Biomarkers, methods and kits for the diagnosis of rheumatoid arthritis

Abstract

The present invention relates to peptides biomarkers that are specifically recognized by autoantibodies present in the sera of patients with Rheumatoid Arthritis (RA). More specifically, the invention provides epitopes of PAD4, of BRAF, and of calpastatin as well as methods and kits for using these sequences for the diagnosis of RA, in particular for the diagnosis of RA in CCP-negative subjects.

1 - 21 . (canceled) 
     
     
         22 . A method for diagnosing rheumatoid arthritis in a subject, said method comprising steps of:
 contacting a biological sample obtained from the subject with at least one biomarker for a time and under conditions allowing a biomarker-antibody complex to form, wherein the at least one biomarker is selected from the group consisting of:
 PAD4-peptides having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, analogues thereof, and fragments thereof, 
 BRAF-peptides having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, analogues thereof, and fragments thereof, and 
 any combination thereof; and 
   detecting any biomarker-antibody complex formed,   
       wherein detection of a biomarker-antibody complex is indicative of rheumatoid arthritis in the subject. 
     
     
         23 . The method of  claim 22 , said method comprising steps of:
 contacting a biological sample obtained from the subject with at least two biomarkers for a time and under conditions allowing a biomarker-antibody complex to form, wherein the at least two biomarkers are selected from the group consisting of:
 PAD4-peptides having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, analogues thereof, and fragments thereof, 
 BRAF-peptides having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, analogues thereof, and fragments thereof, 
 calpastatin-peptides having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, analogues thereof, and fragments thereof, and 
 any combination thereof; and 
   detecting any biomarker-antibody complex formed,   
       wherein detection of a biomarker-antibody complex is indicative of rheumatoid arthritis in the subject. 
     
     
         24 . The method of  claim 23 , wherein the at least two biomarkers comprise three PAD4-peptides, three BRAF-peptides and two calpastatin-peptides and wherein the three PAD4-peptides comprise or consist of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, the three BRAF-peptides comprise or consist of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and the two calpastatin-peptides comprise or consist of SEQ ID NO: 11 and SEQ ID NO: 12. 
     
     
         25 . The method of  claim 22 , wherein the subject is CCP-negative. 
     
     
         26 . The method of  claim 22 , further comprising measuring, in a biological sample obtained from said subject, the concentration of at least one marker selected from the group consisting of C-reactive protein, serum amyloid A, interleukin 6, S100 proteins, osteopontin, rheumatoid factor, matrix metalloprotease 1, matrix metalloprotease 3, hyaluronic acid, sCD14, angiogenesis markers, and products of bone, cartilage, or synovium metabolism. 
     
     
         27 . A method for detecting the presence of anti-PAD4 autoantibodies or anti-BRAF autoantibodies in a biological sample, said method comprising steps of:
 contacting the biological sample with at least one biomarker for a time and under conditions allowing a biomarker-antibody complex to form,
 wherein the at least one biomarker is a PAD4 peptide having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, analogues thereof, fragments thereof, and any combination thereof, or 
 wherein the at least one biomarker is a BRAF-peptide having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, analogues thereof, fragments thereof, and any combination thereof; and 
   detecting any biomarker-antibody complex formed,   
       wherein detection of a PAD4 peptide-antibody complex is indicative of the presence of anti-PAD4 autoantibodies in the biological sample, and wherein detection of a BRAF peptide-antibody complex is indicative of the presence of anti-BRAF autoantibodies in the biological sample. 
     
     
         28 . A kit for the diagnosis of rheumatoid arthritis in a subject, said kit comprising:
 at least two biomarkers selected from the group consisting of:
 PAD4-peptides having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, analogues thereof, and fragments thereof, 
 BRAF-peptides having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, analogues thereof, and fragments thereof, 
 calpastatin-peptides having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, analogues thereof, and fragments thereof, and 
 any combination thereof; and 
   at least one reagent for detecting a biomarker-antibody complex formed between the biomarker and an autoantibody present in a biological sample obtained from the subject, wherein detection of a biomarker-antibody complex is indicative of rheumatoid arthritis in the subject.   
     
     
         29 . The kit according to  claim 28  for the in vitro diagnosis of rheumatoid arthritis in a CCP-negative subject, wherein the at least two biomarkers comprise three PAD4-peptides, three BRAF-peptides and two calpastatin-peptides and wherein the three PAD4-peptides comprise or consist of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, the three BRAF-peptides comprise or consist of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and the two calpastatin-peptides comprise or consist of SEQ ID NO: 11 and SEQ ID NO: 12. 
     
     
         30 . An array for diagnosing rheumatoid arthritis in a subject, said array comprising, attached to its surface at least two biomarkers, wherein the at least two biomarkers are selected from the group consisting of:
 PAD4-peptides having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, analogues thereof, and fragments thereof,   BRAF-peptides having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, analogues thereof, and fragments thereof,   calpastatin-peptides having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, analogues thereof, and fragments thereof, and   any combination thereof.   
     
     
         31 . The array according to  claim 30  for diagnosing rheumatoid arthritis in a CCP-negative subject, wherein the at least two biomarkers comprise three PAD4-peptides, three BRAF-peptides and two calpastatin-peptides and wherein the three PAD4-peptides comprise or consist of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, the three BRAF-peptides comprise or consist of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and the two calpastatin-peptides comprise or consist of SEQ ID NO: 11 and SEQ ID NO: 12. 
     
     
         32 . The array of  claim 30  further comprising, attached to its surface, at least one additional biomarker for detecting the presence of RA-specific autoantibodies in a biological sample, preferably antinuclear antibodies and anti-CCP antibodies. 
     
     
         33 . A biomarker of rheumatoid arthritis, wherein the biomarker is:
 a PAD4-peptide having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, analogues thereof, and fragments thereof, and wherein the biomarker is recognized by an anti-PAD4 autoantibody, or   a BRAF-peptide having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, analogues thereof, and fragments thereof, and wherein the biomarker is recognized by an anti-BRAF autoantibody, or   a calpastatin-peptide having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, analogues thereof, and fragments thereof, and wherein the biomarker is recognized by an anti-calpastatin autoantibody.