PEPTIDE AND BIOMARKER ASSOCIATED WITH INFLAMMATORY DISORDERS, AND USES THEREOF

Abstract

The invention relates to a method of identifying a subject suspected of having or being susceptible to an autoimmune disease, such as rheumatoid arthritis (RA), comprising: contacting a sample of bodily fluid obtained from the subject with (i) a binding pair member having a binding affinity for citrullinated tenascin (cTNC) or a fragment thereof or (ii) a cTNC peptide; determining in a sample of bodily fluid obtained from the subject the presence or amount of (i) a citrullinated peptide derived from tenascin or (ii) an anti cTNC antibody; comparing the presence or amount of (i) the citrullinated peptide derived from tenascin or (ii) the anti cTNC antibody with a pre-defined threshold value; and assigning a diagnosis of RA or a future likelihood of developing RA when the presence or amount of (i) cTNC or (ii) an antibody against cTNC is detected or exceeds the threshold; and associated kits, peptides, binding members and uses thereof.

Claims

1. A method of identifying a subject suspected of having or being susceptible to an autoimmune disease, such as rheumatoid arthritis (RA), comprising: contacting a sample of bodily fluid obtained from the subject with (i) a binding pair member having a binding affinity for citrullinated tenascin (cTNC) or a fragment thereof or (ii) a cTNC peptide; determining in a sample of bodily fluid obtained from the subject the presence or amount of (i) a citrullinated peptide derived from tenascin or (ii) an anti cTNC antibody; comparing the presence or amount of (i) the citrullinated peptide derived from tenascin or (ii) the anti cTNC antibody with a pre-defined threshold value; and assigning a diagnosis of RA or a future likelihood of developing RA when the presence or amount of (i) cTNC or (ii) an antibody against cTNC is detected or exceeds the threshold.

2. The method of claim 1 wherein the binding pair member having an affinity for cTNC is a monoclonal antibody, a polycloncal antibody, or functional binding fragments of each thereof including but not limited to Fab, Fab2, Fv, ScFv, Fc, dAb, Fd, diabodies.

3. The method of claim 1 or 2 wherein the binding pair member is purified from a mammal or is expressed by recombinant DNA technology.

4. The method of claim 1 to 3 wherein the binding pair member has specificity for cTNC in the presence of non-citrullinated TNC.

5. The method of claim 1 wherein the cTNC is cTNC5 as defined in table 1.

6. The method of claim 1 wherein the cTNC has sequence comprising RcitXXXXRcitXXXXRcitRcit, where Rcit is citrulline and X is any amino acid.

7. The method of claim 1 wherein the cTNC is selected from the group comprising RcitXXXXRcitXXXXRcitX1; RcitXXXXRcitXXXXX1Rcit; RcitXXXXX1XXXXRcitRcit; or X1XXXXRcitXXXXRcitRcit; wherein: Rcit is a citrullinated arginine residue; X is any amino acid; and X1 is a non-citrullinated arginine, or any other amino acid.

8. The method of claim 1 wherein the cTNC has sequence comprising RcitPSNFRcitNLEGRcitRcit.

9. The method of claim 1 wherein the cTNC has sequence comprising EHSIQFAEMKLRcitPSNFRcitNLEGRcitRcitKR.

10. The method of claim 1 wherein the cTNC has sequence comprising EHSIQFAEMKLRcitPSNFRcitNLEGRcitRcitKRcit.

11. The method of claim 1 wherein the cTNC has sequence comprising EHSIQFAEMKLRcitPSNFRcitNLEGRcitRcitKRA.

12. The method of claim 1 wherein the cTNC has sequence comprising EHSIQFAEMKLRcitPSNFRcitNLEGRcitRcitKRcitA.

13. The method of any preceding claim wherein the RA is erosive RA.

14. The method of claim 1 to 13 wherein the step of determining the presence or amount of a citrullinated peptide derived from tenascin comprises: (i) performing a sandwich immunoassay configured with a first binding pair member for cTNC associated with a solid phase and a second binding pair member with a detectable label capable of simultaneous binding to cTNC; (ii) performing a competitive immunoassay configured with a binding pair member and labelled cTNC analog capable of competing with cTNC for binding to the binding pair member; (iii) performing a homogeneous immunoassay comprising a binding pair member for cTNC associated with a particle, wherein the presence of cTNC results in formation of aggregates that increase turbidity of the sample; (iv) detecting changes in the presence or amount of detectable label associated with a binding pair member or labelled cTNC analog in steps (i), (ii) or (iii); and (v) correlating changes in presence or amount of detectable label with the presence or amount of cTNC in the sample.

15. The method of claim 1 to 13 wherein the step of determining the presence or amount of an anti cTNC antibody comprises: (i) performing a sandwich immunoassay configured with a first binding pair member for the anti-cTNC antibody associated with a solid phase and a second binding pair member with a detectable label capable of simultaneous binding to the anti-cTNC antibody; (ii) performing a competitive immunoassay configured with a labelled binding pair member and cTNC immobilised on a solid phase, wherein the anti-cTNC antibody competes with the binding pair member for binding to the immobilised cTNC; (iii) performing a homogeneous immunoassay comprising a cTNC associated with a particle and a binding pair member for the anti-cTNC antibody, wherein the presence of anti-cTNC antibody results in formation of aggregates that increase the turbidity of the sample; (iv) detecting changes in the presence or amount of detectable label associated with a binding pair member or cTNC; and (v) correlating changes in presence or amount of detectable label with the presence or amount of anti-cTNC antibody in the sample.

16. A peptide comprising or consisting of the sequence R.sub.citPSNFR.sub.citNLEGR.sub.citR.sub.cit, or a variant thereof, wherein R.sub.cit is a citrullinated arginine residue.

17. The peptide according to claim 16, wherein the peptide comprises or consists of the sequence EHSIQFAEMKLR.sub.citPSNFR.sub.citNLEGR.sub.citR.sub.citKR, or a variant peptide thereof, wherein R.sub.cit is a citrullinated arginine residue.

18. The peptide according to claim 16, wherein the peptide comprises or consists of the sequence EHSIQFAEMKLR.sub.citPSNFRNLEGR.sub.citR.sub.citKR.sub.cit, or a variant peptide thereof, wherein R.sub.cit is a citrullinated arginine residue.

19. The peptide according to claim 16, wherein the peptide comprises or consists of the sequence: EHSIQFAEMKLR.sub.citPSNFR.sub.citNLEGR.sub.citR.sub.citKRA: or EHSIQFAEMKLR.sub.citPSNFR.sub.citNLEGR.sub.citR.sub.citKR.sub.citA; or a variant peptide thereof, wherein R.sub.cit is a citrullinated arginine residue.

20. The peptide of claim 16, wherein the peptide is a variant peptide comprising or consisting of the sequence R.sub.citXXXXR.sub.citXXXXR.sub.citR.sub.cit, wherein R.sub.cit is a citrullinated arginine residue, and X is any amino acid.

21. The peptide of claim 16, wherein the peptide is a variant peptide comprising or consisting of any one of the sequences of R.sub.citXXXXR.sub.citXXXXR.sub.citX.sub.1; R.sub.citXXXXR.sub.citXXXXX.sub.1R.sub.cit; R.sub.citXXXXX.sub.1XXXXR.sub.citR.sub.cit; or X.sub.1XXXXR.sub.citXXXXR.sub.citR.sub.cit; wherein: R.sub.cit is a citrullinated arginine residue; X is any amino acid; and X.sub.1 is a non-citrullinated arginine, or any other amino acid.

22. The peptide of claim 16, wherein the peptide is a variant peptide comprising or consisting of any one of the sequences of: EHSIQFAEMKLR.sub.citPSNFR.sub.citNLEGR.sub.citRKR; EHSIQFAEMKLR.sub.citPSNFR.sub.citNLEGRR.sub.citKR; EHSIQFAEMKLR.sub.citPSNFRNLEGR.sub.citR.sub.citKR; or EHSIQFAEMKLRPSNFR.sub.citNLEGR.sub.citR.sub.citKR; wherein R.sub.cit is a citrullinated arginine residue.

23. The peptide of claim 16, wherein the peptide is a variant peptide comprising or consisting of any one of the sequences of: EHSIQFAEMKLR.sub.citPSNFR.sub.citNLEGR.sub.citRKRA; EHSIQFAEMKLR.sub.citPSNFR.sub.citNLEGRR.sub.citKRA; EHSIQFAEMKLR.sub.citPSNFRNLEGR.sub.citR.sub.citKRA; or EHSIQFAEMKLRPSNFR.sub.citNLEGR.sub.citR.sub.citKRA; wherein R.sub.cit is a citrullinated arginine residue.

24. The peptide of claim 16, wherein the peptide is a variant peptide comprising or consisting of any one of the sequences of: EHSIQFAEMKLR.sub.citPSNFR.sub.citNLEGR.sub.citRKR.sub.cit; EHSIQFAEMKLR.sub.citPSNFR.sub.citNLEGRR.sub.citKR.sub.cit; EHSIQFAEMKLR.sub.citPSNFRNLEGR.sub.citR.sub.citKR.sub.cit; or EHSIQFAEMKLRPSNFR.sub.citNLEGR.sub.citR.sub.citKR.sub.cit; wherein R.sub.cit is a citrullinated arginine residue.

25. The peptide of claim 16, wherein the peptide is a variant peptide comprising or consisting of any one of the sequences of: EHSIQFAEMKLR.sub.citPSNFR.sub.citNLEGR.sub.citRKR.sub.citA; EHSIQFAEMKLR.sub.citPSNFR.sub.citNLEGRR.sub.citKR.sub.citA; EHSIQFAEMKLR.sub.citPSNFRNLEGR.sub.citR.sub.citKR.sub.citA; or EHSIQFAEMKLRPSNFR.sub.citNLEGR.sub.citR.sub.citKR.sub.citA; wherein R.sub.cit is a citrullinated arginine residue.

26. The peptide according to claim 2 16 to 25, wherein one or more non-citrullinated amino acid residues are removed or added, such that the spacing between the citrullinated arginine residues is varied.

27. The peptide according to claims 16 to 26, wherein the peptide comprises a N-terminal cysteine and a C-terminal cysteine.

28. The peptide according to claim 16, wherein the peptide is cTNC5 peptide described herein.

29. A biomarker for determining the inflammatory disorder status, of a subject wherein the biomarker comprises: (i) citrullinated tenascin-C or a fragment thereof which is citrullinated at at least three arginine residues of residue numbers 2187, 2192, 2197, 2198, and 2200; and/or (ii) autoantibodies with specificity for an epitope of citrullinated tenascin-C or a fragment thereof wherein the epitope comprises at least three citrullinated arginine residues of 2187, 2192, 2197, 2198 and 2200.

30. The biomarker of claim 29, wherein the citrullinated tenascin-C or a fragment thereof is citrullinated at at least three arginine residues of residue numbers 2187, 2192, 2197, and 2198.

31. The biomarker of claim 29, wherein the epitope comprises at least three citrullinated arginine residues of 2187, 2192, 2197, and 2198.

32. The biomarker of claim 29 or 30, wherein the biomarker comprises citrullinated tenascin-C or a fragment thereof which is citrullinated at arginine residues 2187, 2192, 2197 and 2198.

33. The biomarker of any of claims 29 to 32, wherein the biomarker comprises citrullinated tenascin-C or a fragment thereof which is citrullinated at arginine residues 2187, 2192, 2197, 2198 and 2200.

34. The biomarker of claims 29 or 31, wherein the biomarker comprises autoantibodies with specificity for an epitope of citrullinated tenascin-C or a fragment thereof wherein the epitope comprises citrullinated arginine residues 2187, 2192, 2197 and 2198.

35. The biomarker of any of claims 29, 31 or 34, wherein the biomarker comprises autoantibodies with specificity for an epitope of citrullinated tenascin-C or a fragment thereof wherein the epitope comprises citrullinated arginine residues 2187, 2192, 2197, 2198, and 2200.

35. The biomarker of any of claims 29 to 35, wherein the inflammatory disorder is rheumatoid arthritis or pre-rheumatoid arthritis.

36. A method of determining the inflammatory disorder status of a subject comprising detecting the presence or absence, or the level, of a biomarker in a sample from said subject, wherein the biomarker comprises: (i) citrullinated tenascin-C or a fragment thereof which is citrullinated at at least three arginine residues of residue numbers 2187, 2192, 2197 2198 and 2200; and/or (ii) detecting the presence or absence, or the level, of autoantibodies with specificity for an epitope of citrullinated tenascin-C or a fragment thereof wherein the epitope comprises at least three citrullinated arginine residues of 2187, 2192, 2197 2198 and 2200.

37. The method according to claim 36, wherein the citrullinated tenascin-C or fragment thereof is citrullinated at at least three arginine residues of residue numbers 2187, 2192, 2197, and 2198.

38. The method according to claim 36, wherein the epitope comprises at least three citrullinated arginine residues of 2187, 2192, 2197, and 2198.

39. The method according to any of claims 36 to 2438 wherein the citrullinated tenascin-C or fragment thereof is citrullinated at arginine residues 2187, 2192, 2197 and 2198, and/or or the epitope comprises citrullinated arginine residues 2187, 2192, 2197 and 2198.

40. The method according to any of claims 36 to 39, wherein the citrullinated tenascin-C or fragment thereof is citrullinated at arginine residues 2187, 2192, 2197, 2198 and 2200, and/or or the epitope comprises citrullinated arginine residues 2187, 2192, 2197, 2198 and 2200.

41. The method according to any of claims 36 to 40, wherein the epitope is on a fragment of cTNC.

42. The method according to any of claims 36 to 41, wherein the method comprises detecting the presence or absence, or the level, of autoantibodies with specificity for cTNC5 described herein.

43. The method of any of claims 36 to 42, wherein the level of the biomarker detected in the sample is compared with one or more reference values.

44. The method of any of claims 36 to 43, wherein the presence of the biomarker in a sample from said subject is sufficient to conclude the subject has an inflammatory disorder.

45. The method of any of claims 36 to 44, wherein the inflammatory disorder is RA.

46. A method of monitoring the progression of an inflammatory disease or monitoring the efficacy of a treatment administered to a subject comprising detecting the level of (i) citrullinated tenascin-C or a fragment thereof which is citrullinated at at least three arginine residues of residue numbers 2187, 2192, 2197 2198 and 2200; and/or (ii) autoantibodies with specificity for an epitope of citrullinated tenascin-C or a fragment thereof wherein the epitope comprises at least three citrullinated arginine residues of 2187, 2192, 2197 2198 and 2200; wherein the detection is in a sample from said subject, and comparing the levels to normal and/or reference values.

47. The method according to claim 46, wherein the citrullinated tenascin-C or fragment thereof is citrullinated at at least three arginine residues of residue numbers 2187, 2192, 2197, and 2198.

48. The method according to claim 46, wherein the epitope comprises at least three citrullinated arginine residues of 2187, 2192, 2197, and 2198.

49. The method according to claim 46, wherein the citrullinated tenascin-C or fragment thereof is citrullinated at arginine residues 2187, 2192, 2197 and 2198 or the epitope comprises citrullinated arginine residues 2187, 2192, 2197 and 2198.

50. The method according to any of claims 46 to 49, wherein the citrullinated tenascin-C or fragment thereof is citrullinated at arginine residues 2187, 2192, 2197, 2198 and 2200, and/or the epitope comprises citrullinated arginine residues 2187, 2192, 2197, 2198 and 2200.

51. The method of any of claims 46 to 50, wherein the reference values are the initial levels in the subject, or the levels in the subject when they were previously tested, or both.

52. A kit for use in determining the inflammatory disorder status of a subject comprising at least one agent for detecting the presence, or the level, of (i) citrullinated tenascin-C or a fragment thereof which is citrullinated at at least three arginine residues of residue numbers 2187, 2192, 2197 2198 and 2200; and/or (ii) autoantibodies with specificity for an epitope of citrullinated tenascin-C or a fragment thereof wherein the epitope comprises at least three citrullinated arginine residues of 2187, 2192, 2197 2198 and 2200; wherein the detection is in a sample provided by the subject.

53. The kit of claim 52 wherein the agent comprises the biomarker according to any of claims 29-35.

54. The kit according to any of claim 52 to 53, wherein the citrullinated tenascin-C or fragment thereof is citrullinated at at least three arginine residues of residue numbers 2187, 2192, 2197, and 2198.

55. The kit according to any of claim 52 to 54, wherein the epitope comprises at least three citrullinated arginine residues of 2187, 2192, 2197, and 2198.

56. The kit according to claim 52, wherein the citrullinated tenascin-C or fragment thereof is citrullinated at arginine residues 2187, 2192, 2197 and 2198 or the epitope comprises citrullinated arginine residues 2187, 2192, 2197 and 2198.

57. The kit according to any of claim 52 to 56, wherein the citrullinated tenascin-C or fragment thereof is citrullinated at arginine residues 2187, 2192, 2197, 2198 and 2200, and/or the epitope comprises citrullinated arginine residues 2187, 2192, 2197, 2198 and 2200.

58. The kit according to any of claims 52 to 57, wherein the agent comprises a peptide according to any of claims 1 to 13.

59. The kit according to any of claims 52 to 58, wherein the autoantibodies have specificity for cTNC5 described herein.

60. The kit according to any of claims 52 to 59, wherein the kit further comprises a panel of peptides and/or antibodies for detecting a panel of biomarkers for the inflammatory condition.

61. Use of the determination of the presence, or the level, of the biomarker according to any of claims 29 to 35 in a sample obtained from a subject, as a means of assessing the inflammatory disorder status in the subject.

62. Use of: (i) citrullinated tenascin-C or a fragment thereof which is citrullinated at at least three arginine residues of residue numbers 2187, 2192, 2197 2198 and 2200; and/or (ii) autoantibodies with specificity for an epitope of citrullinated tenascin-C or a fragment thereof wherein the epitope comprises at least three citrullinated arginine residues of 2187, 2192, 2197 2198 and 2200; as a biomarker for an inflammatory disorder.

63. The use according to claim 61 or claim 62, wherein the citrullinated tenascin-C or fragment thereof is citrullinated at at least three arginine residues of residues 2187, 2192, 2197 and 2198 or the epitope comprises at least three citrullinated arginine residues at residues 2187, 2192, 2197 and 2198.

64. The use according to claim 61 or claim 62, wherein the citrullinated tenascin-C or fragment thereof is citrullinated at arginine residues 2187, 2192, 2197 and 2198 or the epitope comprises citrullinated arginine residues 2187, 2192, 2197 and 2198.

65. The use according to any of claims 61 or 62, wherein the citrullinated tenascin-C or fragment thereof is citrullinated at arginine residues 2187, 2192, 2197, 2198 and 2200, and/or the epitope comprises citrullinated arginine residues 2187, 2192, 2197, 2198 and 2200.

66. The method, kit, biomarker or use of any preceding claim wherein the inflammatory disorder is selected from the group comprising rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases (including Crohn's disease and ulcerative colitis), nonhealing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythematosus (including systemic lupus erythematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, psoriatic arthritis, ankylosing spondylitis, myositis and cardiovascular disease.

67. The method, kit, biomarker or use of claim 66 wherein the inflammatory disorder is rheumatoid arthritis.

68. The method, kit, biomarker or use of any of claims 16 to 67 wherein the presence of the biomarker is diagnostic of an inflammatory condition; or prognostic for RA in the presence of synovial inflammation.

69. The method, kit, biomarker or use of any of claims 16 to 68 wherein the presence of the biomarker is prognostic of an inflammatory condition at least 5 years before onset of the condition.

70. The method, kit, biomarker or use of claim 69 wherein the inflammatory condition is rheumatoid arthritis.

71. The method, kit, biomarker or use of any of claims 16 to 70 wherein the sample is blood, serum, plasma, synovial fluid and/or joint tissue derived from the subject.

72. The method, kit, biomarker or use of any of claims 16 to 71 wherein the sample is pre-RA serum.

73. A binding member capable of specifically binding to a peptide according to any of claims 16 to 28, or a biomarker according to any of claims 29 to 35.

74. The binding member according to claim 73, wherein the binding member competes for binding with an autoantibody with specificity for an epitope of citrullinated tenascin-C or a fragment thereof wherein the epitope comprises at least three citrullinated arginine residues of 2187, 2192, 2197 2198 and 2200.

75. The binding member according to claim 73, wherein the binding member competes for binding with an autoantibody with specificity for an epitope of citrullinated tenascin-C or a fragment thereof wherein the epitope comprises at least three citrullinated arginine residues of 2187, 2192, 2197 and 2198.

76. The binding member according to claims 73 or 74, wherein the binding member competes for binding with an autoantibody with specificity for an epitope of citrullinated tenascin-C or a fragment thereof wherein the epitope comprises citrullinated arginine residues 2187, 2192, 2197 and 2198.

77. The binding member according to any of claims 73 to 76, wherein the binding member competes for binding with an autoantibody with specificity for an epitope of citrullinated tenascin-C or a fragment thereof wherein the epitope comprises citrullinated arginine residues 2187, 2192, 2197, 2198 and 2200.

78. The binding member according to any of claims 73 to 77, wherein the binding member is an antibody, antibody fragment or mimetic thereof.

79. The binding member according to any of claims 73 to 78, wherein the binding member has at least 10-fold higher affinity for binding to (i) a peptide according to any of claims 16 to 28 relative to an equivalent non-citrullinated peptide; or (ii) a citrullinated tenascin-C, or fragment thereof, biomarker according to any of claims 29 to 35 relative to an equivalent non-citrullinated tenascin-C or fragment thereof.

80. The binding member according to claim 78, wherein the affinity is at least 100-fold higher.

81. Use of the binding member according to any of claims 73 to 80, for the detection of the peptide according to any of claims 1 to 13, or the biomarker according to any of claims 14 to 21.

82. The use according to claim 81, wherein the detection is in a sample from a mammal, or in vivo in a mammal.

83. The use according to claim 81, wherein the mammal is human.

84. A peptide, method, kit, biomarker, binding member or use substantially as described herein, optionally with reference to the accompanying figures.

Description

[0273] Examples embodying an aspect of the invention will now be described with reference to the following figures:

[0274] FIG. 1: Citrullination of FBG by rPAD2.

[0275] Coomassie stained SDS gels (A) and immunoblot probed with an AMC antibody (B) of FBG citrullinated by rPAD2. FBG incubated in buffer with rPAD2 but without Calcium (Ca.sup.2+) or without enzyme (-PAD) served as negative controls. Untreated native FBG was loaded in the last well. (C) Arginine residues citrullinated by rPAD2, hPAD2 and hPAD4 were determined by LC-MS/MS. Arginine residues that were modified to citrulline are highlighted in blue (marked), all non-citrullinated arginines are shown in green (marked *). (D) The super secondary structure of native and citrullinated FBG (3 hours, 20 U rPAD2/mg FBG) was analysed by circular dichroism. (E) The melting temperature of native and citrullinated FBG (3 hours, 20 U rPAD2/mg FBG) was analysed by differential scanning fluorimetry. Results are shown as mean+/SD from 3 independent experiments.

[0276] FIG. 2: Identifying the citrullinated antibody epitope.

[0277] IgG response to citrullinated FBG peptides (cTNC) and arginine containing control peptides (rTNC) in patients with rheumatoid arthritis (RA; n=20) and healthy controls (HC, n=20). Mann-Whitney U test was used to calculate p values for differences between groups (ns=no significant difference, *=p<0.05 and **=p<0.01, ***=p<0.001, ****=p<0.001).

[0278] FIG. 3. Anti-cTNC antibody cross-reactivity with CEP-1, cVIM and cFIB.

[0279] Sera, double-reactive with peptides cTNC1 or cTNC5 and CEP1, cVIM or cFIB3 respectively, were pre-incubated with increasing concentrations of the indicated peptides, and the IgG response to cTNC1 (A) and cTNC5 (B) was measured.

[0280] FIG. 4: Anti-cTNC antibody cross-reactivity with homologous fibrinogen peptides.

[0281] (A) Multiple Sequence Alignment (Clustal Omega) of Tenascin-C, fibrinogen chain and fibrinogen chain. Arginines found citrullinated in vitro are highlighted in red, citrullinated arginines described as ACPA epitopes are underlined. (B and C) IgG response to cTNC1, cTNC5 and homologous citrullinated fibrinogen peptides was measured in sera positive for cTNC1 (B) or cTNC5 (C). (D) Sera, double-reactive to peptides cTNC1 or cTNC5 and the homologous fibrinogen peptides were pre-incubated with increasing concentrations of the indicated peptides, and IgG response to cTNC1 and cTNC5 was measured.

[0282] FIG. 5: Anti-cTNC5 antibody response in RA and pre-RA sera.

[0283] IgG response to cTNC5 and rTNC5 in serum samples from (A) 101 pre-RA individuals (pre-RA) and 326 controls, and (B) from 1985 RA patients (RA) and 150 healthy controls (HC) from the EIRA cohort. (C) IgG response to cTNC5 in the EIRA cohort in dAU when controlled for binding to arginine control peptide. dAU was calculated as described in Material and Methods. (D) IgG response to cTNC5 and rTNC5 in 287 serum samples from RA patients (RA) and 330 serum samples from osteoarthritis patients (OA) from an U.S. cohort. The red line indicates the mean. Blue dotted lines indicate cut-off for positivity. Mann-Whitney U test was used to calculate p values for differences between groups (n.s.=no significant difference, ****=p<0.001).

[0284] FIG. 6: Anti-cTNC antibody levels correlate with CCP2 levels.

[0285] IgG response to cTNC5 in (A) CCP2 positive (CCP2+, n=1255) and CCP2 negative (CCP2, n=730) sera of RA patients of the EIRA cohort, (B) CCP2 positive (n=240) and CCP2 negative (n=47) sera of RA patients of the U.S. cohort and (C) in CCP2 positive (n=27) and CCP2 negative (n=74) sera of pre-RA individuals. (D and E) Heat maps showing the presence (blue) or absence (red) of an IgG antibody response to ACPA epitopes in RA sera from the EIRA cohort (D) or pre-RA sera (E).

[0286] FIG. 7: Mass spectrometry analysis of citrullinated peptides derived from the FGB domain of Tenascin C (accession number P24821). The MS/MS spectra of tryptic peptides 2031-2041, 2042-2051, 2097-2012, 2143-2151, 2148-2157, 2186-2192, 2186-2197, 2193-2199 and 2186-2198 are shown, where r refers to the citrullinated position, indicating that R2033, R2050, R2098, R2147, R2151, R2187, R2192, R2197 and R2198. The matched fragment ions of the y-type (red) and b-type (blue) are shown. The MS/MS spectrum in the bottom panel indicates matches of predominant fragment ions to the peptide sequence.

[0287] FIG. 8: Relative quantitation of citrullinated peptide occurrence in FBG samples citrullinated with 2 Units of rPAD2, hPAD2 and hPAD4 for 24 hours. (No peptide TR(+0.98)YKLK was detected in the sample citrullinated with hPAD4, however this peptide was detected when citrullinated with 20 U of enzyme, data not shown).

[0288] FIG. 9: Correlation of anti-cTNC5 IgG levels with IgG levels of other ACPA in RA (A) and pre-RA (B) sera. Numbers in corners are percentage of positivity; numbers in parenthesis are percentage of positivity within the CCP2-positive group. The dotted lines indicate cut-off for positivity.

[0289] FIG. 10: Anti-cTNC5 antibody response in sera from patients with early synovitis. IgG response to cTNC5 and rTNC5 in serum samples from people with CCP negative RA (CCP RA)(n=53), CCP positive RA (CCP+ RA)(n=48), persistent non-RA arthritis (PNRA)(n=66) and resolving RA (resolving)(n=96). The solid line indicates the mean, and the black dotted lines indicate the cut-off for positivity. AU, arbitrary units; cTNC, citrullinated tenascin-C; rTNC, non-citrullinated tenascin-C.

IDENTIFICATION OF AN IMMUNODOMINANT PEPTIDE FROM CITRULLINATED TENASCIN-C AS A MAJOR TARGET FOR AUTOANTIBODIES IN RHEUMATOID ARTHRITIS

[0290] It was investigated whether citrullinated tenascin-C(cTNC), an extracellular matrix protein expressed at high levels in the joints of rheumatoid arthritis (RA) patients, is a target for the autoantibodies in RA.

[0291] Citrullinated sites were mapped by mass spectrometry in the fibrinogen-like globe (FBG) domain of tenascin-C treated with peptidyl arginine deiminases (PAD) 2 and 4. Antibodies to cyclic peptides containing citrullinated sites were screened in sera from RA patients by ELISA. Potential cross-reactivity with well-established anti-citrullinated protein antibody (ACPA) epitopes was tested by inhibition assays. The autoantibody response to one immunodominant cTNC peptide was then analysed in 101 pre-RA sera (median 7 years before onset) and two large independent RA cohorts.

[0292] Nine arginine residues within FBG were citrullinated by PAD2 and PAD4. Two immunodominant peptides cTNC1 (VFLRRKNG-cit-ENFYQNW) and cTNC5 (EHSIQFAEMKL-cit-PSNF-cit-NLEG-cit-cit-KR) were identified. Antibodies to both showed limited cross-reactivity with ACPA epitopes from a-enolase, vimentin and fibrinogen, and no reactivity with citrullinated fibrinogen peptides sharing sequence homology with FBG. cTNC5 antibodies were detected in 18% of pre-RA sera, and in 47% of 1,985 Swedish RA patients and 51% of 287 North American RA patients. The specificity was 98% compared to 160 healthy controls and 330 osteoarthritis patients.

[0293] There are multiple citrullination sites in the FBG domain of tenascin-C. Amongst these, one epitope is recognized by autoantibodies that are detected years before disease onset, and which can serve as a useful biomarker to identify ACPA-positive patients with high sensitivity and specificity in established disease.

[0294] The arginine rich domain of tenascin-C as a novel autoantigen is characterised herein by epitope mapping the FBG domain with a screening panel of RA sera, and examining the antigen specificity of two immunodominant epitopes by inhibition studies. An ELISA assay was standardised and used it to detect antibodies in pre-RA serum samples and two large independent patient cohorts with early and established RA.

[0295] All RA cases fulfilled the 1987 American College of Rheumatology (ACR) classification criteria [32]. 4 cohorts were examined, all from previously published studies with informed consent and ethical approval. (i) The screening cohort comprised 20 British RA patients and 20 healthy individuals[13]. (ii) The 101 pre-RA cases and 326 matched controls were identified in a nested case-control study in four Southern European cohorts[33]. (iii) 1,985 cases of RA and 160 controls were from the Swedish population-based case-control study EIRA (Epidemiological Investigation of RA)[34]. (iv) 287 patients with RA and 330 control patients with osteoarthritis (OA) were from the United States[35].

Citrullination Reaction

[0296] Recombinant human FBG[26] was incubated with rabbit skeletal muscle PAD2 (rPAD2), or recombinant human PAD2 or PAD4 (hPAD2, hPAD4), resolved on a SDS gel and stained with Coomassie-blue or Western blotted with a monoclonal human anti-modified citrulline (AMC) antibody (Modiquest Research, clone C4, 1:500).

LC-MS/MS Analyses

[0297] Citrullinated FBG was resolved on a 12% SDS gel, Coomassie-stained protein bands were excised and in gel-digestion performed as described[36]. Peptides were analysed by LC-MS/MS.

Peptides, ELISAs and Cross-Reactivity Assay

[0298] ELISAs were used to detect antibodies against citrullinated peptides in human sera as described[13]. Briefly, 96-well plates were coated with 10 g/ml peptide, blocked with 2% BSA and incubated with sera diluted 1:100. Bound antibodies were detected with an HRP conjugated anti-human IgGFc monoclonal antibody (Jacksonfor EIRA study, Stratechfor all other ELISAs). A standard curve of positive sera was used to calculate relative antibody titres in arbitrary units (AU) for each sample. Subtraction of the OD450 of the native peptide from the OD450 of the citrullinated peptide was used to correct reactivity and dOD450 values were transformed into arbitrary units using the standard curve (dAU).

EIRA Cohort

[0299] EIRA controls were randomly selected from the population registry, to match EIRA cases on age-, gender- and residential area [34]. Smoking data was collected by questionnaire at baseline. Subjects were categorized as ever-smokers or never-smokers [34]. HLA-DRB1 subtyping and genotyping of the protein tyrosine phosphatase gene (PTPN22 rs2476601) was described before [57,58].

Citrullination Reaction

[0300] Recombinant human FBG [26] was incubated with rabbit skeletal muscle PAD2 (rPAD2) (Sigma Aldrich), or recombinant human PAD2 or PAD4 (hPAD2, hPAD4) (Modiquest Research) in citrullination buffer (50 mM Tris pH 8, 150 mM NaCl, 10 mM CaCl.sub.2) with 5 mM DTT for 3, 8 or 24 hours at 37 C. 1 g of each sample was resolved on a 12% SDS gel and stained with Coomassie-blue or Western blotted with a monoclonal human anti-modified citrulline (AMC) antibody (Modiquest Research, clone C4, 1:500), following chemical modification of the membrane (0.0125% FeCl.sub.3, 2.3 M H.sub.2SO.sub.4, 1.52 M H.sub.3PO.sub.4, 0.25 M Acetic Acid, 0.25% 2, 3-butanedione monoxime, 0.125% antipyrine).

LC-MS/MS Analyses

[0301] Peptides were analysed on a LC-MS/MS workflow comprising a Dionex Ultimate 3000 nLC system coupled to a Q-Exactive mass spectrometer (Thermo Scientific) [59,51]. Briefly, chromatographic separation was achieved using a 50 cm nEASY spray column (Thermo Scientific, (PepMAP C18, 75 m500 mm, 2 m particle size) and a linear acetonitrile gradient from 2-35% in 5% DMSO and 0.1% formic acid. Precursor peptides were detected with a resolution of 70000 at 200 m/z followed by the selection of up to 15 precursor ions. Raw data was imported into Progenesis QI (Waters, V4.1.4832.42146) for label-free quantitation and alignment, and peptides were identified with PEAKS (Bioinformatics Solutions, V7). The abundance of citrullinated peptides was normalised to the abundance of their non-citrullinated counterparts, which value was further normalised to the citrullinated peptide abundance detected in a non-citrullinated FBG control sample.

Differential Scanning Fluorimetry and Circular Dichroism

[0302] Five M FBG was mixed with 5SYPRO orange protein gel stain (Life Technologies) in citrullination buffer and melting curves from 38 C. up to 99 C. recorded on the Applied Biosystems ViiA 7 Real-Time PCR System (excitation 550 nm, emission, 586 nm). The melting temperature (Tm) of each sample was defined as the maximum of the first derivate of the fluorescence. Circular dichroism spectra of FBG samples (200 g/ml) were recorded using a Jasco J-815 CD Spectrometer, between 210 and 270 nm.

Peptides

[0303] Peptides from tenascin-C(accession number P24821) and fibrinogen b (P02675) and g (P02679) chains (Table 1 and 2) were synthesized at a purity >90-95%, with C- and N-terminal cysteines (Pepceuticals) and solubilized in water at 10 mg/ml. CEP-1, cVIM and cFIBP peptides used for cross-reactivity assays are described elsewhere[33].

Crossreactivity ELISA

[0304] Cross-reactivity was analysed in human sera that were reactive to both cTNC1 or cTNC5 and CEP-1, cVIM, cFIB or citrullinated homologous fibrinogen peptides. Sera were diluted 1:100, incubated with 1, 10 and 100 g/ml of peptides for 2 hours, centrifuged at 10,000 g for 10 min and the supernatant added to peptide-coated plates for analysis by ELISA as described in Material and Methods.

Statistical Analysis and Software

[0305] Mann-Whitney U tests were used to calculate p values for differences between groups (GraphPad Prism). The 98% percentile of healthy control samples was used to define the cut-off for positive antibody levels. Chi-square tests were used to compare frequency distributions of categorical variables while t-tests and Wilcoxon rank-sum tests were used to compare all continuous variables. To determine the association of smoking, HLA-DRB1 SE (subtypes DRB1*01 (except DRB1*0103), DRB1*04 and DRB1*10), HLA-DRB1*13 and PTPN22 with different RA subsets, odds ratios (OR) with 95% Confidence Intervals (95% CI) were calculated using unconditional logistic regression models, with unexposed cases and controls as reference group. Analyses were adjusted for age, gender and residential area. All analyses were performed using SAS version 9.3. Heatmaps were generated using G-ProX and three-dimensional models were created using Pymol (Schrodinger, LLC).

FBG is Citrullinated In Vitro by PAD2 and PAD4

[0306] FBG was citrullinated by rPAD2, demonstrated by a small increase in the molecular weight on Coomassie-stained SDS-PAGE and Western Blotting with an AMC antibody (FIG. 1A, B). Mass spectrometry analysis of citrullinated FBG covered 14 of the 17 arginines present in this domain of tenascin-C, of which 9 were citrullinated (FIG. 1C, FIG. 7). rPAD2, hPAD2 and hPAD4 each citrullinated the same sites within FBG with no major difference in the degree of citrullination observed (FIG. 8). Circular dichroism showed comparable spectra between native FBG and citrullinated FBG (FIG. 1D) indicating that citrullination of FBG does not impact the secondary structure of the protein. Differential scanning fluorimetry however revealed a significantly lower melting temperature of citrullinated FBG (46.5+/0.2) compared to FBG (54.3+/0.1) (FIG. 1E), demonstrating that citrullination leads to partial protein unfolding.

cTNC1 and cTNC5 are the Primary Epitopes Recognised by ACPA in RA Patients

[0307] Five tenascin-C cyclic peptides encompassing the citrullinated residues identified by mass-spectrometry, together with their arginine-containing controls (Table 1), were used to map antibody response in a screening panel of serum samples from 20 patients with RA, and from 20 healthy subjects, by ELISA. Antibodies to citrullinated tenascin-C (cTNC) peptides cTNC1 and cTNC5 were detected in serum samples from 35% and 40% of patients respectively, but not in control sera, with no response against the arginine-containing control peptides (rTNC). There was little or no reactivity with the other three peptides tested (FIG. 2). Therefore cTNC1 and cTNC5 were selected for further study.

Anti-cTNC Antibodies Show Limited Cross-Reactivity with Other ACPA Epitopes

[0308] To examine epitope specificity and potential cross-reactivity of anti-cTNC antibodies with already identified ACPA antigens, inhibition experiments were performed with the well-defined peptides of CEP-1 (.sup.5KIHA-cit-EIFDS-cit-GNPTVE.sup.21), cVIM (.sup.59VYAT-cit-SSAV-cit-L-cit-SSVP.sup.74) and cFIBP (.sup.36NEEGFFSA-cit-GHRPLDKK.sup.52). Absorption by the homologous peptides was more efficient for cTNC5 than cTNC1. There was no cross-reactivity between anti-cTNC1 and cVIM and cFIB3, though there was some inhibition by the CEP-1 peptide (17 to 70% inhibition) (FIG. 3A). In contrast, there was no cross-reactivity between anti-cTNC5 and CEP-1, while these antibodies showed limited cross-reactivity with cVIM and cFIB3 in one serum sample (inhibition by 58% and 50%, respectively) (FIG. 3B).

[0309] Because the FBG domain of tenascin-C exhibits some sequence homology with fibrinogen we also analysed whether anti-cTNC antibodies cross-react with epitopes on citrullinated peptides containing similar sequences of fibrinogen chain (cFib.sup.281-296) and fibrinogen chain (cFib.sup.474-491, cFib.sup.409-426) (FIG. 4A). From 17 sera reactive with cTNC1, 7 also reacted with cFib.sup.281-296 (FIG. 4B), from 19 sera reactive with cTNC5, 14 also reacted with cFib.sup.474-491, and 9 with cFib.sup.409-426 (FIG. 4C). To examine whether this dual positivity was due to true cross-reactivity, 4 samples that were double positive for cTNC and citrullinated fibrinogen peptide IgG were tested by inhibition experiments. No reduced reactivity to cFBG epitopes was observed when sera were pre-incubated with citrullinated fibrinogen and peptides (FIG. 4D).

Anti-cTNC5 is Detected in Pre-RA Sera and with Moderate-to-High Sensitivity in Early and Established RA

[0310] In 101 pre-RA sera (median 7 years before diagnosis), 18% of pre-RA sera were positive for anti-cTNC5 antibodies (FIG. 5A) compared to 2% of 326 sera from controls. No antibodies against cTNC1 were detected. Therefore, because cTNC5 appeared to have better antigen specificity in our absorption experiments and a higher frequency of antibodies in both pre-RA and established RA, further analysis focused on cTNC5.

[0311] In the EIRA cohort, 47% of 1,985 RA sera and 2% of 160 healthy control sera were positive for antibodies to cTNC5, indicating a diagnostic sensitivity of 47% and specificity of 98%. Within the RA sera, 2.5% also bound the arginine-control peptide rTNC5 (FIG. 5B), and when controlling for binding to the arginine control peptide the sensitivity remained moderately high at 41% (FIG. 5C). This figure was higher than the frequency of citrulline-specific antibodies to CEP-1 (35%), anti-cVIM (37%) and anti-cFIB3 (37%), measured in the same cohort by ELISA[14].

[0312] We confirmed the moderate-to-high diagnostic sensitivity of cTNC5 (51%) in an independent US cohort of 287 sera from RA patients and 330 sera from osteoarthritis disease controls (FIG. 5D). In this cohort the binding to rTNC5 was not increased compared to OA.

[0313] Anti-cTNC5 reactivity was significantly higher in CCP2 positive patients compared to CCP2 negative patients in both RA (FIG. 6A, B) and pre-RA samples (FIG. 6C). Anti-cTNC5 antibody largely overlapped with other ACPA (FIG. 6D, E, FIG. 9) in the RA and pre-RA cohort. However in the EIRA cohort 5.4% of the serum samples exclusively reacted with the cTNC5 peptide alone. In the anti-CCP2 negative samples 4.9% reacted with cTN5.

[0314] In EIRA cTNC5 positive RA was associated with smoking (OR 1.65 vs 1.26) and HLA-DRB1 shared epitopes (OR 4.98 vs 1.68), but not with PTPN22 (OR 1.77 vs 1.44) when compared to the cTNC5 positive/CCP2 negative RA subset (Table 9). We also analysed whether cTNC5 antibodies are associated with specific HLA-DRB1 epitopes and found that cTNC5 antibodies did not associate with DRB1*10 alleles, but with HLA-DRB1*01 and DRB1*04 alleles (Table 4). Antibodies against cTNC5 negatively associated with HLA-DR13 (Table 5).

[0315] In the US cohort, cTNC5 antibody positivity was significantly associated with disease activity (DAS 28-CRP), but did not associate with other analysed clinical parameters (disease duration, swollen and tender joints, sharp score and erosion score) (Table 6).

[0316] In this study, we describe a novel citrullinated peptide from the FBG domain of tenascin-C. The citrullinated residues can be generated by either PAD2 or PAD4, yielding epitopes that are recognised by antibodies in approximately 1 of every 5 individuals with pre-clinical RA and with a moderate-to-high diagnostic sensitivity in early and established disease. Inhibition assays and analysis of antibodies to other well-characterised peptides indicate that anti-cTNC5 antibody status is independent of reactivity to other citrullinated peptides. Even though a large number of antigenic citrullinated peptides have been described as reactive with ACPA in previous reports, few have been examined with the stringent criteria used in this study. Therefore, our findings suggest that cTNC5 is a novel and independent addition to the relatively small number of citrullinated peptides which are genuinely targeted by ACPA, and which may have a role in both clinical diagnosis and investigating pathogenesis in RA.

[0317] The FBG domain of tenascin-C was citrullinated in vitro by PAD2 and PAD4. Whilst these enzymes have different substrate specificities[37], both modified the same 9 arginines in FBG to a similar degree. Lack of citrullination of 5 other arginines in FBG by any PAD reflects the specificity of this modification, likely due to hindered accessibility of these residues, or unfavourable neighbouring amino acids. Citrullinated arginines were located at 5 distinct sites within FBG, of which two, cTNC1 and cTNC5, were reactive with sera from RA patients. However, antibodies to only one, cTNC5, were detected in sera of pre-RA cases.

[0318] The different associations of cTNC1 and cTNC5 suggest that cTNC5 may be important in priming the ACPA response, whereas antibodies to cTNC1 may arise as a result of epitope spreading in more established disease. These data also reflect that the autoantibody response in RA is not citrulline-specific; instead it depends on the whole epitope around the modified residue including neighboring amino acids and the three dimensional structure[10]. It is well documented that distinct ACPA responses to different citrullinated epitopes within one protein exist, as described for example for citrullinated -enolase[13] or fibrinogen[38]. The peptide sequence of cTNC5 is predicted to form a very distinct, exposed structure at the very C-terminus of tenascin-C, potentially rendering it more easily accessible to ACPA than cTNC1. In addition, four sites are citrullinated within TNC5, compared to only a single citrullinated site within cTNC1, which may also contribute to the higher frequency of cTNC5 ACPA observed. The frequency of anti-cTNC5 antibodies in the pre-RA cohort (18%) is comparable to antibody frequencies described for other ACPA in the same cohort, including cFIB (18%) and CEP-1 (15%)[33]. Analysis of a large cohort of patients with early RA, demonstrated moderate-to-high sensitivity of RA samples for cTNC5 (47%). This is the highest recorded sensitivity for any single antigenic peptide in this cohort, in this case compared to a 35%-37% sensitivity for antibodies to each of the three other antigenic peptides[14]. We also found reactivity to cTNC5 at a similar frequency (51%) in a second cohort of RA sera from U.S. patients.

[0319] ACPA generally show limited cross-reactivity[14,39]. In line with these reports, we showed that antibodies to cTNC exhibited little cross-reactivity with citrullinated enolase, vimentin and fibrinogen peptides, and are distinct from antibodies reacting with peptides from homologous regions in fibrinogen. cTNC5 ACPA positive sera were mostly found within the anti-CCP2 antibody positive RA population with cTNC5 antibody levels highest in the anti-CCP2 antibody positive subgroup, as described for other ACPA[14]. 4.9% of RA patients within the anti-CCP2 antibody negative group were also anti-cTNC5 ACPA positive, demonstrating that not all ACPA-positive patients can be detected by testing for CCP reactivity. Moreover, a subset of CCP positive RA patients was single-positive for cTNC5 antibodies (5.4%), revealing cTNC5 as a distinct ACPA fine specificity in RA sera and indicating that assaying this ACPA alone would be helpful in diagnosing patients that might otherwise be missed. Combined testing for several specific ACPAs has been shown to increase both diagnostic sensitivity and specificity[0]. Together these data suggest that both the addition of cTNC5 to an assay combining multiple ACPA, as well as analysis of anti-cTNC5 alone, might be beneficial approaches in diagnosing RA.

[0320] HLA-DRB1 SE alleles are associated with ACPA-positive RA[41]. We found a strong association of anti-cTNC5 antibodies with SE positivity, as has been described for antibodies to other citrullinated antigens, like CEP-1 and cVIM[14,15]. cTNC5 antibodies mainly associated with HLA-DRB1 subtypes DRB1*04 as described for other ACPA[15,39]. HLA-DR13 alleles protect against ACPA positive RA[42], and we show here that it is also protective against cTNC5 positive RA. However, we did not observe a statistical significant association of PTPN22, another genetic risk factor for RA[43], with anti-cTNC5 positive RA. Smoking is a well-established environmental risk factor for ACPA positive RA[34,44] and here we describe a positive association of cTNC5 positive RA with smoking in the EIRA cohort, similarly as it has been described for antibodies against CEP-1 and cVIM[14,15]. Smoking-induced inflammation, in the context of chronic obstructive pulmonary disease (COPD), is associated with enhanced citrullination and may contribute to the generation of ACPA[45,46]. Interestingly, high tenascin-C expression was detected in lungs of COPD patients compared to non-smokers[4-8]. Another risk factor for RA, is periodontitis[49]. Porphyromonas gingivalis is a major periodontal pathogen and possesses a unique bacterial PAD enzyme (PPAD) which citrullinates both bacterial and endogenous host peptides[50,51]. Tenascin-C is also expressed in periodontal tissue, and tenascin-C fragments were detected in gingival crevicular fluid of periodontitis patients[52]. Our results and these studies therefore reveal potential mechanisms for the generation of antigenic cTNC peptides in RA.

[0321] ACPAs are produced locally within the RA joint and may contribute directly to disease pathogenesis[53]. For example immune complexes containing citrullinated fibrinogen stimulate cytokine synthesis in macrophages via activation of Fc-receptor and TLR4 and, due to the homology of fibrinogen and the FBG domain of tenascin-C, it is conceivable that immune complexes containing citrullinated tenascin-C may contribute to disease pathogenesis through a similar mechanism. Furthermore, citrullinated proteins themselves can be pathogenic, as described for citrullinated fibrinogen [54]. It will be interesting to see if ACPA for cTNC5 bind to citrullinated tenascin-C found within the RA joint and trigger cytokine formation in the form of immune complexes, or whether citrullination of the FBG domain enhances its activation of TLR4[26]. The citrullinated FBG peptide previously detected in RA synovial fluid[31] comprised the sequence we found in cTNC1. However, further citrullinated sites and ACPA epitopes are likely to be found in other domains of tenascin-C. For example the fibronectin type-III like repeats that share sequence homology with fibronectin, a molecule also found in synovial fluid and which is targeted by the autoimmune response in RA[55].

[0322] The CCP positive subset of RA patients is linked with a more severe disease development and worse prognosis[5,7,8]. However, no association of specific ACPAs with clinical parameters has been described so far[56]. Here, we found that cTNC5 antibodies do not correlate with a number of clinical parameters, however, there was a significant association of cTNC5 antibodies with disease activity (DAS28-CRP), suggesting that cTNC5 antibodies may be useful tool for predicting clinical outcome.

[0323] In conclusion, we describe an immunodominant peptide from tenascin-C, which is distinct from the other major antigenic citrullinated peptides described to date, and superior in terms of diagnostic sensitivity and specificity when used as an antigen in ELISA. Furthermore, previous demonstrations of the pro-inflammatory effects of tenascin-C and its detection at site of inflammation suggest that immune responses to the FBG domain may be important in the aetiology and pathogenesis of RA.

Abbreviations

[0324] American College of Rheumatology (ACR) [0325] anti-citrullinated protein antibody (ACPA) [0326] anti-modified citrulline (AMC) [0327] arbitrary units (AU) [0328] chronic obstructive pulmonary disease (COPD) [0329] citrullinated -enolase peptide 1 (CEP-1) [0330] citrullinated fibrinogen (cFib) [0331] citrullinated vimentin (cVim) [0332] cyclic-citrullinated peptide (CCP) [0333] extracellular matrix (ECM) [0334] Epidemiological Investigation of Rheumatoid Arthritis (EIRA) [0335] fibrinogen-like globe (FBG) [0336] melting temperature (Tm) [0337] odds ratio (OR) [0338] osteoarthritis (OA) [0339] peptidyl arginine deiminases (PAD) [0340] Porphyromonas gingivalis PAD (PPAD) [0341] rheumatoid arthritis (RA) [0342] shared epitope (SE) [0343] toll-like receptor 4 (TLR4)

TABLE-US-00001 TABLE1 SequencesofFBGpeptidesusedforELISAs. FBGpeptides Sequence rTNC1(aa2026-2040) CVFLRRKNG-R-ENFYQNWC cTNC1 CVFLRRKNG-cit-ENFYQNWC rTNC2(aa2042-2058) CAYAAGFGD-R-REEFWLGLC cTNC2 CAYAAGFGD-cit-REEFWLGLC rTNC3(aa2091-2106) CFSVGDAKT-R-YKLKVEGYC cTNC3 CFSVGDAKT-cit-YKLKVEGYC rTNC4(aa2141-2157) CKGAFWY-R-NCH-R-VNLMGRC cTNC4 CKGAFWY-cit-NCH-cit-VNLMGRC rTNC5(aa2183-2200) CEMKL-R-PSNF-R-NLEG-R-R-KRC cTNC5 CEMKL-cit-PSNF-cit-NLEG- cit-cit-KRC

TABLE-US-00002 TABLE2 Fibrinogenpeptideshomologous tocitrullinatedFBGpeptides. Fibrinogen peptides Sequence cFIB.sup.281-296 CVIQN-cit-QDGSVDFG-cit-KWC cFIB.sup.474-491 CWYSMRKMSMKI-cit-PFFPQQC cFIB.sup.409-426 CTMKIIPFN-cit-LTIGEGQQHC

TABLE-US-00003 TABLE 3 Association between smoking, any SE, PTPN22 and different RA subgroups (in EIRA). Exposure p-value Subgroup Never smokers Ever smokers OR (95% CI) (+/+ vs +/) Controls 2114(43.46) 2750(56.54) 1.0 ref. CCP2/cTNC5 / 85(41.87) 118(58.13) 0.86 (0.64-1.17) /+ 12(30.00) 28(70.00) 1.45 (0.72-2.93) +/ 201(33.84) 393(66.16) 1.26 (1.03-1.53) +/+ 309(27.01) 835(72.99) 1.65 (1.41-1.93) 0.0049 None SE Any SE Controls 959(49.87) 964(50.13) 1.0 ref. CCP2/cTNC5 / 94(46.31) 108(53.20) 1.15 (0.85-1.56) /+ 15(35.71) 27(64.29) 1.75 (0.91-3.34) +/ 218(36.64) 368(61.85) 1.68 (1.37-2.06) +/+ 191(16.68) 941(82.18) 4.98 (4.11-6.04) <0.0001 None PTPN22 Any PTPN22 Controls 1533(79.18) 403(20.82) 1.0 ref. CCP2/cTNC5 / 126(62.07) 50(24.63) 1.65 (1.13-2.41) /+ 24(57.14) 12(28.57) 2.15 (1.03-4.49) +/ 371(62.35) 136(22.86) 1.44 (1.12-1.86) +/+ 682(59.56) 314(27.42) 1.77 (1.44-2.18) 0.08

TABLE-US-00004 TABLE 4 Associations between DR*01, *04 and *10 and different RA subgroups with the combination of presence or absence of CCP and cTNC5. OR were adjusted for age, gender, residential area, smoking, alcohol consumption and other SE. p-value Exposure (+/+ Subgroup None Any OR**(95% CI) vs +/) DR*01 Controls 375(59.43) 256(40.57) 1.0ref. CCP2/cTNC5 / 62(30.54) 45(22.17) 0.91(0.40-2.11) /+ 17(40.48) 10(23.81) NA +/ 238(40.00) 129(21.68) 1.00(0.62-1.63) +/+ 627(54.76) 310(27.07) 1.81(1.26-2.60) 0.01 DR*04 Controls 220(34.87) 411(65.13) 1.0ref. CCP2/cTNC5 / 41(20.20) 66(32.51) 0.77(0.33-1.84) /+ 13(30.95) 14(33.33) NA +/ 110(18.49) 257(43.19) 1.29(0.78-2.15) +/+ 192(16.77) 745(65.07) 3.46(2.35-5.11) <0.0001 DR*10 Controls 609(96.51) 22(3.49) 1.0ref. CCP2/cTNC5 / 102(50.25) 5(2.46) 1.18(0.36-3.87) /+ 24(57.14) 3(7.14) NA +/ 351(58.99) 16(2.69) 1.53(0.71-3.29) +/+ 898(78.43) 39(3.41) 2.11(1.15-3.88) 0.13 NAnot analysed.

TABLE-US-00005 TABLE 5 Association between DR13 and different RA subgroups with the combination of presence or absence of CCP and cTNC5. OR were adjusted for age, gender, residential area, smoking, alcohol consumption and any SE. Exposure Subgroup No DR13 Any DR13 OR (95% CI) p-value Controls 503(79.71) 128(20.29) 1.0ref. CCP2/cTNC5 / 86(42.36) 21(10.34) 0.84(0.49-1.46) /+ 25(59.52) 2(4.76) 0.31(0.07-1.34) (/+ vs /) 0.16 +/ 315(52.94) 52(8.74) 0.67(0.47-0.97) +/+ 867(75.72) 70(6.11) 0.30(0.22-0.42) .sup.(+/+ vs .sup.+/) 0.0003

TABLE-US-00006 TABLE 6 Association of cTNC5 antibodies with RA disease characteristics. Numbers in brackets are s.d. cTNC5 cTNC5 Total positive negative Characteristic (N = 287) (N = 145) (N = 142) p-value Age, years 60 (12) 58 (11) 0.252 Male gender 68 58 0.084 Dis. duration, years 13 (10) 12 (9) 0.189 Swollen joints 3.9 (4.7) 3.2 (3.8) 0.212 Tender joints 3.4 (4.8) 2.9 (4.4) 0.366 Patient global (0-10) 4.5 (2.8) 3.9 (2.5) 0.067 DAS-28-CRP 4.1 (1.4) 3.8 (1.3) 0.044 Sharp score 21 (23) 17 (22) 0.061 Erosion score 5 (8) 4 (8) 0.632 Anti-CCP, U/ml 187 (123) 101 (117) <0.001

TABLE-US-00007 TABLE7 Peptide Protein Aminoacids AminoAcidsequence CEP-1/Eno5-21 -Enolase 5-21 CKIHA(cit)EIFDS(cit)GNPTVEC(cyclic) Vim60-75 Vimentin 60-75 VYAT(cit)SSAV(cit)L(cit)SSVP Vim2-17 Vimentin 2-17 ST(cit)SVSSSSY(cit)(cit)MFGG CCP1/Fil307-324 Filaggrin 307-324 SHQEST(cit)GRSRGRSGRSGS(cyclic) Fib36-52 Fibrinogen-chain 36-52 NEEGFFSA(cit)GHRPLDKK Fib563-583 Fibrinogen-chain 563-583 HHPGIAEFPS(cit)GKSSSYSKQF Fib580-600 Fibrinogen-chain 580-600 SKQFTSSTSYN(cit)GQSTFESKS Fib62-81a.sup.a Fibrinogen-chain 62-81 APPPISGGGY(cit)ARPAKAAAT Fib62-81b.sup.b Fibrinogen-chain 62-81 APPPISGGGYRA(cit)PAKAAAT Fib60-74 Fibrinogen-chain 60-74 (cit)PAPPPISGGGY(cit)A(cit) Fib621-635 Fibrinogen-chain 621-635 (cit)GHAKS(cit)PV(cit)GIHTS citCI/CII359-369 CollagentypeII 359-369 (GPO)S-GA(cit)GLTG(cit)PGDA(GPO)2-GKKYG

TABLE-US-00008 Sequences TNCsequence-AccessionNo.P24821 >sp|P24821|TENA_HUMANTenascinOS= Homosapiens GN= TNCPE= 1SV= 3 SEQIDNO:1 MGAMTQLLAGVFLAFLALA1EGGVLKKVIRHKRQSGVNATLPEENQPVV FNHVYNIKLPVGSQCSVDLESASGEKDLAPPSEPSESFQEHTVDGENQI VFTHRINIPRRACGCAAAPDVKELLSRLEELENLVSSLREQCTAGAGCC LQPATGRLDTRPFCSGRGNFSTEGCGCVCEPGWKGPNCSEPECPGNCHL RGRCIDGQCICDDGFTGEDCSQLACPSDCNDQGKCVNGVCICFEGYAGA DCSREICPVPCSEEHGTCVDGLCVCHDGFAGDDCNKPLCLNNCYNRGRC VENECVCDEGFTGEDCSELICPNDCFDRGRCINGTCYCEEGFTGEDCGK PTCPHACHTQGRCEEGQCVCDEGFAGVDCSEKRCPADCHNRGRCVDGRC ECDDGFTGADCGELKCPNGCSGHGRCVNGQCVCDEGYTGEDCSQLRCPN DCHSRGRCVEGKCVCEQGFKGYDCSDMSCPNDCHQHGRCVNGMCVCDDG YTGEDCRDRQCPRDCSNRGLCVDGQCVCEDGFTGPDCAELSCPNDCHGQ GRCVNGQCVCHEGFMGKDCKEQRCPSDCHGQGRCVDGQCICHEGFTGLD CGQHSCPSDCNNLGQCVSGRCICNEGYSGEDCSEVSPPKDLVVTEVTEE TVNLAWDNEMRVTEYLVVYTPTHEGGLEMQFRVPGDQTSTIIQELEPGV EYFIRVFAILENKKSIPVSARVATYLPAPEGLKFKSIKETSVEVEWDPL DIAFETWEIIFRNMNKEDEGEITKSLRRPETSYRQTGLAPGQEYEISLH IVKNNTRGPGLKRVTTTRLDAPSQIEVKDVTDTTALITWFKPLAEIDGI ELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPDTEYEVSLISRRGDMSS NPAKETFTTGLDAPRNLRRVSQTDNSITLEWRNGKAAIDSYRIKYAPIS GGDHAEVDVPKSQQATTKTTLTGLRPGTEYGIGVSAVKEDKESNPATIN AATELDTPKDLQVSETAETSLTLLWKTPLAKFDRYRLNYSLPTGQWVGV QLPRNTTSYVLRGLEPGQEYNVLLTAEKGRHKSKPARVKASTEQAPELE NLTVTEVGWDGLRLNWTAADQAYEHFIIQVQEANKVEAARNLTVPGSLR AVDIPGLKAATPYTVSIYGVIQGYRTPVLSAEASTGETPNLGEVVVAEV GWDALKLNWTAPEGAYEYFFIQVQEADTVEAAQNLTVPGGLRSTDLPGL KAATHYTITIRGVTQDFSTTPLSVEVLTEEVPDMGNLTVTEVSWDALRL NWTTPDGTYDQFTIQVQEADQVEEAHNLTVPGSLRSMEIPGLRAGTPYT VTLHGEVRGHSTRPLAVEVVTEDLPQLGDLAVSEVGWDGLRLNWTAADN AYEHFVIQVQEVNKVEAAQNLTLPGSLRAVDIPGLEAATPYRVSIYGVI RGYRTPVLSAEASTAKEPEIGNLNVSDITPESFNLSWMATDGIFETFTI EIIDSNRLLETVEYNISGAERTAHISGLPPSTDFIVYLSGLAPSIRTKT ISATATIEALPLLENLTISDINPYGFTVSWMASENAFDSFLVTVVDSGK LLDPQEFTLSGTQRKLELRGLITGIGYEVMVSGFTQGHQTKPLRAEIVT EAEPEVDNLLVSDATPDGFRLSWTADEGVFDNFVLKIRDTKKQSEPLEI TLLAPERTRDITGLREATEYEIELYGISKGRRSQTVSAIATTAMGSPKE VIFSDITENSATVSWRAPTAQVESFRITYVPITGGTPSMVTVDGTKTQT RLVKLIPGVEYLVSIIAMKGFEESEPVSGSFTTALDGPSGLVTANITDS EALARWQPAIATVDSYVISYTGEKVPEITRTVSGNTVEYALTDLEPATE YTLRIFAEKGPQKSSTITAKFTTDLDSPRDLTATEVQSETALLTWRPPR ASVTGYLLVYESVDGTVKEVIVGPDTTSYSLADLSPSTHYTAKIQALNG PLRSNMIQTIFTTIGLLYPFPKDCSQAMLNGDTTSGLYTIYLNGDKAEA LEVFCDMTSDGGGWIVFLRRKNGRENFYQNWKAYAAGFGDRREEFWLGL DNLNKITAQGQYELRVDLRDHGETAFAVYDKFSVGDAKTRYKLKVEGYS GTAGDSMAYHNGRSFSTFDKDTDSAITNCALSYKGAFWYRNCHRVNLMG RYGDNNHSQGVNWFHWKGHEHSIQFAEMKLRPSNFRNLEGRRKRA
Detection of Antibodies to Citrullinated Tenascin-C in Patients with Early Synovitis is Associated with the Development of Rheumatoid Arthritis

[0344] Early treatment of rheumatoid arthritis (RA) results in more effective disease suppression and can be key to a successful patient response. However, not all patients with early synovitis develop RA; for example, in some, synovial inflammation resolves spontaneously. The factors that drive RA development remain unclear and clinical tools to predict RA development are imperfect.

[0345] Tenascin-C is a pro-inflammatory matrix molecule that is absent from healthy joints but highly expressed in the joints of RA patients. We identified an immunodominant peptide in citrullinated tenascin-C, cTNC5, antibodies against which are detected in around half of RA patients, and can be found years before disease onset in some patients. Here, we sought to determine if anti-cTNC5 antibodies can discriminate amongst patients with early synovial inflammation those who develop RA and those with other outcomes.

[0346] Sera from 263 patients in the Birmingham early arthritis cohort were analysed. Patients were DMARD nave with clinically apparent synovitis of 1 joint and with inflammatory joint symptoms of 3 months duration. At 18 month follow-up patients were assigned to the following outcome categories: RA according to ACR 2010 criteria (Arnett F C, et al. Arthritis and rheumatism. 1988; 31(3):315-24.) (RA, n=101), persistent non-RA arthritis (PNRA, n=66) and resolving arthritis (no clinically apparent joint swelling, no DMARD/steroid use in the previous 3 months, n=96). Demographic and clinical parameters were recorded, and RA patients divided into anti-CCP antibody positive and negative subsets (Raza K, et al. The Journal of rheumatology. 2005; 32(2):231-8; and Raza K, et al. Arthritis research & therapy. 2005; 7(4):R784-95). Antibodies recognizing cTNC5 or a non-citrullinated peptide (rTNC5) were analysed by ELISA as described (Schwenzer A, et al. Annals of the rheumatic diseases. 2015).

[0347] Anti-cTNC5 antibodies were found in 40.6% of people with early synovitis that went on to develop RA, and were significantly more prevalent in anti-CCP antibody +ve compared to anti-CCP antibody ve RA patients (81.3 vs. 3.8%, p<0.0001). Anti-cTNC5 antibodies were detected in a low proportion of people who developed PNRA (6.1%), or whose disease resolved (3.1%). No significant antibody response to rTNC5 was detected (p=0.527) (Table 8, FIG. 10). Anti-cTNC5 levels were higher in anti-CCP antibody +ve RA patients (193.1449.8 AU) compared to patients with in anti-CCP antibody ve RA (3.563.30 AU), PNRA (19.42122.7 AU) and resolving arthritis (6.6028.02 AU) ANOVA p<0.0001).

[0348] Whilst anti-cTNC5 was not better at predicting the development of RA than anti-CCP antibody (specificity; sensitivity: 40.6%; 95.7% (cTNC5), 47.5%; 98.8% (CCP), anti-cTNC5 did detect a subset of patients that developed RA who were not anti-CCP antibody positive (3.8%). Anti-cTNC5 antibody positive RA patients were more frequently anti-CCP antibody and RF positive than anti-cTNC5 antibody negative patients (Table 9).

[0349] In addition to anti-cTNC5 predicting the development of RA, cTNC5 positive individuals had significantly higher CRP and ESR levels, higher disease activity scores, and higher tender and swollen joint counts than cTNC5 negative individuals (Table 10); there has previously been reported no difference in clinical phenotype between CCP+ve or CCPve RA patients (Cader M Z, et al. BMC musculoskeletal disorders. 2010; 11:187).

[0350] Together these data reveal that detection of anti-cTNC5 antibodies in the sera of patients with early synovitis is associated with the development of RA, and particularly with high levels of disease activity. This study therefore highlights a potential role for citrullinated tenascin-C in the biological pathways underlying the differentiation of early synovitis towards RA and away from disease resolution.

TABLE-US-00009 TABLE 8 Demographic, clinical and laboratory characteristics of patients in each outcome group. Anti-CCP Anti-CCP Persistent Resolving negative RA positive RA non-RA arthritis (n = 53) (n = 48) (n = 66) (n = 96) P-Value Female, n (%) 27 (50.9) 31 (64.6) 37 (56.1) 46 (47.9) 0.274 Age (years) 55.6 15.7 55.5 14.4 52.1 18.9 45.9 16.8 <0.0001 Disease duration (days) 52.4 21.4 55.3 21.7 56.4 21.5 45.3 20.8 0.005 CRP (mg/dl) 10 (0-39) 17.5 (6-43.8) 20.5 (7.5-35.3) 7 (0-17) <0.0001 ESR (mm/hour) 18 (11.5-44.5) 27.5 (18.3-51.3) 21.5 (7.8-45.8) 12.5 (5-27) <0.0001 DAS28 (CRP) 4.4 1.4 4.4 1.4 3.6 1.2 2.8 1.3 <0.0001 DAS28 (ESR) Smoking n (%) 4.6 1.5 4.7 1.6 3.6 1.8 2.9 1.5 <0.001 Ever smoker 28/49 (57-1) 27/47 (57.4) 26/64 (40.6) 35/89 (39.3) 0.07 Never smoker 21/49 (42.9) 20/47 (42.6) 38/64 (59.4) 54/89 (60.7) Anti-CCP positive, n (%) 0 (0) 48 (100) 1 (1.5) 1 (1.0) <0.0001 cTNC5 positive, n (%) 2 (3.8) 39 (81.3) 4 (6.1) 3 (3.1) <0.0001 rTNC5 positive, n (%) 1 (1.9) 1 (2.1) 3 (4.5) 1 (1.0) 0.527 Data are shown as number (percentage), mean +/ SD, or median (IQR) as appropriate. CCP, cyclic citrullinated peptide; CRP, C reactive protein; DAS, disease activity score; ESR, erythrocyte sedimentation rate; RA, rhematoid arthritits; RF, rheumatoid factor; v cTNC, citrullinated tenascin-C.

TABLE-US-00010 TABLE 9 Characteristics of RA patients with and without anti-cTNC5 antibodies. Anti-cTNC5 Anti-cTNC5 negative RA positive RA (n = 60) (n = 41) P value Female, n (%) 33 (55) 25 (60.1) 0.682 Age (years) 55.2 16.1 56.1 13.3 0.785 Symptom 52.3 21.5 56 21.5 0.400 duration (days) CRP (mg/dl) 10.5 (0-43) 18 (6-39) 0.062 ESR (mm/hour) 18 (11-45) 25 (19-46) 0.372 DAS28 (CRP) 4.26 1.4 4.55 1.4 0.320 DAS28 (ESR) 4.51 1.5 4.82 1.6 0.320 28 TJC 7.22 6.5 9.1 10.4 0.267 28 SJC 7.6 7.2 6.9 5.5 0.595 Smoking, n (%) Ever smoker 34/56 (60.7) 21/40 (52.5) 0.682 Never smoker 22/56 (39.3) 19/40 (47.5) 0.374 Anti-CCP 9 (15) 39 (95.1) <0.0001 positive, n (%) Data are shown as number (percentage), mean +/ SD, or median (IQR) as appropriate. Comparisons have been performed with 2, T test and Mann Whiteny U test for categorical, parametric continuous and non-parametric continuous data, respectively. cTNC, citrullinated tenascin-C; CRP, C reactive protein; ESR, erythrocyte sedimentation rate; DAS, disease activity score; TJC, tender joint count; SJC, swollen joint count; CCP, cyclic citrullinated peptide.

TABLE-US-00011 TABLE 10 Characteristics of early synovitis patients with and without anti-cTNC5 antibodies. cTNC5 cTNC5 negative positive P value Female, n (%) 112 (52.1) 29 (60.4) 0.188 Age (years) 50.4 17.7 54.6 13.7 0.123 Disease 50.6 21.7 54.9 21.6 0.124 duration (days) CRP (mg/dl) 10 (0-28) 18 (6-43.8) 0.021 ESR (mm/hour) 17 (7-38) 25 (18.3-44.8) 0.002 DAS28 (CRP) 3.4 1.4 4.4 1.4 <0.0001 DAS28 (ESR) 3.6 1.7 4.6 1.7 <0.0001 28 TJC 4.2 5.5 8.1 9.9 <0.0001 28 SJC 3.9 5.3 6.5 5.4 0.002 Smoking, n (%) Ever smoker 90/202 (44.5) 21/47 (44.7) 0.343 Never smoker 112/202 (55.5) 26/47 (55.3) 0.374 Anti-CCP 10 (4.7) 40 (83.3) <0.0001 positive, n (%) Data are shown as number (percentage), mean +/ SD, or median (IQR) as appropriate. cTNC, citrullinated tenascin-C; CRP, C reactive protein; ESR, erythrocyte sedimentation rate; DAS, disease activity score; TJC, tender joint count; SJC, swollen joint count; CCP, cyclic citrullinated peptide.

REFERENCES

[0351] 1. Kinloch A, Lundberg K, Wait R, et al. Synovial fluid is a site of citrullination of autoantigens in inflammatory arthritis. Arthritis Rheum. 2008; 58:2287-95. [0352] 2. Vossenaar E R, Smeets T J, Kraan M C, et al. The presence of citrullinated proteins is not specific for rheumatoid synovial tissue. Arthritis Rheum. 2004; 50:3485-94. [0353] 3. Girbal-Neuhauser E, Durieux J J, Arnaud M, et al. The epitopes targeted by the rheumatoid arthritis-associated antifilaggrin autoantibodies are posttranslationally generated on various sites of (pro)filaggrin by deimination of arginine residues. J Immunol. 1999; 162:585-94. [0354] 4. Schellekens G A, de Jong B A, van den Hoogen F H, et al. Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J Clin Invest. 1998; 101:273-81. [0355] 5. Kroot E J, de Jong B A, van Leeuwen M A, et al. The prognostic value of anti-cyclic citrullinated peptide antibody in patients with recent-onset rheumatoid arthritis. Arthritis Rheum. 2000; 43:1831-5. [0356] 6. Nielen M M, van Schaardenburg D, Reesink H W, et al. Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors. Arthritis Rheum. 2004; 50:380-6. [0357] 7. Rantapaa-Dahlqvist S, de Jong B A, Berglin E, et al. Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum. 2003; 48:2741-9. [0358] 8. Vencovsky J, Machacek S, Sedova L, et al. Autoantibodies can be prognostic markers of an erosive disease in early rheumatoid arthritis. Ann Rheum Dis. 2003; 62:427-30. [0359] 10. Wegner N, Lundberg K, Kinloch A, et al. Autoimmunity to specific citrullinated proteins gives the first clues to the etiology of rheumatoid arthritis. Immunol Rev. 2010; 233:34-54. [0360] 13. Lundberg K, Kinloch A, Fisher B A, et al. Antibodies to citrullinated alpha-enolase peptide 1 are specific for rheumatoid arthritis and cross-react with bacterial enolase. Arthritis Rheum. 2008; 58:3009-19. [0361] 14. Lundberg K, Bengtsson C, Kharlamova N, et al. Genetic and environmental determinants for disease risk in subsets of rheumatoid arthritis defined by the anticitrullinated protein/peptide antibody fine specificity profile. Ann Rheum Dis. 2013; 72:652-8. [0362] 19. Midwood K S, Orend G. The role of tenascin-C in tissue injury and tumorigenesis. J Cell Commun Signal. 2009; 3:287-310. [0363] 20. Udalova I A, Ruhmann M, Thomson S J, et al. Expression and immune function of tenascin-C. Crit Rev Immunol. 2011; 31:115-45. [0364] 21. Chevalier X, Groult N, Larget-Piet B, et al. Tenascin distribution in articular cartilage from normal subjects and from patients with osteoarthritis and rheumatoid arthritis. Arthritis Rheum. 1994; 37:1013-22. [0365] 22. Goh F G, Piccinini A M, Krausgruber T, et al. Transcriptional regulation of the endogenous danger signal tenascin-C: a novel autocrine loop in inflammation. J Immunol. 2010; 184:2655-62. [0366] 23. Hasegawa M, Nakoshi Y, Muraki M, et al. Expression of large tenascin-C splice variants in synovial fluid of patients with rheumatoid arthritis. J Orthop Res. 2007; 25:563-8. [0367] 24. Salter D M. Tenascin is increased in cartilage and synovium from arthritic knees. Br J Rheumatol. 1993; 32:780-6. [0368] 25. Page T H, Charles P J, Piccinini A M, et al. Raised circulating tenascin-C in rheumatoid arthritis. Arthritis Res Ther. 2012; 14:R260. [0369] 26. Midwood K, Sacre S, Piccinini A M, et al. Tenascin-C is an endogenous activator of Toll-like receptor 4 that is essential for maintaining inflammation in arthritic joint disease. Nat Med. 2009; 15:774-80. [0370] 30. Monach P A, Hueber W, Kessler B, et al. A broad screen for targets of immune complexes decorating arthritic joints highlights deposition of nucleosomes in rheumatoid arthritis. Proc Natl Acad Sci USA. 2009; 106:15867-72. [0371] 31. Tutturen A E, Fleckenstein B, de Souza G A. Assessing the citrullinome in rheumatoid arthritis synovial fluid with and without enrichment of citrullinated peptides. J Proteome Res. 2014; 13:2867-73. [0372] 32. Arnett F C, Edworthy S M, Bloch D A, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988; 31:315-24. [0373] 33. Cartwright A J, Quirke A-M, de Pablo P, et al. Smoking Affects Immune Response to Citrullinated Autoantigens Before Clinical Onset of Rheumatoid Arthritis in a Southern European Cohort [abstract]. Rheumatology (Oxford). 2014; 53:i36. [0374] 34. Stolt P, Bengtsson C, Nordmark B, et al. Quantification of the influence of cigarette smoking on rheumatoid arthritis: results from a population based case-control study, using incident cases. Ann Rheum Dis. 2003; 62:835-41. [0375] 35. Mikuls T R, Payne J B, Yu F, et al. Periodontitis and Porphyromonas gingivalis in patients with rheumatoid arthritis. Arthritis Rheumatol. 2014; 66:1090-100. [0376] 36. Xu D, Suenaga N, Edelmann M J, et al. Novel MMP-9 substrates in cancer cells revealed by a label-free quantitative proteomics approach. Mol Cell Proteomics. 2008; 7:2215-28. [0377] 37. Assohou-Luty C, Raijmakers R, Benckhuijsen W E, et al. The human peptidylarginine deiminases type 2 and type 4 have distinct substrate specificities. Biochim Biophys Acta. 2014; 1844:829-36. [0378] 38. van Beers J J, Raijmakers R, Alexander L E, et al. Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance. Arthritis Res Ther. 2010; 12:R219. [0379] 39. Snir O, Widhe M, von Spee C, et al. Multiple antibody reactivities to citrullinated antigens in sera from patients with rheumatoid arthritis: association with HLA-DRB1 alleles. Ann Rheum Dis. 2009; 68:736-43. [0380] 40. Wagner C A, Sokolove J, Lahey L J, et al. Identification of anticitrullinated protein antibody reactivities in a subset of anti-CCP-negative rheumatoid arthritis: association with cigarette smoking and HLA-DRB1 shared epitope alleles. Ann Rheum Dis. 2015; 74:579-86. [0381] 41. Huizinga T W, Amos C I, van der Helm-van Mil A H, et al. Refining the complex rheumatoid arthritis phenotype based on specificity of the HLA-DRB1 shared epitope for antibodies to citrullinated proteins. Arthritis Rheum. 2005; 52:3433-8. [0382] 42. van der Woude D, Lie B A, Lundstrom E, et al. Protection against anti-citrullinated protein antibody-positive rheumatoid arthritis is predominantly associated with HLA-DRB1*1301: A meta-analysis of HLA-DRB1 associations with anti-citrullinated protein antibody-positive and anti-citrullinated protein antibody-negative rheumatoid arthritis in four European populations. Arthritis Rheum. 2010; 62:1236-45. [0383] 43. Plenge R M, Padyukov L, Remmers E F, et al. Replication of putative candidate-gene associations with rheumatoid arthritis in >4,000 samples from North America and Sweden: association of susceptibility with PTPN22, CTLA4, and PADI4. Am J Hum Genet. 2005; 77:1044-60. [0384] 44. Silman A J, Newman J, MacGregor A J. Cigarette smoking increases the risk of rheumatoid arthritis. Results from a nationwide study of disease-discordant twins. Arthritis Rheum. 1996; 39:732-5. [0385] 45. Klareskog L, Catrina A I. Autoimmunity: lungs and citrullination. Nat Rev Rheumatol. 2015; 11:261-2. [0386] 46. Lugli E B, Correia R E, Fischer R, et al. Expression of citrulline and homocitrulline residues in the lungs of non-smokers and smokers: implications for autoimmunity in rheumatoid arthritis. Arthritis Res Ther. 2015; 17:9. [0387] 47. Annoni R, Lancas T, Yukimatsu Tanigawa R, et al. Extracellular matrix composition in COPD. Eur Respir J. 2012; 40:1362-73. [0388] 48. Lofdahl M, Kaarteenaho R, Lappi-Blanco E, et al. Tenascin-C and alpha-smooth muscle actin positive cells are increased in the large airways in patients with COPD. Respir Res. 2011; 12:48. [0389] 49. Rosenstein E D, Greenwald R A, Kushner L J, et al. Hypothesis: the humoral immune response to oral bacteria provides a stimulus for the development of rheumatoid arthritis. Inflammation. 2004; 28:311-8. [0390] 50. Wegner N, Wait R, Sroka A, et al. Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and alpha-enolase: implications for autoimmunity in rheumatoid arthritis. Arthritis Rheum. 2010; 62:2662-72. [0391] 51. Montgomery A B, Kopec J, Shrestha L, et al. Crystal structure of Porphyromonas gingivalis peptidylarginine deiminase: implications for autoimmunity in rheumatoid arthritis. Ann Rheum Dis. 2015. [0392] 52. Ruggiero S, Cosgarea R, Potempa J, et al. Cleavage of extracellular matrix in periodontitis: gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C. Biochim Biophys Acta. 2013; 1832:517-26. [0393] 53. Kuhn K A, Kulik L, Tomooka B, et al. Antibodies against citrullinated proteins enhance tissue injury in experimental autoimmune arthritis. J Clin Invest. 2006; 116:961-73. [0394] 54. Sanchez-Pernaute O, Filkova M, Gabucio A, et al. Citrullination enhances the pro-inflammatory response to fibrin in rheumatoid arthritis synovial fibroblasts. Ann Rheum Dis. 2013; 72:1400-6. [0395] 55. van Beers J J, Willemze A, Stammen-Vogelzangs J, et al. Anti-citrullinated fibronectin antibodies in rheumatoid arthritis are associated with human leukocyte antigen-DRB1 shared epitope alleles. Arthritis Res Ther. 2012; 14:R35. [0396] 56. Fisher B A, Plant D, Brode M, et al. Antibodies to citrullinated alpha-enolase peptide 1 and clinical and radiological outcomes in rheumatoid arthritis. Ann Rheum Dis. 2011; 70:1095-8. [0397] 57. Kallberg H, Padyukov L, Plenge R M, et al. Gene-gene and gene-environment interactions involving HLA-DRB1, PTPN22, and smoking in two subsets of rheumatoid arthritis. Am J Hum Genet. 2007; 80:867-75. [0398] 58. Padyukov L, Silva C, Stolt P, et al. A gene-environment interaction between smoking and shared epitope genes in HLA-D R provides a high risk of seropositive rheumatoid arthritis. Arthritis Rheum. 2004; 50:3085-92. [0399] 59. Fischer R, Kessler B M. Gel-aided sample preparation (GASP)a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells. Proteomics. 2015; 15:1224-9.