# US 2012/0083423 A1 · Biomarkers, Methods and Kits for the Diagnosis of Rheumatoid Arthritis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2012/0083423 A1 - Document ID: US20120083423A1 - Status: Published application - Kind: A1 - Application number: 13/259,645 - Filed: 2010-03-29 - Published: 2012-04-05 - Earliest claimed: 2009-03-30 - Assignees: Universite De La Mediterranee - Aix-Marseille 11 - Inventors: Isabelle Auger; Jean Roudier - Web page: https://patentgrep.com/patent/US20120083423A1 ## CPC - G01N 33/564: for pre-existing immune complex or autoimmune disease {, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9} - C07K 14/4713: {Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens} - C07K 14/81: Protease inhibitors - C07K 14/8139: {Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin} - C07K 14/82: Translation products from oncogenes - C12N 9/12: transferring phosphorus containing groups, e.g. kinases (2.7) - C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases} - C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5) - G01N 33/6854: {Immunoglobulins} - C12Y 207/11001: Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase - C12Y 305/03015: Protein-arginine deiminase (3.5.3.15) - G01N 2800/102: Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints - G01N 2800/60: Complex ways of combining multiple protein biomarkers for diagnosis - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES ## 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. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The method of claim 1, 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. ### Claim 3 (depends on 2) The method of claim 2, 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. ### Claim 4 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. ### Claim at position 5 5-8. (canceled) ### Claim 9 A kit for the in vitro 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. ### Claim 10 (depends on 9) The kit according to claim 9 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. ### Claim 11 A kit for detecting the presence of anti-PAD4 autoantibodies or anti-BRAF autoantibodies in a biological sample, said kit comprising: at least one biomarker, 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 at least one reagent for detecting a biomarker-antibody complex formed between the biomarker and an autoantibody present in the biological sample, wherein the autoantibody is an anti-PAD4 autoantibody or an anti-BRAF autoantibody that is indicative of rheumatoid arthritis. ### Claim at position 9 12-13. (canceled) ### Claim 14 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. ### Claim 15 (depends on 14) The array according to claim 14 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. ### Claim 16 (canceled) ### Claim 17 (depends on 14) The array of claim 14 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. ### Claim 18 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 ### Claim at position 15 19-21. (canceled) ### Claim 22 (depends on 1) The method of claim 1, wherein the subject is CCP-negative. ### Claim 23 (depends on 1) The method of claim 1, 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.