for only about 30% of the genetic burden of Rheumatoid Arthritis (Plenge RM, Rheumatoid arthritis genetics: 2009 update. Current Rheumatololgy Reports 11:351-356, 2009) ). The major histocompatibility complex HLA-DR genes are associated with Rheumatoid Arthritis and participate in antigen presentation to T cells. The association of these genes with HLA-DR cells with Rheumatoid Arthritis is currently the strongest evidence of the role of genetics in the pathogenesis and frequency of disease (Angeles-Han S, Prahalad S. The Genetics of Juvenile Idiopathic Arthritis: What Is New in 2010? Current Rheumatology Reports 12:87-93, 2010). Similarly in Juvenile Idiopathic Arthritis, it has been estimated that HLA-DR represents only about 17% of the genetic burden of the disease, suggesting that other variants within and outside the MHC complex may play a role in susceptibility (Prahalad S. et al., Juvenile rheumatoid arthritis: linkage to HLA demonstrated by allele sharing in affected sibpairs. Arthritis & Rheumatism 43:2335-2338, 2000).

[0007] To date, the HLA complex variants and the PTPN22 locus have been confirmed to be indisputably associated with Juvenile Idiopathic Arthritis by several works. Recently, several other candidates have been added to the list of disease-associated loci, including STAT4, TNFAIP3, IL2RA, TRAF1/C5, and VTCN1 (Angeles-Han S, Prahalad S. The Genetics of Juvenile Idiopathic Arthritis: What Is New in 2010? Current Rheumatology Reports 12:87-93, 2010). It should be noted that different subtypes of this disease have different phenotypes, and it is extremely important that such subgroups be stratified in genetic studies (Macaubas C. et al., Oligoarticular and polyarticular JIA: epidemiology and pathogenesis. This review describes the epidemiology, genetics, and pathogenesis of JIA. Nature Reviews Rheumatology 5:616-626, 2009). However, the lack of adequate samples and consequent statistical power prevents the detection of important genetic associations with the subtypes of the disease. These challenges highlight the need for international multicenter studies to obtain well-phenotyped samples (Prahalad S. et al., Juvenile rheumatoid arthritis: linkage to HLA demonstrated by allele sharing in affected sibpairs. Arthritis & Rheumatism 43:2335-2338, 2000).

[0008] The ideal treatment of Juvenile Idiopathic Arthritis should start with the education of those responsible for the patient about the disease, about the risks and joint deformities and loss of functional capacity, as well as guidance on the risks and benefits of available therapeutic options (Kwoh CK. American College of Rheumatology Subcommittee on rheumatoid arthritis guidelines. Guidelines for the management of rheumatoid arthritis. Arthritis & Rheumatism 46:328-346, 2002). Therefore, the treatment of children with Juvenile Idiopathic Arthritis should focus not only on achieving optimal joint function, but also on preserving normal child development, minimizing negative impacts on the patient's family. Treating people with Juvenile Idiopathic Arthritis is challenging and complex, and patients and their families can have psychological problems, financial difficulties and take time to deal with all the different aspects of therapy (Dannecker GE, Quartier P. Juvenile Idiopathic Arthritis : Classification, Clinical Presentation and Current Treatments. Hormone Research in Pediatrics 72:4-12, 2009).

[0009] Rheumatic diseases of childhood are not always benign or of short duration (Minden K, et. al., A. Longterm outcome in patients with juvenile idiopathic arthritis. Arthritis & Rheumatism 46:2392-2401, 2002). Given the complexity of the disease, therapy may be needed for many years and a multidisciplinary pediatric rheumatology team is crucial for optimal treatment of these children. In addition to the pediatric rheumatologist, representatives from different specialties (eg, an orthopedist, ophthalmologist, oral surgeon, physical and occupational therapists, social workers and psychologists) should be included as members of a multifunctional treatment team (Dannecker GE, Quartier P. Juvenile Idiopathic Arthritis: Classification, Clinical Presentation and Current Treatments. Hormone Research in Pediatrics 72:4-12, 2009).

[0010] The treatment of Juvenile Idiopathic Arthritis aims at both controlling the underlying inflammatory process and preventing complications. Until a few years ago, therapy was essentially based on the use of non-steroidal anti-inflammatory drugs, corticosteroids (systemic and intra-articular) and disease-modifying anti-rheumatic drugs, such as methotrexate. However, some patients, especially those with systemic and polyarticular presentation, were refractory to this approach (Petty RE. Prognosis in children with rheumatic diseases: justification for consideration of new therapies. Rheumatology 38:739-742, 1999).

[0011] The increase in knowledge of the inflammatory process underlying Juvenile Idiopathic Arthritis and its main actors, allowed the establishment of new therapeutic targets. In fact, treatment options have improved with the development of drugs directed against these inflammatory mediators, namely tumor necrosis factor alpha (anti-TNF) antagonists. Several articles have demonstrated a favorable response to these agents in patients with JIA refractory to conventional treatment (Horneff G, et. al., The German etanercept registry for treatment of juvenile idiopathic arthritis. Annals of the Rheumatic Diseases 63:1638-1644, 2004 ; Quartier P, et. al., Efficacy of etanercept for the treatment of juvenile idiopathic arthritis according to the onset type. Arthritis & Rheumatism 48:1093-1101, 2003; Lovell DJ, et. al., Safety and efficacy of up to eight years of continuous etanercept therapy in patients with juvenile rheumatoid arthritis. Arthritis & Rheumatis 58:1496-1504, 2008). After several years of using anti-tumour necrosis factor (anti-TNFs) in the treatment of arthritis, approximately one-third of patients stop using the medication due to loss of efficacy or adverse effects (Scheinberg M, et. al., Retrospective study evaluating dose standards for infliximab in patients with rheumatoid arthritis at Hospital Israelita Albert Einstein, São Paulo, Brazil Clinical Rheumatology 27:1049-1052, 2008; Grinblat B, Scheinberg M. The enigmatic development of psoriasis and psoriasiform lesions during anti-TNF therapy: a review. Seminars in Arthritis and Rheumatism 37:251-255, 2008). Other cytokines, in addition to TNF, participate in the etiopathogenic mechanisms of arthritis: this is the case of interleukin-1 (IL-1) and interleukin-6 (IL-6). Several studies show that blocking IL-1 and IL-6 may have therapeutic benefits in these patients. Tocilizumab is a humanized monoclonal antibody against the soluble and cellular interleukin-6 receptor, also significantly altering vascular endothelial growth factor levels. Although its mechanism of action is not yet fully understood, several studies in the medical literature indicate that its benefits are evident and suggest that it will be a new anticytokine treatment for inflammatory arthritis (Nishimoto N, et. al., Interleukin-6. In: Smolen J , Lipsky P, editors. Targeted Therapy in Rheumatology p:231-241, 2003; Takagi N, et al., Blockage of interleukin-6 receptor ameliorates joint disease in murine collagen-induced arthritis. , 1998).

[0012] Although no drug has the power to cure Juvenile Idiopathic Arthritis, treatment has improved dramatically in recent years. Disease control can be achieved more effectively and faster with fewer side effects. This encouraging evolution is due to major advances in pharmacological options, especially biological agents (Dannecker GE, Quartier P. Juvenile Idiopathic Arthritis: Classification, Clinical Presentation and Current Treatments. Hormone Research in Pediatrics 72:4-12, 2009).

[0013] The diagnosis of Juvenile Idiopathic Arthritis is, so far, based on clinical history and physical examination, and the use of complementary methods, mainly directed to the exclusion of other diseases, there are no laboratory tests considered specific for its diagnostic definition . Biochemical laboratory tests and serological markers are useful to aid in differential diagnosis, classify subtype, assess extent of inflammation, determine prognosis and response to therapy (Cassidy JP, Petty RE. Juvenile rheumatoid arthritis. In: Cassidy JT, Petty RE, editors. Textbook of Pediatric Rheumatology. 4th ed. Philadelphia: WB Saunders Co. p:214-221, 2001). The diagnosis of serologic positivity for Juvenile Idiopathic Arthritis is restricted to patients with later-onset polyarthritis who have IgM-FR+ detected by nephelometry in 7 to 10% of cases. Antinuclear factors (ANA), or antinuclear antibodies, can be found in 2 to 70% of populations with Juvenile Idiopathic Arthritis, and this variability is probably due to the different substrates used in their measurement and the different subtypes of the disease (Nakamura RN. Progress in the use of biochemical and biological markers for the evolution of rheumatoid arthritis. Journal of Clinical Laboratory Analysis 14:305-313, 2000).

[0014] The clinical symptoms of Juvenile Idiopathic Arthritis can be variable. Several symptoms that indicate arthritis are not necessarily a diagnosis of Juvenile Idiopathic Arthritis and may have multiple etiologies that can only be differentiated by careful study of the patient's history (Boros C, Whitehead D. Juvenile idiopathic arthritis. Australian Family Physician 39:630 -636, 2010). Polyarticular Juvenile Idiopathic Arthritis has a multifactorial etiology, and it can present as a viral disease or it can be the beginning of a chronic disease, and it can evolve over a day or sometimes weeks, making the diagnosis difficult at the time of the consultation. Thus, to accurately diagnose Juvenile Idiopathic Arthritis, the first step is to rule out arthritis with known etiologies (Singh S, Mehra S. Approach to Polyarthritis. Indian Journal of Pediatrics 77:1005-1010, 2010).

[0015] As briefly mentioned above, the diagnosis of Juvenile Idiopathic Arthritis is essentially clinical, and no laboratory test available today can confirm its presence. However, laboratory studies can be used to provide evidence of inflammation, support clinical diagnosis, monitor treatment toxicity, and better understand the pathogenesis of the disease (Kim KH, Kim DS. Juvenile idiopathic arthritis: Diagnosis and differential Diagnosis Korean Journal of Pediatrics 53:931-935, 2010). Haematological abnormalities often reflect the extent of inflammatory disease. Laboratory tests that can help to better diagnose Juvenile Idiopathic Arthritis include inflammatory markers such as ESR and C-reactive protein (CRP). However, ESR and CRP levels in patients with active Juvenile Idiopathic Arthritis are variable. In general, VHS is occasionally useful in monitoring therapeutic efficacy (Cassidy JP, Petty RE. Juvenile rheumatoid arthritis. In: Cassidy JT, Petty RE, editors. Textbook of Pediatric Rheumatology. 4th ed. Philadelphia: WB Saunders Co. p: 214-221, 2001).

[0016] Conventional radiographs are the most accessible, quick and cheapest methods for evaluating joints. Ultrasonography is often the best way to identify intra-articular fluid, particularly in joints such as the hip and shoulder where fluid may be difficult to detect clinically (Fedrizzi MS, et. al., Ultrasonography in the early diagnosis of hip joint involvement in juvenile rheumatoid arthritis. The Journal of Rheumatology 24:820-1825, 1997). The increased use of MRI and advances in more functional MRI techniques in the last ten years have improved the assessment of joint disease in Juvenile Idiopathic Arthritis. Compared to ultrasonography or radiography, magnetic resonance imaging provides better results in detecting inflammation and damage to joints and cartilage. Magnetic resonance imaging is also able to accurately assess the posterior manifestations of Juvenile Idiopathic Arthritis, including erosions, loss of joint space, cartilage and ligament damage (Lamer S, Sebag GH. MRI and ultrasound in children with juvenile chronic arthritis. European Journal of Radiology 33:85-93, 2000).

[0017] Recent studies have shown that there is a great benefit in response to treatment when disease-modifying anti-rheumatic drugs (DMARDs) are started at an early stage and more aggressively (Egsmose C, et. al.. Patients with rheumatoid arthritis benefit from early second line therapy: 5-year follow-up of a prospective double-blind placebo-controlled study. The Journal of Rheumatology 22:2208-2213, 1995; Van der Heide A, et. al., The effectiveness of early treatment with "second-line" antirheumatic drugs. A randomized, controlled trial, Annals of Internal Medicine 124:699-707, 1996; Lard LR, et. al., Early versus delayed treatment in patients with recent-onset rheumatoid arthritis : comparison of two cohorts who received different treatment strategies. American Journal of Medicine 111:446-451, 2001; Mottonen T, et. al., Delay to institution of therapy and induction of remission using single-drug or combination-disease-modifying antirheumatic drug therapy in early rheumatoid arthritis. Arthritis & Rheumatism 46:894898, 2002; Nell et. al., Benefit of very early referral and therapy with disease-modifying antirheumatic drugs in patients with early rheumatoid arthritis. Rheumatology 43:906-914 2004; Breedvel FC, Kalden JR. Appropriate and effective management of rheumatoid arthritis. Annals of the Rheumatic Diseases 63:627-633, 2004). In the past, the classic therapeutic pyramid that consisted of conservative treatment in the initial phase of the disease gave many patients a chance to have more erosions and irreversible deformities before using more effective drugs. The ineffectiveness of the therapeutic scheme proposed by the therapeutic pyramid for arthritis led to a change in that treatment paradigm (Pincus T, Callahan LF. Remodeling the pyramid or remodeling the paradigms concerning rheumatoid arthritis: lessons from Hodgkin's disease and coronary artery disease. The Journal of Rheumatology 17 :1582-1585, 1990).

[0018] The use of methodologies that scan the cells of the immune system, without prior knowledge of the proteins present in them, provides the identification of new markers for various diseases. The technique of libraries displayed on the surface of phages allows the use not only of a peptide, but of a vast library of peptides against the set of antibodies of the patient with Juvenile Idiopathic Arthritis, with great potential to immunodiagnose the disease. Since its inception, the Phage Display technique has been used to identify recombinant peptides that can bind hormone receptors (Cwirla SE, et al., Peptide agonist of the thrombopoietin receptor as potent as the natural cytokine. Science 276:1696 -1699, 1997), proteins (Dennis MS, et al., Peptide exosite inhibitors of factor VIIa and anticoagulants. Nature 404:465-470, 2000) and inorganic components (Whaley SR, et al., Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly. Nature 405:665-668, 2000) and thus elucidate its target molecules.

[0019] The use of serum samples in the selection of peptides using the Phage Display technique is an attractive strategy that can be applied for immunodiagnosis or development of vaccines against various diseases, in an attempt to identify specific sequences that bind to antibodies present in such samples. This procedure can lead to the development of new simpler and more sensitive compounds, in addition to allowing the understanding of the participation of known or unknown antigens in the immune response mechanisms (Manoutcharian K, et al., Characterization of cerebrospinal fluid antibodies specificities in neurocysticercosis using Phage Display Peptide Library. Clinical Immunology 91:117-121, 1999). The. Display of peptides on the surface of filamentous phages may be effective for the isolation of clones encoding autoantigens identified in the synovial fluid or serum of patients with Rheumatoid Arthritis, Juvenile Idiopathic Arthritis or with other autoimmune diseases (Niwa M, et al., Efficient isolation). of cDNA clones encoding rheumatoid arthritis autoantigens by lambda phage surface display. Journal of Biotechnology 114:55-58, 2004).

[0020] Obtaining new biomarkers that serve both as diagnostic and therapeutic targets for Juvenile Idiopathic Arthritis along with existing methodologies for this purpose can significantly improve the quality of life of children with this pathology.

[0021] In the search for diagnostic biomarkers, patent PI1003746-2A2 reports the use of the phage display technique to isolate immunoglobulin G-binding peptides from patients with ovarian cancer, and which can be used in immunodiagnosis and in vaccine compositions for prevention and treatment of ovarian cancer.

[0022] The patent WO2007120651A2 reports the use of the phage display technique in the isolation of antibodies against the human cytokine TNF-α in order to neutralize it, and these selected antibodies can be used in the treatment of Juvenile Idiopathic Arthritis. This invention was the first involving fully human anti-TNF-α monoclonal antibodies to be approved by the US Department of Health and Human Services. This invention is now marketed for the treatment of Juvenile Idiopathic Arthritis, Rheumatoid Arthritis and other autoimmune diseases.

[0023] The patent US20110212470A1 reports the use of the phage display technique in the isolation of polypeptides for the diagnosis of autoimmune diseases, especially for the diagnosis of Rheumatoid Arthritis. When tested, the selected polypeptides showed greater reactivity against antibodies present in the serum of patients with Rheumatoid Arthritis compared to antibodies present in the serum of healthy individuals.

[0024] The patent WO1997002342A1 reports the use of the phage display technique in the isolation of antibodies or antibody fragments binding peptides derived from the MHC complex for diagnosis or treatment of autoimmune diseases, infectious diseases and cancer. The. Patents US6306365-B1, US6296832-B1, US6068829-A report the use of the phage display technique in the isolation and identification of peptides binding to various organs of the human body, isolated by injecting peptide libraries into living people. These peptides can be used in the diagnosis of different pathologies. B. WO9946284-A; EP1062232-A; WO9946284-A2; AU9930783-A; EP1062232-A2; US6174687-B1; US6232287-B1; JP2002506079-W; AU762991-B; US6610651-B1; US6784153-B1; US2005074812-A1 and US6933281-B2 report the use of the phage display technique in the isolation and identification of peptides binding to various organs in mice.

[0025] Patents US7083945, US2006068421, US2005202512, GB2408332, US2003104604, EP1452599 and US2006035223 report the use of the phage display technique in order to express recombinant polypeptides with affinity for the ligand, in addition to proposing methodologies that make the technique more efficient.

[0026] In the search for a fast, efficient methodology capable of providing a more accurate diagnosis of Juvenile Idiopathic Arthritis, as well as the use of specific ligands as substance carriers, the present invention proposes the use of the Phage Display technique for selection and identification of recombinant peptides specific to Juvenile Idiopathic Arthritis, through the interaction of a combinatorial peptide library displayed on the surface of filamentous phages. The Phage Display technique in combination with other technologies makes this invention readily accepted, as the new technology has high sensitivity and flexibility of use in relation to other current technologies for identifying new markers for diagnosis, monitoring, prognosis and use as drug carriers for the treatment of Juvenile Idiopathic Arthritis, as well as other pathologies. Selected peptides that mimic juvenile idiopathic arthritis autoantigens can be used as probes in in vitro immunoassays to detect the presence of binding molecules that are present in the serum, such as circulating antibodies and other proteins, that react against components of the immune system or molecules involved directly or indirectly in autoimmune processes. Immunodetection can be carried out through in vitro immunological assays already routinely used in laboratories, such as immunoenzymatic tests ELISA, Western Blot, immunohistochemistry, immunocytochemistry, immunofluorescence, EIA, MEIA, tests to measure immunoagglutination, tests that use these peptides in electrochemical sensors, tests that use these peptides in any other form of detection related directly or indirectly in samples of body fluids such as saliva, urine, serum or blood plasma.

[0027] Thus, the present invention proposes the use of peptide sequences, fused or not to carriers, such as filamentous phages, drugs and/or fluorescent/radioactive substances, or to any other type of carrier and its use in detection and identification of substances present in Juvenile Idiopathic Arthritis or other arthritis-like diseases for diagnostic testing.

[0028] The invention may be better understood through the description of the results obtained. The experimental procedures involved are detailed at the end of this descriptive report. The methodology used for the selection, characterization and use of recombinant peptides that interact with antibodies from patients with Juvenile Idiopathic Arthritis was applied to select, from immunoglobulins G purified from the serum of mice with collagen-induced arthritis, mimetic peptides to Arthritis autoantigens Juvenile Idiopathic Arthritis that were later validated through tests using patient serum, proving the efficiency of its use in the diagnosis of Juvenile Idiopathic Arthritis.

Example 1:

[0029] This example refers to the selection and characterization of recombinant peptides and protein motifs that bind to components present in the serum of patients with Juvenile Idiopathic Arthritis/Rheumatoid Arthritis and/or molecules involved directly or indirectly in the development of autoimmune diseases. Several parameters can be used to indicate the successful selection of each peptide. These same parameters were previously used by Rodi D.J. et al. Quantitative assessment of peptide sequence diversity in M13 combinatorial peptide Phage Display Libraries. Journal of Molecular Biology 322: 1039-1052, 2002.

[0030] The sequences identified by Seq. ID Nos. 1 to 14 refer to peptides selected as linkers to components present in the serum of patients with Juvenile Idiopathic Arthritis (Ph.D.-12). As there is the possibility of binding the peptides in their target in the carboxy terminal to amino terminal direction, we have also included the sequences in their reverse direction, identified as their respective inverted sequences (Seq. ID Nos. 25 to 28). For example, Seq. ID #1 has the reverse sequence Seq. ID No. 15, Seq. ID No. 2 has the inverse sequence Seq. ID No. 16, and so on, as shown in TABLE 1. TABLE 1

Example 2:

[0031] The immunoassay ELISA was used for pre-selection of the phages to be studied in more detail, considering their immunoreactivity with Immunoglobulin G present in the serum of the tested individuals.

[0032] FIGURE 1 shows the representative graph of the readings at 492nm of the ELISA assay showing the immunoreactivity of fourteen phage clones detected by anti-human IgG antibody labeled with peroxidase against sera from patients with Juvenile Idiopathic Arthritis and healthy controls. All results obtained in the analysis of absorbance differences obtained in the comparison of sick and healthy were statistically significant, although with different profiles between the tested phages.

[0033] The peptide Seq. ID N° 09, showed the highest immunoreactivity and was selected for a more detailed study, as shown in FIGURE 2A. FIGURE 2B shows the ROC curve for the peptide Seq. ID N° 09, proving its 90% sensitivity and 100% specificity to discriminate sick and healthy patients and those with other autoimmune diseases. These data, added to other analyzes performed, suggest that this peptide Seq. ID N° 09 could be a potential serological biomarker for use in diagnostics and therapeutic processes as a drug carrier.

Example 3:

[0034] The 14 peptides selected by Phage Display were subjected to similarity analysis with some proteins reported in previous studies as possible autoantigens of mice and humans in Rheumatoid Arthritis/Juvenile Idiopathic Arthritis. The five proteins were chosen for similarity analysis with the peptides due to their relevance and contribution to the evolution of arthritis, and showed alignment with them. FIGURE 3 shows the bioinformatics analyzes obtained for the proteins taken as chosen rheumatoid autoantigens, with the respective peptides that aligned with them in the 3D analyses: A, calreticulin; B, glycoprotein 39; C, α-Enolase; D, PADI4.

[0035] The technology adopted here identified new and potential autoantigens of Juvenile Idiopathic Arthritis/Rheumatoid Arthritis, which may allow a better characterization of the disease with regard to immunodiagnosis and delineate new therapeutic targets. The selection by Phage Display resulted in the isolation of a highly specific marker (Seq. ID No. 09) for the diagnosis of Juvenile Idiopathic Arthritis, while when tested by the ELISA assay against the serum of healthy children or with other autoimmune diseases that are not Juvenile Idiopathic Arthritis or Rheumatoid Arthritis, a low interaction of the peptide with immunoglobulin G present in the serum of the individuals tested was observed. This biomarker may offer a new tool for a better characterization of Juvenile Idiopathic Arthritis and Rheumatoid Arthritis inflammatory diseases, since the system currently adopted is descriptive and based on morphological and clinical changes.

[0036] For a better understanding of the examples used, below is a detailed description of the experimental procedures adopted:

[0037] To obtain binding peptides to Immunoglobulin G present in Rheumatoid Arthritis and Juvenile Idiopathic Arthritis, an animal model was used due to the need for a live model with arthritis that resembles that observed in humans. The animal model used was the DBA/1J mouse strain, which is genetically modified to have a predisposition to develop arthritis after stimulation by type II collagen. DBA/1J models are particularly very powerful tools for genetic studies of rheumatoid arthritis, as they are pure strains of genetically homogeneous mice and therefore lack the heterogeneity of the human genetic complex [Kunz, M; Ibrahim, S.M. Cytokines and Cytokine Profiles in Human Autoimmune Diseases and Animal Models of Autoimmunity. Mediators of Inflammation 2009:1-20, 2009].

[0038] All experiments related to the handling of mice were performed at the Animal Experimentation Laboratory Animal Laboratory at the Federal University of Uberlândia (LEA - UFU). All experimental procedures involving selection analysis and biomarker characterization were performed at the Nanobiotechnology Laboratory of the Institute of Genetics and Biochemistry of the Federal University of Uberlândia. This project was approved by the Committee on Ethics in the use of animals (CEUA), with its registration number 020/12, and also by the Ethics and Research Committee (CEP), with its registration number being 262/09. The blood collection procedure from children with Juvenile Idiopathic Arthritis was performed within the routine of the Clinical Analysis Laboratory of the Hospital de Clinicas of the Federal University of Uberlândia, Uberlândia-MG, Brazil, without causing additional discomfort to the children. Peripheral blood samples (5mL) were collected in vacutainerTM tubes containing 7.2mg K2EDTA, and in tubes without the addition of anticoagulant (5mL). The serum was transferred to another tube and stored at -80°C.

[0039] For the process of inducing arthritis in mice of the DBA1/J lineage, a group of three animals at 12 weeks of age was immunized with 62.5 ul containing 100ug of type II bovine collagen (BD Biosciences) homogenized with adjuvant of Complete Freund (Sigma Chemical Co) in a 1:1 ratio, used as a highly irritating immunopotentiator, containing mycobacteria to emulsify the solution, being injected at the base of the tail. After immunization, the mice were then kept under daily observation, in cages lined with wood shavings, feed and water ad libitum, in a monitored environment with a 12-hour light cycle and a room temperature of 25°C. The animals were not subjected to situations of stress or pain, only the natural course of manifestation of arthritis was observed. Another group containing two mice of the DBA1/J lineage was used as a negative control, receiving only PBS1x with CFA for immunization.

[0040] After a certain period of immunization, approximately 2-4 weeks, the animals in the positive and negative group had their joints measured by using a caliper for analysis of inflammation, as proposed by Brand D.D., et al. Collagen-induced arthritis. Nature Protocols 2:5, 2007. After confirming the manifestations of Arthritis in the joints of mice, they were sacrificed and had peripheral blood and knees removed for the step of selection of peptides taken as autoantigens of Rheumatoid Arthritis/Juvenile Idiopathic Arthritis. The purification of Immunoglobulin G from the serum of mice immunized with collagen type II (with arthritis) and non-immunized (control) occurred through magnetic beads (magnetic beads) conjugated with protein G (GE Healthcare), following the manufacturer's manual. The joints of mice with arthritis and control mice were macerated in crucibles containing liquid nitrogen and then total protein extraction buffer (20mM Tris-HCL pH7.2, 10mM EDTA, 2mM EGTA, 250mM sucrose, DTT) was added 1mM, 1mM Benzamidine). The material resulting from the aforementioned process was collected in microtubes, which were centrifuged for 30 minutes at 20,000 g at 4°C, collecting the supernatant. In order to optimize the amount of proteins obtained, the remaining pellet from the first centrifugation of the tubes was again macerated and the centrifugation repeated, collecting the supernatant again (where the proteins are found, which will be used in the process of selection of peptides by the phage display technique). The collected supernatant was subjected to quantification of the proteins obtained, for this, this quantification was performed by the method of Bradford (Bradford MM. Anal Biochem 72: 248-254, 1976).

[0041] For the selection of binding phages to Arthritis autoantigens, a commercial library of peptides displayed in M-13 phage was used, consisting of 12 amino acids: Ph.D.-12 (complexity: 2.7 x 109 transformants), with peptides fused to pIII protein (5 copies per phage particle) from New England BioLabs, Schwalbach, Germany.

[0042] 10 μl (1x1012 phage particles) of the aforementioned library diluted in 190 ul of TBS-Tween 0.1% were used for the selection of immunoglobulin G (IgG's) binders from DBA/1J mice. Initially, in order to remove IgG-binding phages from the serum of healthy mice, the phage library was incubated under shaking for 30 minutes at room temperature with 20 μl of the microspheres coupled to IgG's from healthy control mice and then the microspheres were affinity precipitated to a magnetic apparatus. For positive selection, the phage supernatant, after previous incubation with IgG's purified from the serum of healthy mice, was transferred to another tube in which they contained 20 μl of magnetic microspheres coupled to IgG's purified from the serum of mice immunized with type II collagen who presented manifestations of arthritis and this was incubated under agitation for 30 minutes at room temperature. Then, the supernatant was discarded and the beads-antibody plus phage system was washed 10 times with 0.1% TBS-T, and the selected phages were eluted with 500ul of glycine pH 2.0 and neutralized with 75ul of Tris 1M pH 9.1.

[0043] After selection, amplification of glycine-eluted phages was done by inoculating a Luria Bertani medium (LB- Tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L), containing tetracycline, with a colony isolated from Escherichia coli strain ER2738. The medium was incubated with shaking at 37°C until the early-log phase (OD 600 ~0.3). Upon reaching this stage, the bacterial culture was inoculated with 500 ul of the phage eluate and incubated at 37°C for 4-5 hours under strong agitation. Then, the culture was centrifuged at 4°C at 10000 rpm for 10 minutes and the supernatant was transferred to a tube and again centrifuged at 4°C at 10000 rpm for 10 minutes and this supernatant was placed in a sterile tube containing a solution of PEG/NaCl (20% of Polyethylene Glycol 8000 and 2.5 M of NaCl - sterile solution) in the amount of 1/6 of the volume of the supernatant. The solution was incubated for 12-16 hours at 4°C for phage precipitation and later centrifuged at 10000 rpm for 15 minutes at 4°C to discard the supernatant. The precipitate was then suspended in 1mL of TBS, transferred to another tube, centrifuged again at 14000 rpm for 10 minutes at 4°C and the supernatant was placed in another tube with 1/6 of the volume of PEG/NaCl remaining for 1 hour on ice. It was centrifuged at 14000 rpm for 10 minutes at 4°C and the supernatant was discarded. The precipitate was resuspended in 200 uL of TBS, thus obtaining the amplified eluate, which was later titrated and stored at 4°C. Titration is a necessary procedure to determine the number of entry and exit of viral particles during the “Biopanning” cycles.

[0044] At each biopanning cycle, the stringency of washes ranged from 0.1 to 0.5% in each cycle, in addition, in the rounds following the first elution of the selected phages proceeded with the extract of total proteins from mice knees to ensure that the peptides obtained were actually mimetic to arthritis autoantigens, 500 uL (715 μg) of this extract being used in the elution in the second round and only 10 uL (14 μg) in the third round.

[0045] The phage solution containing the third round eluate to be titrated was subjected to serial dilutions of 10 times in LB medium. For non-amplified eluates, dilutions of 10-1 to 10-4 were used; for solutions with amplified phages the dilution range used was 10-8 to 10-11. Each dilution was added with 200ml of the ER2738 culture in the mid-log phase (OD600 ~0.5). The mixture was shaken briefly and incubated for 5 minutes at room temperature. The cells, now infected, were transferred to culture tubes containing 3ml of Agarose Top at 45°C and spread on a Petri dish containing solid LB medium, with IPTG/X-Gal and tetracycline. For each dilution a plate was prepared.

[0046] The plates were incubated at 37°C for 16 hours, after which, the colonies of the plates that had approximately 100 colonies were counted. Each number was multiplied by the dilution factor of each plate to obtain the phage titer.

[0047] After titration of the phages obtained in the third round of selection, 96 clones were randomly chosen from the titration plate and transferred to wells of a culture plate (deepwell type), which already contained 1.2 ml of in each well. early-log phase ER2738 culture (OD 600 ~ 0.3) for amplification and further extraction of phage DNA. Subsequently, the plate was sealed with perforated adhesive and incubated at 37°C for 24 hours under strong agitation. Then, the plate was centrifuged at 3700 rpm at 20°C for a period of 30 minutes, thus obtaining the phage isolated from the bacteria. Then, 800 μL of the supernatant was transferred to another plate and, in addition to 350 μL of PEG/NaCl, they were incubated for 10 minutes at room temperature. After the incubation time, the plate was centrifuged at 3700 rpm at 20°C for 40 minutes in order to precipitate the phages. The supernatant was discarded and then 100 μL of iodide buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA and 4M NaI) was added to each well of the plate. The plate was then shaken for five minutes and 500 µL of absolute ethanol were added per well of the plate. After a 10-minute incubation at room temperature, the plate was centrifuged at 3700 rpm at 20°C for 40 minutes. The supernatant was later discarded and then 150 μL of 70% ethanol was added to the phage DNA, and the plate was again centrifuged at 3700 rpm at 20°C for 10 minutes. Then, the supernatant was discarded and the plate was left to dry for a period of 10 minutes. Finally, the DNA precipitated on the plate was diluted in 20 μL of Milli-Q water. To check the quality of the DNA, electrophoresis was performed on a 0.8% agarose gel stained with ethidium bromide solution.

[0048] For the sequencing reaction, 2 μL of DNA, 3 μL of water, 1 μL of primer -96 gIII (5'-OH CCC TCA TAG TTA GCG TAA CG-3' - Biolabs) and 4 μL of Premix (DYEnamic ET Dye Terminator Cycle Kit. - Amersham Biosciences).

[0049] The 35-cycle reaction was performed in a plate thermocycler (MasterCycler-Eppendorf) under the following conditions: Denaturation (at 95°C for 20 seconds); primer annealing (at 50°C for 15 seconds) and extension (at 60°C for one minute).

[0050] The sequenced DNA was precipitated with 1.0 μL of ammonium acetate and 27.5 μL of absolute ethanol. Then, the plate was centrifuged at 3700 rpm at 4°C for 45 minutes and the supernatant was discarded. Then, 150 μL of 70% ethanol was added to the precipitated DNA and the plate was centrifuged again at 3700 rpm at 4°C for 15 minutes. The supernatant was discarded and the plate was inverted on a paper towel and thus pulsed at 800 rpm for one second. The plate was wrapped with aluminum foil and left to rest for 10 minutes in order to evaporate the remaining ethanol. Afterwards, 10 μL of Loading Buffer were added to each well of the plate, which was again protected from light with aluminum foil and vortexed for approximately 10 minutes. The sequencing reading was performed in the MegaBace 1000 automatic sequencer (Amersham Biosciences) at the Nanobiotechnology Laboratory of the Federal University of Uberlândia.

[0051] The bioinformatics program Sequence Manipulacion Suite (http://www.bioinformatics.org/sms/rev_comp.html) was used to supply the complementary reverse sequences and these were translated by the ExPAsy web site program (http:// ca.expasy.org/tools/dna.html). For the analysis of similarities between mouse autoantigens and human autoantigens, the programs Pepitope Server (http://pepitope.tau.ac.il/index.html) and Immune Epitope Database and Analysis Resource (http://www.immuneepitope) were used .org/).

[0052] To perform the ELISA test, a high affinity plate (Costar-Corning 3590, NY, USA) was sensitized with the 14 phages that presented distinct sequences after the DNA sequencing procedure. The ELISA test in this case was performed in order to verify whether the selected peptides, binding to immunoglobulin G from mice with arthritis, would be recognized by immunoglobulin G present in the serum of patients with Juvenile Idiopathic Arthritis. For this, a 96-well ELISA plate was sensitized with 100 μL/well of a solution of each of the 14 phages with 0.1 M carbonate bicarbonate buffer pH 9.4 and remained so at 4°C overnight. The following day, the liquid was discarded from the plate and then 300 µL/well of 5% TBS-BSA blocking buffer was added, and the plate was blocked for 1 hour at 37°C. After blocking, the plate was washed once with 1x TBS and, subsequently, 100 μL/well of serum from patients with Juvenile Idiopathic Arthritis diluted 1:100 in 5% TBS-BSA containing 0.5% Tween 20 was added for 1 hour at 37°C. Serum from healthy children was also used under the same circumstances as a control. Then, the plate was washed three times with TBS buffer 0.5% Tween, then 100 µL/well of peroxidation labeled anti-human IgG solution (Sigma) diluted 1:5000 in TBS-BSA 5 was added. % containing 0.05% Tween. The plate was again incubated for 1 hour at 37°C. Finally, the plate was washed three more times with TBS 0.5% Tween and, subsequently, to detect antigen-antibody binding, the ortho-phenylenediamine buffer ( OPD) (Sigma) was added to the plate. The reaction was stopped by the addition of 4N sulfuric acid. Reactivity was obtained in a plate reader (Titertek Multiskan Plus, Flow Laboratories, USA) by reading at 492 nm.

[0053] The 14 peptides tested in the ELISA test selected from a previous incubation with Immunoglobulin G from mice with arthritis, showed statistical difference when incubated with serum from 40 individuals with Juvenile Idiopathic Arthritis compared with the serum from 22 healthy individuals.

Claims (3)

1. Recombinant peptides mimetic to juvenile idiopathic arthritis autoantigens and their reverse sequences characterized by the fact that they comprise the sequences Seq ID N° 1 to Seq ID N° 14. 2. Use of peptides and their reverse sequences as defined in claim 1 characterized in that they are used as probes for in vitro detection of the presence of circulating antibodies or other binding molecules against autoimmune and/or inflammatory diseases, preferably Juvenile Idiopathic Arthritis. 3. Use according to claim 2, characterized in that the detection of binding molecules to the peptides is carried out through immunological assays, such as immunoenzymatic tests (such as ELISA, Western Blot, immunofluorescence, immunohistochemistry, EIA, MEIA and others), tests by immunoagglutination, electrochemical sensors, or any other form of detection related directly or indirectly in samples of body fluids, such as saliva, urine, blood, preferably blood.

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