WO2026020127A2

WO2026020127A2 - Methods of assessing citrullination and activity of pad2 modulators

Info

Publication number: WO2026020127A2
Application number: PCT/US2025/038299
Authority: WO
Inventors: Ashok Dongre; Mary Adams; Sium HABTE; Lauren LAHEY; Laurence Celine MENARD; Kofi Mensah; Anh REYNOLDS; Ari SALINGER; Rasa Santockyte; Wei Sun; Ali Ekrem YESILKANAL; Tai WANG; Qing Xiao; Qihong Zhao
Original assignee: Bristol Myers Squibb Co
Current assignee: Bristol Myers Squibb Co
Priority date: 2024-07-19
Filing date: 2025-07-18
Publication date: 2026-01-22
Anticipated expiration: 2027-01-19

Abstract

The present application relates to methods for quantifying citrullination and methods that may be used to assess the activity of agents including PAD2 (peptidyl arginine deiminase 2) modulators, such as small molecules or antibodies, for example. In certain embodiments, the methods comprise assessing citrullination of one or more citrullination sites on a protein or a peptide fragment thereof present in a biological sample. In some embodiments, citrullination sites herein are citrullinated by PAD2 under physiological conditions. In some cases the citrullination sites are citrullinated by PAD2 as well as at least one other PAD enzyme such as PAD4 under physiological conditions. In some cases the citrullination sites are citrullinated by PAD2 but are not citrullinated by PAD4 under physiological conditions.

Description

METHODS OF ASSESSING CITRULLINATION AND ACTIVITY OF PAD2 MODULATORS

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority of United States Provisional Application No. 63/673,332, filed July 19, 2025, which is incorporated by reference herein in its entirety.

REFERENCE TO THE SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on July 18, 2025, is named “01275-0069-00PCT.xml” and is 323,461 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

FIELD

The present application relates to methods involving assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in a biological sample from a subject. The methods may be used for, inter alia, assessing the citrullination of sites that are modified by PAD2, and for assessing the modifying activity of agents including PAD2 (peptidyl arginine deiminase 2) modulators, such as small molecules or antibodies, for example. In some embodiments, citrullination sites herein are citrullinated by PAD2 under physiological conditions. In some cases the citrullination sites are citrullinated by PAD2 as well as at least one other PAD enzyme such as PAD4 under physiological conditions. In some cases the citrullination sites are citrullinated by PAD2 but are not citrullinated by PAD4 under physiological conditions.

BACKGROUND

PAD2 (peptidyl arginine deiminase 2) is a PAD enzyme found in synovial tissues of subjects with rheumatoid arthritis (RA), and is also associated with other autoimmune diseases, such as lupus, lupus nephritis, vasculitis, thrombosis (e.g., venous thrombosis), inflammatory bowel disease (IBD), and others. PAD2 is an enzyme that catalyzes conversion of arginine to citrulline. Various large and small molecule compounds have been developed that act as PAD2 modulators (i.e., that modulate the activity of PAD2), sometimes in conjunction with other peptidyl arginine deiminase enzymes such as PAD4, and sometimes being specific for PAD2. For example, several PAD2 inhibitors may be useful for treating autoimmune diseases such as rheumatoid arthritis (RA) and others.

Accordingly, methods for assessing the activity of PAD2 modulators are useful, for example, for monitoring treatment with existing PAD2 modulators and for assessing novel PAD2 modulators in development, among other uses.

SUMMARY

The present disclosure provides methods of assessing citrullination of proteins or peptides, and methods of assessing the activity of PAD2 modulators or other therapeutic agents that can affect citrullination, by assessing citrullination of one or more citrullination sites on a protein or a peptide fragment thereof present in a biological sample.

The present disclosure relates, inter alia, to embodiments including the following, for example:

1. A method of determining citrullination of a protein or peptide fragment thereof, comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof in a biological sample from a subject, wherein the citrullination site is selected from one or more of the following:

R342 of carboxypeptidase B2 (CPB2; corresponding to R342 of Q96IY4), R239 of apolipoprotein A-l (APO Al; corresponding to R239 of P02647), R198 of apolipoprotein E (APOE; corresponding to R198 of P02649),

R337 of alpha-2-HS-glycoprotein (AHSG; corresponding to R337 of P02765),

R212 of apolipoprotein A-l (APO Al; corresponding to R212 of P02647), R173 of apolipoprotein A-l (AP0A1; corresponding to R173 of P02647), R108 of apolipoprotein E (APOE; corresponding to R108 of P02649), R383 of prothrombin (F2; corresponding to R383 of P00734),

R362 of vitronectin (VTN; corresponding to R362 of P04004),

R155 of apolipoprotein A-IV (AP0A4; corresponding to R155 of P06727),

R306 of apolipoprotein A-IV (AP0A4; corresponding to R306 of P06727),

R101 of vitamin K-dependent plasma glycoprotein (PROS1; corresponding to RIO 1 ofP07225),

R496 of albumin (ALB; corresponding to R496 of P02768), R194 of clusterin (CLU; corresponding to R194 and/or R198 of P10909), or

R198 of clusterin (CLU; corresponding to R194 and/or R198 of Pl 0909).

2. The method of embodiment 1, wherein the biological sample has been exposed to a PAD2 modulator such as a PAD2 inhibitor.

3. A method of assessing the activity of a PAD2 modulator, the method comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample from a subject, wherein the biological sample has been exposed to the PAD2 modulator, wherein the citrullination site is selected from one or more of the following:

R342 of carboxypeptidase B2 (CPB2; corresponding to R342 of Q96IY4), R239 of apolipoprotein A-l (APO Al; corresponding to R239 of P02647), R198 of apolipoprotein E (APOE; corresponding to R198 of P02649), R337 of alpha-2-HS-glycoprotein (AHSG; corresponding to R337 of P02765), R212 of apolipoprotein A-l (APO Al; corresponding to R212 of P02647), R173 of apolipoprotein A-l (APOA1; corresponding to R173 of P02647), R108 of apolipoprotein E (APOE; corresponding to R108 of P02649), R383 of prothrombin (F2; corresponding to R383 of P00734), R362 of vitronectin (VTN; corresponding to R362 of P04004),

R155 of apolipoprotein A-IV (APOA4; corresponding to R155 of P06727), R306 of apolipoprotein A-IV (APOA4; corresponding to R306 of P06727), R101 of vitamin K-dependent plasma glycoprotein (PROS1; corresponding to RIO 1 ofP07225),

R496 of albumin (ALB; corresponding to R496 of P02768),

R315 of complement C3 (C3; corresponding to R315 of P01024),

R194 of clusterin (CLU; corresponding to R194 and/or R198 of P10909), or R198 of clusterin (CLU; corresponding to R194 and/or R198 of Pl 0909).

4. The method of embodiment 3, wherein the PAD2 modulator is a PAD2 inhibitor.

5. The method of embodiment 3 or 4, wherein the PAD2 modulator reduces citrullination at the citrullination site in a dose-dependent manner.

6. The method of any one of embodiments 1-5, wherein the method comprises assessing citrullination of a peptide fragment of the protein, wherein the peptide fragment comprises the citrullination site. 7. The method of any one of embodiments 2-6, wherein the biological sample has been exposed to a PAD2 inhibitor in vivo in a subject as a result of the PAD2 inhibitor being administered to the subject.

8. The method of any one of embodiments 2-7, wherein the biological sample has been exposed to the PAD2 inhibitor by contacting the biological sample with the PAD2 inhibitor ex vivo.

9. The method of any one of embodiments 1-8, wherein the assessing citrullination of the citrullination site comprises mass spectrometry (MS).

10. The method of any one of embodiments 1-8, wherein the assessing citrullination of the citrullination site comprises liquid chromatography and mass spectrometry (LC-MS).

11. The method of any one of embodiments 1-8, wherein the assessing citrullination comprises selective reaction monitoring and chromatographic separation.

12. The method of any one of embodiments 1-11, wherein assessing citrullination of the citrullination site comprises measuring, in the biological sample, a first concentration of the citrullinated protein or peptide fragment thereof and a second concentration of corresponding total protein.

13. The method of embodiment 12, wherein assessing citrullination of the citrullination site comprises determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.

14. The method of embodiment 13, wherein the method comprises comparing the citrullination ratio to a reference citrullination ratio.

15. The method of embodiment 14, wherein the reference citrullination ratio is a citrullination ratio determined for a control biological sample.

16. The method of embodiment 15, wherein the control biological sample is a biological sample that: (a) has not been exposed to a PAD2 modulator or PAD2 inhibitor, (b) is from the same subject, and/or (c) is from the same subject prior to treatment with the PAD2 modulator or PAD2 inhibitor.

17. The method of any one of embodiments 14-16, wherein the assessing comprises determining a difference between the citrullination ratio for the biological sample and the reference citrullination ratio.

18. The method of any one of embodiments 1-17, wherein the method comprises contacting the biological sample with exogenous PAD2.

19. A method comprising:

(i) contacting a biological sample from a subject with exogenous PAD2, and (ii) assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample; wherein the citrullination site is selected from one or more of the following:

R337 of alpha-2-HS-glycoprotein (AHSG; corresponding to R337 of P02765),

R362 of vitronectin (VTN; corresponding to R362 of P04004),

R155 of apolipoprotein A-IV (AP0A4; corresponding to R155 of P06727),

R306 of apolipoprotein A-IV (AP0A4; corresponding to R306 of P06727),

R496 of albumin (ALB; corresponding to R496 of P02768),

R315 of complement C3 (C3; corresponding to R315 of PO 1024),

R194 of clusterin (CLU; corresponding to R194 and/or R198 of P10909), or

R198 of clusterin (CLU; corresponding to R194 and/or R198 of Pl 0909).

20. The method of embodiment 18 or 19, wherein a concentration of 1-5 nM, 2-4 nM, 1 nM, 2 nM, 3 nM, 4 nM, or 5 nM exogenous PAD2 is added to the biological sample.

21. The method of embodiment 18 or 19 or 20, wherein the method comprises incubating the exogenous PAD2 with the biological sample.

22. The method of embodiment 21, wherein the method comprises incubating the exogenous PAD2 with the biological sample at a temperature of 36-38 °C.

23. The method of embodiment 21 or 22, wherein the incubating is for 1-3 hours.

24. The method of any one of embodiments 21-23, wherein the method comprises adding EDTA to the biological sample after the incubation with exogenous PAD2.

25. The method of any one of embodiments 1-24, wherein the method comprises incubating the biological sample for an incubation period before assessing citrullination.

26. The method of embodiment 25, wherein the incubating is performed at 35-40°C.

27. The method of embodiment 25 or 26, wherein the incubation period is 48 to 96 hours. 28. The method of any one of embodiments 1-27, comprising preparing the biological sample for assessment prior to the assessing.

29. The method of embodiment 28, wherein the preparing comprises enzymatically digesting proteins in the biological sample.

30. The method of embodiment 28 or 29, wherein the preparing comprises depleting proteins from the biological sample that are not targeted by the assessing.

31. The method of any one of embodiments 28-30, wherein the preparing comprises diluting the biological sample.

32. The method of any one of embodiments 28-31, wherein the preparing comprises denaturing proteins in the biological sample.

33. The method of any one of embodiments 28-32, wherein the preparing comprises enriching the biological sample for a protein or peptide fragment thereof that comprises the citrullination site to be assessed.

34. The method of embodiment 33, wherein the preparing comprises contacting the biological sample with an antibody that specifically binds to citrullinated and noncitrullinated forms of the protein or peptide fragment thereof that comprises the citrullination site to be assessed.

35. The method of any one of embodiments 28-34, wherein the method comprises enriching the biological sample for membrane-bound proteins and peptide fragments thereof prior to the assessing.

36. The method of any one of embodiments 28-35, wherein the method comprises contacting the biological sample with a strong anion exchange (SAX) chromatography medium.

37. The method of embodiment 36, wherein the SAX chromatography medium comprises particles.

38. The method of embodiment 36, wherein the particles comprise magnetic particles.

39. The method of any one of embodiments 36-38, wherein the SAX chromatography medium enriches membrane-bound proteins and peptide fragments thereof in the sample.

40. The method of any one of embodiments 36-39, wherein the method further comprises eluting proteins or peptide fragments thereof bound to the SAX chromatography medium and assessing citrullination of the eluted proteins or peptide fragments thereof.

41. The method of any one of embodiments 35-40, comprising enzymatically digesting proteins in the biological sample following the enriching or the contacting of the biological sample with the SAX chromatography medium or following the eluting of the proteins or peptide fragments thereof from the SAX chromatography medium. 42. The method of any one of embodiments 1- 1, wherein the method comprises freezing and thawing the biological sample before assessing citrullination of the citrullination site.

43. The method of any one of embodiments 1-42, comprising:

(a) immunoenriching the sample for the protein or peptide fragment thereof to form an immunoenriched sample, wherein the immunoenriching comprises contacting the sample with an antibody that binds to both citrullinated and noncitrullinated forms of the protein and/or to both citrullinated and noncitrullinated forms of the peptide fragment thereof comprising the citrullination site,

(b) enzymatically digesting proteins in the sample either before or after the immunoenriching to form the peptide fragment, and

(c) assessing citrullination of the peptide fragment at the citrullination site.

44. The method of embodiment 43, wherein the immunoenriching comprises contacting the sample with an immobilized antibody that binds to both citrullinated and noncitrullinated forms of the protein and/or to both citrullinated and noncitrullinated forms of the peptide fragment thereof comprising the citrullination site, and eluting protein and/or peptide bound to the immobilized antibody.

45. The method of embodiment 43 or 44, wherein the enzymatically digesting is conducted before the immunoenriching.

46. The method of embodiment 43 or 44, wherein the enzymatically digesting is conducted after the immunoenriching.

47. The method of any one of embodiments 43-46, wherein the immunoenriching comprises incubating the sample with the immobilized antibody for at least 30 minutes.

48. The method of embodiment 47, wherein the incubating is at a temperature of 22-28°C.

49. The method of embodiment 47 or 48, wherein the incubating is for a period of 30 to 90 minutes.

50. The method of any one of embodiments 43-49, wherein the immunoenriching comprises shaking during the incubating.

51. The method of any one of embodiments 44-50, wherein the immunoenriching comprises removing the immobilized antibody from the sample and washing the immobilized antibody prior to the eluting.

52. The method of any one of embodiments 44-51, wherein the eluting comprises washing the immobilized antibody with an elution composition under acidic conditions.

53. The method of embodiment 52, wherein the elution composition comprises a detergent.

54. The method of embodiment 53, wherein the detergent is a zwitterionic detergent. 55. The method of any one of embodiments 44-54, the method comprises neutralizing eluted protein in a buffer prior to assessing, and optionally prior to enzymatically digesting.

56. The method of any one of embodiments 43-55, wherein the antibody is immobilized by attachment to a solid surface.

57. The method of any one of embodiments 43-56, wherein the assessing citrullination comprises measuring a first concentration of citrullinated peptide in the digested peptides and a second concentration of signature peptide in the digested peptides, and optionally determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.

58. The method of any one of embodiments 43-57, comprising denaturing proteins in the immunoenriched sample prior to the assessing.

59. The method of any one of embodiments 44-58, comprising denaturing the eluted protein prior to the assessing.

60. The method of embodiment 59, comprising denaturing the eluted protein prior to enzymatically digesting the eluted protein.

61. The method of any one of embodiments 43-60, comprising diluting the plasma or serum sample prior to the immunoenriching and performing the immunoenriching on the sample that has been diluted.

62. The method of embodiment 61, comprising diluting the plasma or serum sample by 2 to 1000 fold.

63. The method of embodiment 61 or 62, wherein the sample that has been diluted has a volume of at least 5 pl.

64. The method of any one of embodiments 1-63, wherein the sequence of the peptide fragment comprises one of the following sequences, wherein the citrullination site is designated by underlining of R residues and wherein modifications of non-arginine residues are designated by underlining of the modified residues: DHEELSLVASEAVRAIEK;

AKPALEDLRQGLLPVLESFK; ERLGPLVEQGR; HTFMGVVSLGSPSGEVSHPRKT; ENGGARLAEYHAK; LSPLGEEMRDR; SELEEQLTPVAEETRAR; TPVSDRVTK;

IVEGSDAEIGMSPWQVMLFRK; IYISGMAPRPSLAK; RQLTPYAQR;

RVEPYGENFNK; SFQTGLFTAARQSTNAYPDLR;

VLLDGVQNPRAEDLVGKSLYVSATVILHSGSDMVQAER; or ASSIIDELFQDRFFTREPQDTYHYLPFSLPHR (corresponding to SEQ ID NOs: 14, 6, 10, 11, 7, 8, 9, 12, 15, 16, 17, 18, 19, 20, or 13, respectively). 65. The method of any one of embodiments 1-64, wherein the biological sample comprises whole blood, plasma, serum, or blood supernatant.

66. The method of any one of embodiments 1-64, wherein the biological sample comprises synovial fluid.67. The method of any one of embodiments 64-66, wherein the peptide fragment comprises the sequence of: DHEELSLVASEAVRAIEK;

AKPALEDLRQGLLPVLESFK; ERLGPLVEQGR; or HTFMGVVSLGSPSGEVSHPRKT (SEQ ID NOs: 14, 6, 10, or 11, respectively).

68. The method of any one of embodiments 1-67, wherein the method comprises assessing citrullination of two or more proteins or peptide fragments thereof.

69. The method of any one of embodiments 1-68, wherein the method further comprises assessing citrullination of at least one additional citrullination site listed in Table 1.

70. The method of any one of embodiments 1-68, wherein the method further comprises assessing citrullination of at least one additional citrullination site listed in Table 11.

71. A method comprising:

(a) immunoenriching a biological sample for a protein of interest or a peptide fragment thereof to form an immunoenriched sample, wherein the immunoenriching comprises contacting the sample with an antibody that specifically binds to both citrullinated and noncitrullinated forms of the protein of interest or that specifically binds to both citrullinated and noncitrullinated forms of a peptide fragment of the protein of interest, the protein of interest comprising a citrullination site, and

(b) assessing citrullination in the immunoenriched sample at the citrullination site, wherein the biological sample has been exposed to a PAD2 modulator such as a PAD2 inhibitor.

72. A method comprising:

(a) immunoenriching a biological sample for a protein of interest or a peptide fragment thereof to form an immunoenriched sample, wherein the immunoenriching comprises contacting the sample with an antibody that specifically binds to both citrullinated and noncitrullinated forms of the protein of interest or that specifically binds to both citrullinated and noncitrullinated forms of peptide fragments of the protein of interest, the protein of interest comprising a citrullination site,

(b) enzymatically digesting the immunoenriched sample to form peptide fragments from the protein of interest, wherein parts (a) and (b) may be performed in any order, and

(c) assessing citrullination of a peptides at the citrullination site, wherein the biological sample has been exposed to a PAD2 modulator such as a PAD2 inhibitor.

73. A method comprising

(a) immunoenriching a biological sample by contacting the sample with an immobilized antibody that specifically binds to both citrullinated and noncitrullinated forms of a protein of interest, the protein of interest comprising a citrullination site,

(b) eluting protein bound to the immobilized antibody,

(c) enzymatically digesting the eluted protein to form peptide fragments of the eluted protein, and

(d) assessing citrullination of the peptides at the citrullination site, wherein the biological sample has been exposed to a PAD2 modulator such as a PAD2 inhibitor.

74. A method comprising

(a) enzymatically digesting proteins in a biological sample to form peptide fragments thereof,

(b) immunoenriching the enzymatically digested biological sample by contacting the sample with an immobilized antibody that specifically binds to both citrullinated and noncitrullinated forms of a peptide fragment of a protein of interest, the peptide fragment comprising a citrullination site,

(c) eluting peptides bound to the immobilized antibody, and

75. The method of any one of embodiments 71-74, wherein the immunoenriching comprises incubating the sample with the immobilized antibody for at least 30 minutes.

76. The method of embodiment 75, wherein the incubating is at a temperature of 22-28°C.

77. The method of embodiment 75 or 76, wherein the incubating is for a period of 30 to 90 minutes.

78. The method of any one of embodiments 71-77, wherein the immunoenriching comprises shaking during the incubating.

79. The method of any one of embodiments 73-77, wherein the immunoenriching comprises removing the immobilized antibody from the sample and washing the immobilized antibody prior to the eluting. 80. The method of any one of embodiments 73-79, wherein the eluting comprises washing the immobilized antibody with an elution composition under acidic conditions.

81. The method of embodiment 80, wherein the elution composition comprises a detergent.

82. The method of embodiment 81, wherein the detergent is a zwitterionic detergent.

83. The method of any one of embodiments 80-82, the method comprises neutralizing eluted protein or peptide in a buffer prior to assessing, and optionally prior to enzymatically digesting.

84. The method of any one of embodiments 71-83, wherein the antibody is immobilized by attachment to a solid surface.

85. The method of any one of embodiments 71-84, wherein the assessing citrullination comprises measuring a first concentration of a citrullinated peptide and a second concentration of a signature peptide in the enzymatically digested sample, and optionally determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.

86. The method of any one of embodiments 71-85, comprising denaturing proteins or peptides in the immunoenriched sample prior to the assessing.

87. The method of any one of embodiments 73-86, comprising denaturing the eluted protein or peptide prior to the assessing.

88. The method of embodiment 87, comprising denaturing the eluted protein prior to enzymatically digesting the eluted protein.

89. The method of any one of embodiments 71-88, comprising diluting the sample prior to the immunoenriching and performing the immunoenriching on the sample that has been diluted.

90. The method of embodiment 89, comprising diluting the sample by 2 to 1000 fold.

91. The method of embodiment 89 or 90, wherein the sample that has been diluted has a volume of at least 5 pl.

92. The method of any one of embodiments 71-91, wherein the method comprises assessing citrullination of two or more proteins of interest or peptide fragments thereof and the antibody comprises antibodies that specifically bind citrullinated and noncitrullinated forms of each of the two or more proteins of interest or peptide fragments thereof.

93. The method of any one of embodiments 71-92, wherein the biological sample is a serum or plasma sample.94. A method of assessing the activity of a PAD2 modulator, the method comprising

(a) obtaining a biological sample from a subject following treatment of the subject with at least one dose of a PAD2 modulator, and

(b) assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the biological sample.

95. An in vitro method of assessing the activity of a PAD2 modulator, the method comprising

(a) treating a biological sample from a subject with a PAD2 modulator to form a treated biological sample, and after the treating,

96. The method of embodiment 94 or 95, further comprising assessing citrullination of the citrullination site on the protein or a peptide fragment thereof in a control biological sample.

97. The method of embodiment 96, further comprising comparing an outcome of the assessment of citrullination in the biological sample with a corresponding outcome of the assessment of citrullination in the control biological sample.

98. The method of any one of embodiments 84-97, wherein the assessing comprises (i) measuring a first concentration of citrullinated protein or peptide in the sample and (ii) measuring a second concentration of corresponding total protein in the sample, wherein the citrullinated protein or peptide is citrullinated at the citrullination site and the corresponding total protein encompasses modified and unmodified forms of the protein, and optionally (iii) determining a citrullination ratio, which is a ratio of the first concentration to the second concentration.

99. The method of embodiment 98, wherein the method comprises comparing the citrullination ratio of the biological sample to the citrullination ratio of a control biological sample.

100. The method of embodiment 99, wherein the control biological sample is from the same subject as the biological sample.

101. The method of embodiment 100, wherein the control biological sample is a biological sample obtained from the subject prior to treatment with a PAD2 modulator.

102. The method of any one of embodiments 99-101, wherein the control biological sample has not been exposed to the PAD2 modulator.

103. The method of any one of embodiments 99-102, wherein the control biological sample has been exposed to a different treatment than the biological sample.

104. The method of any one of embodiments 94 or 96-103, further comprising (a) selecting or adjusting a dose of a PAD2 modulator based on the outcome of the assessing; or (b) selecting the subject for treatment with a PAD2 modulator based on the outcome of the assessing.

105. An in vitro method of assessing the activity of a PAD2 modulator, the method comprising

(i) dividing a biological sample obtained from a subject into a plurality of biological samples,

(ii) contacting each of the plurality of biological samples with a different dose of the PAD2 modulator, and

(iii) assessing, for each of the plurality of biological samples, citrullination of a PAD2- dependent citrullination site on a protein or peptide fragment thereof that is present in the biological sample.

106. The method of embodiment 105, further comprising calculating an IC50 for the PAD2 modulator based on the outcome of the assessing.

107. The method of any one of embodiments 105-106, further comprising selecting or adjusting a dose of a PAD2 modulator to be administered to the subject based on the outcome of the assessing.

108. The method of any one of embodiments 1-106, further comprising selecting the subject from which the biological sample was derived for treatment of a citrullination-related disease based on the outcome of the assessing.

109. An in vitro method of assessing citrullination by endogenous PAD2, the method comprising

(a) incubating a whole blood sample from a subject at 35-40°C for an incubation period,

(b) after the incubation period, separating plasma or supernatant from the whole blood sample, and

(c) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant.

110. The method of embodiment 109, wherein the incubation period is 48 to 96 hours.

111. The method of any one of embodiments 109 or 110, wherein the incubating is at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C.

112. The method of any one of embodiments 109-111, comprising enzymatically digesting polypeptides in the plasma or supernatant before the assessing.

113. The method of any one of embodiments 109-112, further comprising (i) incubating the plasma or supernatant with a protein depletion resin, and (ii) recovering depleted plasma or supernatant that has flowed through the resin to obtain depleted flowthrough, optionally wherein the recovering comprises centrifugation. 114. The method of embodiment 113, comprising enzymatically digesting polypeptides in the depleted flowthrough before the assessing.

115. The method of embodiment 113 or 114, further comprising cleaning up enzymatically digested plasma or supernatant before the assessing.

116. The method of any one of embodiments 109-115, wherein the whole blood sample and the plasma or supernatant separated therefrom have not been frozen and thawed.

117. The method of any one of embodiments 109-116, wherein the whole blood sample and/or the plasma or supernatant separated therefrom has been frozen and thawed.

118. The method of embodiment 117, wherein the method comprises freezing the plasma or supernatant after the separating and subsequently thawing the plasma or supernatant before the assessing.

119. The method of any one of embodiments 109-118, wherein the whole blood sample has been exposed to a PAD2 modulator.

120 The method of embodiment 119, wherein the whole blood sample has been exposed to a PAD2 modulator in vitro.

121. The method of embodiment 119, wherein the whole blood sample has been exposed to a PAD2 modulator in vivo in the subject as a result of the PAD2 modulator being administered to the subj ect.

122. An in vitro method of assessing changes in citrullination in a subject, the method comprising

(a) incubating a whole blood sample from a subject at 35-40°C for an incubation period, wherein the sample is obtained from the subject following administration of a PAD2 modulator to the subject,

123. The method of embodiment 122, further comprising

(d) incubating a second whole blood sample from the subject at 35-40°C for an incubation period, wherein the second whole blood sample is obtained from the subject before administration of the PAD2 modulator to the subject,

(e) after the incubation period, separating plasma or supernatant from the second whole blood sample, and

(f) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant from the second whole blood sample.

124. The method of embodiment 123, further comprising comparing an outcome of the assessing in step (f) with an outcome of the assessing in step (c).

125. A method of assessing effects of a PAD2 modulator, the method comprising

(a) exposing a whole blood sample from a subject to a PAD2 modulator in vitro,

(b) incubating the whole blood sample at 35-40°C for an incubation period,

(c) after the incubation period, separating plasma or supernatant from the whole blood sample, and

(d) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant.

126. The method of embodiment 125, further comprising

(e) incubating a control whole blood sample at 35-40°C for an incubation period,

(f) after the incubation period, separating control plasma or supernatant from the control whole blood sample, and

(g) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the control plasma or supernatant.

127. The method of embodiment 126, further comprising comparing an outcome of the assessing in step (g) with an outcome of the assessing in step (d).

128. An in vitro method of assessing PAD2-dependent citrullination, the method comprising assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in plasma or supernatant that has been separated from a whole blood sample that has been incubated at 35-40°C for an incubation period.

129. The method of embodiment 128, wherein the plasma or supernatant has been frozen and the method comprises thawing the plasma or supernatant prior to the assessing.

130. A method comprising

(i) contacting a biological sample from a subject with exogenous PAD2 and

(ii) assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample.

131. The method of embodiment 130, wherein the biological sample comprises plasma or serum.

132. The method of embodiment 130 or 131, wherein the method comprises preparing the biological sample for assessment prior to the assessing. 133. The method of embodiment 123, wherein the preparing comprises enzymatically digesting proteins in the biological sample to produce digested peptides and optionally cleaning up the digested peptides.

134. The method of embodiment 132 or 133, wherein the preparing comprises depleting proteins from the biological sample that are not targeted by the assessing.

135. The method of any one of embodiment 134, wherein depleting comprises depletion of human serum albumin.

136. The method of any one of embodiments 130-135, wherein the preparing comprises immunoenriching the biological sample for the protein or peptide fragment thereof using an antibody that binds to citrullinated and non-citrullinated forms of the protein or peptide.

137. The method of any one of embodiments 71-136, wherein the assessing comprises analysis with mass spectrometry (MS).

138. The method of any one of embodiments 71-136, wherein the assessing is performed using liquid chromatography and mass spectrometry (LC-MS).

139. The method of any one of embodiments 71-136, wherein the assessing comprises selective reaction monitoring and chromatographic separation.

140. A method of assessing citrullination at a citrullination site, the method comprising

(i) measuring a first concentration of a citrullinated protein or a citrullinated peptide in a biological sample by mass spectrometry (MS), wherein the citrullinated protein or citrullinated peptide is citrullinated at a citrullination site, and

(ii) measuring a second concentration of corresponding total protein in the biological sample by MS wherein the biological sample has been exposed to a PAD2 modulator such as a PAD2 inhibitor.

141. The method of embodiment 140, wherein the method comprises (i) measuring a first concentration of a citrullinated peptide from a target protein in a biological sample by MS, wherein the citrullinated peptide is citrullinated at a citrullination site, and (ii) measuring the second concentration by measuring the concentration of a signature peptide from the target protein in the biological sample by MS.

142. The method of embodiment 140 or 141, further comprising calculating a ratio of the first concentration to the second concentration

143. The method of any one of embodiments 140-142, wherein the method comprises measuring the first concentration and the second concentration for each of a plurality of different proteins or peptides. 144. The method of embodiment 143, wherein the method comprises measuring the first concentration for each of two different peptides, each of which are nonoverlapping fragments of the same protein and contain different citrullination sites.

145. The method of embodiment 143 or 144, wherein the method comprises calculating a ratio of the first concentration to the second concentration for each of the plurality of different proteins or peptides.

146. The method of any one of embodiments 71-145, wherein the PAD2 modulator is a PAD2 inhibitor.

147. The method of any one of embodiments 71-146, wherein the citrullination site is a site listed in Table 1.

148. The method of any one of embodiments 71-146, wherein the citrullination site is a site listed in Table 11.

149. The method of any one of embodiments 71-146, wherein the citrullination site is selected from one or more of the following:

R337 of alpha-2-HS-glycoprotein (AHSG; corresponding to R337 of P02765),

R362 of vitronectin (VTN; corresponding to R362 of P04004),

R155 of apolipoprotein A-IV (AP0A4; corresponding to R155 of P06727),

R306 of apolipoprotein A-IV (AP0A4; corresponding to R306 of P06727),

R496 of albumin (ALB; corresponding to R496 of P02768),

R315 of complement C3 (C3; corresponding to R315 of PO 1024),

R194 of clusterin (CLU; corresponding to R194 and/or R198 of P10909), or

R198 of clusterin (CLU; corresponding to R194 and/or R198 of Pl 0909).

150. The method of embodiment 149, wherein the method comprises assessing citrullination at one of the following peptide sequences, wherein the citrullination site is designated by underlining: DHEELSLVASEAVRAIEK; AKPALEDLRQGLLPVLESFK; ERLGPLVEQGR; HTFMGVVSLGSPSGEVSHPRKT; ENGGARLAEYHAK;

LSPLGEEMRDR; SELEEQLTPVAEETRAR; TPVSDRVTK;

IVEGSDAEIGMSPWQVMLFRK; IYISGMAPRPSLAK; RQLTPYAQR;

RVEPYGENFNK; SFQTGLFTAARQSTNAYPDLR;

VLLDGVQNPRAEDLVGKSLYVSATVILHSGSDMVQAER; or ASSIIDELFQDRFFTREPQDTYHYLPFSLPHR (corresponding to SEQ ID NOs: 14, 6, 10, 11, 7, 8, 9, 12, 15, 16, 17, 18, 19, 20, or 13, respectively).

151. A method of determining citrullination of a protein or peptide fragment thereof, comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof in a biological sample from a subject, comprising:

(a) contacting the biological sample with a strong anion exchange (SAX) chromatography medium,

(b) enzymatically digesting proteins in the sample either before or after the contacting to form the peptide fragment, and

152. The method of embodiment 151, wherein the enzymatically digesting is conducted after the contacting.

153. The method of embodiment 151 or 152, wherein the SAX chromatography medium comprises particles.

154. The method of embodiment 153, wherein the particles comprise magnetic particles.

155. The method of embodiment 153 or 154, wherein the SAX chromatography medium enriches membrane-bound proteins and peptide fragments thereof in the sample.

156. The method of any one of embodiments 153-155 wherein the contacting comprises incubating the sample with the SAX chromatography medium for at least 30 minutes.

157. The method of any one of embodiments 153-156, wherein the method further comprises eluting proteins or peptide fragments thereof bound to the SAX chromatography medium either before or after the enzymatically digesting, and assessing citrullination of the eluted proteins or peptide fragments thereof.

158. The method of embodiment 157, wherein the method further comprises washing the proteins or peptide fragments thereof bound to the SAX chromatography medium at least once prior to eluting the proteins or peptide fragments thereof from the SAX chromatography medium.

159. The method of embodiment 157 or 158, wherein the enzymatically digesting is conducted after the eluting. 160. The method of any one of embodiments 151-159, wherein the method comprises at least one further chromatography or a filtration step after the enzymatically digesting and prior to the assessing citrullination to separate the protein or peptide fragment thereof from one or more salts, buffers, or small molecules.

161. The method of any one of embodiments 151-160, wherein the assessing citrullination comprises measuring a first concentration of citrullinated peptide in the digested peptides and a second concentration of signature peptide in the digested peptides, and optionally determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.

162. The method of any one of embodiments 151-161, comprising denaturing proteins in the sample prior to the assessing.

163. The method of embodiment 162, comprising denaturing the proteins prior to the enzymatically digesting.

164. A method of determining citrullination of a protein or peptide fragment thereof, comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof in a biological sample from a subject, comprising:

(b) eluting proteins bound to the SAX chromatography medium, wherein the proteins bound to the SAX chromatography medium are optionally washed at least once with a wash buffer prior to the eluting,

(b) enzymatically digesting the eluted proteins to form peptide fragments thereof, optionally wherein the proteins are denatured prior to the enzymatically digesting,

(c) conducting chromatography or filtration on the enzymatically digested proteins to separate the enzymatically digested proteins from one or more salts, buffers, or small molecules, and

(d) assessing citrullination of the peptide fragment at the citrullination site.

165. The method of any one of embodiments 151-164, wherein the assessing citrullination comprises analysis with mass spectrometry (MS) or performing liquid chromatography and mass spectrometry (LC-MS).

166. The method of any one of embodiments 151-165, wherein the biological sample has been exposed to a PAD2 modulator such as a PAD2 inhibitor, a PALM modulator such as a PALM inhibitor, or a PAD2 and PALM modulator such as a PAD2 and PALM inhibitor.. 167. The method of any one of embodiments 151-166, wherein the citrullination site is a site listed in Table 1.

168. The method of any one of embodiments 151-166, wherein the citrullination site is a site listed in Table 11.

169. The method of any one of embodiments 151-166, wherein the sequence of the peptide fragment comprises one of the following sequences, wherein the citrullination site is designated by underlining of R residues and wherein modifications of non-arginine residues are designated by underlining of the modified residues: DHEELSLVASEAVRAIEK;

AKPALEDLRQGLLPVLESFK; ERLGPLVEQGR; HTFMGVVSLGSPSGEVSHPRKT; ENGGARLAEYHAK; LSPLGEEMRDR; SELEEQLTPVAEETRAR; TPVSDRVTK; IVEGSDAEIGMSPWQVMLFRK; IYISGMAPRPSLAK; RQLTPYAQR;

RVEPYGENFNK; SFQTGLFTAARQSTNAYPDLR;

170. The method of any one of embodiments 151-169, wherein the biological sample comprises whole blood, plasma, serum, or blood supernatant.

171. The method of any one of embodiments 151-169, wherein the biological sample comprises synovial fluid.

172. The method of any one of embodiments 151-171, wherein the peptide fragment comprises the sequence of: DHEELSLVASEAVRAIEK; AKPALEDLRQGLLPVLESFK; ERLGPLVEQGR; or HTFMGVVSLGSPSGEVSHPRKT (SEQ ID NOs: 14, 6, 10, or 11, respectively).

173. The method of any one of embodiments 151-172, wherein the method comprises assessing citrullination of two or more, three or more, four or more, 2-150, 2-100, 2-50, 5- 150, 5-100, 5-50, 10-150, 10-100, 10-50, 10-25, 25-50, 50-100, 50-150, 100-150, or 2-25 proteins or peptide fragments thereof.

174. The method of any one of embodiments 1-173, wherein the biological sample is obtained from a subject that is ACPA positive.

175. The method of any one of embodiments 1-174, wherein the biological sample is obtained from a subject that is positive for endogenous PAD antibodies.

176. The method of any one of embodiments 1-175, wherein the biological sample is obtained from a subject that has been diagnosed with a citrullinati on-related disease or is at risk for developing a citrullination-related disease. 177. The method of any one of embodiments 1-176, wherein the method further comprises selecting the subject from which the biological sample was derived for treatment of a citrullination-related disease.

178. The method of embodiment 176 or 177, wherein the citrullination-related disease is an autoimmune disorder.

179. The method of embodiment 176 or 177, wherein the citrullination-related disease is rheumatoid arthritis, lupus (e.g., systemic lupus erythematosus (SLE)), lupus nephritis, vasculitis (e.g., ANCA-associated vasculitis), thrombosis (e.g, venous thrombosis), or inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn’s disease).

180. The method of any one of embodiments 1-176, wherein the biological sample is obtained from a subject that is a normal, healthy subject.

181. The method of any one of embodiments 1-179, wherein the biological sample is obtained from a subject following treatment of the subject with at least one dose of a PAD2 modulator such as a PAD2 inhibitor.

182. The method of any one of embodiments 1-179, wherein the method further comprises administering a PAD2 modulator such as a PAD2 inhibitor to the subject, and optionally assessing citrullination of a citrullination site on a protein or peptide fragment thereof in a sample from the subject collected following administering of the PAD2 modulator or PAD2 inhibitor.

Any of the method embodiments listed above that include a PAD2 modulator may, in further embodiments, also be performed with other therapeutic agents. Nonlimiting examples of such other therapeutic agents include agents that may be used for treatment of citrullination-related diseases, and agents that target molecules involved in biological pathways, such as, for example, NETosis or METosis, which may also involve PAD2. Thus, in the above such methods, a PAD2 modulator may be substituted with such another therapeutic agent. In any of the above embodiments, where a PAD2 modulator is included in the method, the PAD2 modulator may, in some cases, be a PAD2 inhibitor, examples of which are provided throughout the disclosure.

In any of the above embodiments, in some cases the assessing of citrullination may be performed by MS, including by LC-MS, for example. In any of the above embodiments, unless particular citrullination sites or proteins or peptides are recited, citrullination sites for assessment may be as recited, for instance, in embodiments above. In any of the methods herein, unless specifically stated otherwise, biological samples may be any such samples recited herein, including without limitation, biological fluid samples such as blood, plasma, serum, blood supernatant, synovial fluid, pleural fluid, interstitial fluid, lymph, sweat, tears, or urine, as well as tissue samples. As also indicated above, in any of the methods herein, a citrullination ratio, as described below, and/or a concentration of citrullinated protein or peptide and a concentration of corresponding total protein may be determined as part of the method. In addition, in any of the methods, as indicated above, a biological sample may be prepared prior to assessing, in a variety of ways, such as dilution, freezing and thawing, denaturing of proteins, enzymatic digestion of proteins, separation of a supernatant from the sample for analysis, and the like. The above embodiments may also be applied to control samples as well as to biological samples of interest, for instance, to allow for comparisons of citrullination assessments between samples of interest and controls.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims. References cited herein are incorporated by reference in their entirety, and the amino acid sequences of proteins obtainable at www.uniprot.org through their UniProt accession numbers provided herein are also incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A-1D show induction of citrullination by PAD4 and inhibition of citrullination ex vivo in incubated blood by an anti-PAD4 antibody inhibitor. Whole blood samples were treated with the anti-PAD4 antibody hzl3-5 D31E, PBS (control), or isotype control and incubated for 48 h at 37°C for induction of citrullination. PBS-Oh shows citrullination levels before induction. Samples were analyzed using global proteomics. Citrullination of the peptides listed in Table 3 was assessed. The average % citrullination for each peptide, calculated as the average ratio of citrullinated peptide intensity to total protein intensity *100%, wherein the total protein intensity was measured by measuring the intensity of a signature peptide, is shown for 6 donors. Black bar: 1^st blood draw; gray bar: repeat blood draw 1 week later. FIG. 1 A shows the results for the gelsolin (GSN) peptide (SEQ ID NO: 54). FIG. IB shows the results for the complement C3 (C3) peptide (SEQ ID NO: 23). FIG. 1C shows the results for the SerpinCl peptide (SEQ ID NO: 85). FIG. ID shows the results for the fibrinogen A (FGA) peptide (SEQ ID NO: 81).

FIG. 2A-2C show that citrullination of endogenous serum proteins by exogenous PAD4 is inhibited by hzl3-5 D3 IE in a dose dependent manner. Samples were analyzed using targeted proteomic approach on nanoLC-TimsTOF platform. Citrullination of the peptides listed in Table 7 was assessed. The % citrullination is shown and was calculated as described for FIGs. 1 A-1D. FIG. 2A shows inhibition of citrullination of the FGA peptide ESSSHHPGIAEFPSRGK (SEQ ID NO: 26). FIG. 2B shows inhibition of citrullination of the FGA peptide QFTSSTSYNRGDSTFESK (SEQ ID NO: 81). FIG. 2C shows inhibition of citrullination of the PRG4 peptide (SEQ ID NO: 80).

FIG. 3 A-3B show that induction and inhibition of citrullination ex vivo in incubated blood (from 1^st draw) was confirmed using a targeted proteomic approach. Whole blood samples were treated with Ab hzl3-5 D31E, PBS (control), or isotype control and incubated for 48 h at 37°C. PBS-Oh shows citrullination levels before induction. Samples were analyzed with targeted proteomics. Citrullination of GSN and FGA peptides, sequences of which are provided in Table 8, was assessed. The average % citrullination was calculated as described for FIGs. 1 A-1D (N=6 donors). Error bars are standard errors of the mean. FIG. 3 A shows the results for the GSN peptide (SEQ ID NO: 54). FIG. 3B shows results for the FGA peptide (SEQ ID NO: 81).

FIG. 4 shows targeted proteomic analysis of ex vivo citrullinated FGA in TruCulture® samples after immunocapture enrichment. The FGA peptide QFTSSTSYNRGDSTFESK (SEQ ID NO: 81) was measured on the LC-TripleQuad® 7500 system. Representative chromatograms are shown from two different donor samples in which citrullination percentages of 0.5% (left panel) and 2.6% (right panel) were measured. The % citrullination was calculated as described for FIGs. 1 A-1D.

FIGs. 5A-5B show that the hzl3-5 D3 IE antibody maintained potency in the presence of endogenous PAD4 antibodies from RA patients. FIG. 5 A shows PAD4 autoantibodies measured by OD450 in ELISA from purified IgG of serum samples of 21 RA patients and 10 healthy control subjects (NHV). FIG. 5B shows inhibition of H3 citrullination by hzl3-5 D3 IE in the presence of purified IgG from RA (closed squares) or NHV (open squares) serum. Dotted lines represent H3 citrullination by PAD4 in the presence of purified IgG from RA donors (lower line) and NHV donors (upper line) without hzl3-5 D3 IE.

FIG. 6 shows the citrullinated proteoglycan 4 (Cit-PRG4) plasma concentrations in pM from rheumatoid (RA) patents and normal healthy volunteers (NHV). Error bars represent mean ±SD. 30 RA and 30 NHV samples were analyzed for this dataset. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

I. Definitions

Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

In this application, the use of “or” means “and/or” unless stated otherwise. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim in the alternative only. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

Exemplary techniques used in connection with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), enzymatic reactions, and purification techniques are described, e.g., in Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), among other places.

As used herein, the term “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values (e.g., +/-5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

The terms “subject” and “patient” are used interchangeably herein to refer to a human unless expressly indicated otherwise (i.e., a murine subject or the like).

A “biological sample” herein refers to a sample taken from a biological source, such as from a subject. A “biological sample” may comprise fluid and/or tissue, and thus it may be a “biological fluid sample” or a “tissue sample,” and it may be processed, such as centrifuged or mixed with other agents or the like, in order to facilitate analysis. For example, certain biological fluid samples may comprise supernatant following separation of particulate and fluid matter. In some cases, the biological sample comprises, for example, blood or blood components such as whole blood, serum, plasma, or blood supernatant. In some cases, it may comprise synovial fluid. In other cases, the biological sample may be a tissue sample, for instance, a tumor sample or a neoplasia sample. In other cases, a biological sample herein may comprise biological fluid (i.e., a biological fluid sample), such as, without limitation, blood, plasma, serum, blood supernatant, synovial fluid, pleural fluid, interstitial fluid, lymph, sweat, tears, urine, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), or sputum.

The term “polypeptide” refers to a polymer of amino acid residues, and is not limited to a minimum length. A “protein” may comprise one or more polypeptides. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a “polypeptide” or “protein” refers to a polypeptide or protein, respectively, which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site- directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins or errors due to PCR amplification. A protein may comprise two or more polypeptides.

The term “peptide fragment” or “peptide” of a longer polypeptide or protein (for example, a “protein or peptide fragment thereof’) refers to a portion of the polypeptide or protein. In some cases, analysis of a chemical modification of an amino acid residue in a polypeptide, for example, may be performed by analyzing a peptide fragment thereof that includes the amino acid residue in question.

Some amino acids within a protein or its peptide fragment may be “citrullinated.” As used herein, this term means that the protein or its peptide fragment contains at least one amino acid that is converted from one of the 20 naturally occurring amino acids to a citrulline amino acid. For example, certain arginine (Arg, R) residues may be chemically modified to convert their side chains to the side chain of the amino acid citrulline (Cit). Thus, such arginine residues, and the corresponding peptides or proteins on which they are found are referred to as being citrullinated.

The amino acid residue that is converted to citrulline may also be referred to herein as a “citrullination site.” For example, a residue at a particular amino acid position within a protein may be citrullinated. In such a case, that particular amino acid position may be referred to as a “citrullination site.” A citrullination site herein may in some cases be identified with reference to an exemplary, reference amino acid sequence for the protein. In the case where a particular protein has more than one naturally occurring amino acid sequence, one such sequence may be provided herein as a reference sequence for identifying the residue corresponding to a citrullination site, and that site may be identified in other naturally occurring variants of the protein through sequence alignment with the reference sequence provided herein. A given protein may also have one or more such citrullination sites when more than one residue in the protein is found to be citrullinated.

A “PAD” or “protein arginine deiminase” refers to a protein that catalyzes conversion arginine to citrulline in a protein or peptide under certain conditions. Examples of PADs include PAD4 and PAD2, for example.

“PAD2” or “protein arginine deiminase 2” or “peptidyl arginine deiminase 2,” as used herein, refers to human PAD2 (huPAD2; UniProt ID: Q9Y2J8), unless expressly noted otherwise (i.e., murine PAD2, cynomolgus PAD2, or the like). An exemplary human PAD2 amino acid sequence is shown in SEQ ID NO: 4 (human isoform 1), while a truncated PAD2 isoform 2 sequence is provided in SEQ ID NO: 5. PAD2 protein is also known by other acronyms such as PAD-H19, and the associated gene is also known by the acronyms PADI2, PDI2, and KIAA0994 (see www.uniprot.org, under Q9Y2J8).

As used herein, a “PAD2 modulator” refers to a compound (e.g.., a small molecule, large molecule, or biologic) that alters (i.e., modulates) at least one activity or function of PAD2. In some cases, a “PAD2 modulator” is a “PAD2 inhibitor,” which is a compound that reduces at least one activity of PAD2, i.e., a PAD2 antagonist. In other cases, a “PAD2 modulator” is a compound that increases the activity of PAD2, i.e., a PAD2 agonist. The modulation of the activity of PAD2 may be by any mechanism and may be observed in vitro and/or in vivo. In some cases, a PAD2 modulator may modulate the activity of additional proteins in addition to PAD2, such as, for example, additional PAD enzymes such as PAD4; while in other cases, the activity modulation may be specific to PAD2 and the PAD2 modulator does not modulate the activity of other proteins such as, for example, other PAD enzymes such as PAD4.

“PAD4” or “protein arginine deiminase 4” or “peptidyl arginine deiminase 4,” as used herein, refers to human PAD4 (huPAD4; UniProt ID: Q9UM07), unless expressly noted otherwise (i.e., murine PAD4, cynomolgus PAD4, or the like). Exemplary human PAD4 amino acid sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 and SEQ ID NO: 3. As used herein, a “PAD4 modulator” refers to a compound (e.g.., a small molecule, large molecule, or biologic) that alters (i.e., modulates) at least one activity or function of PAD4. In some cases, a “PAD4 modulator” is a “PAD4 inhibitor,” which is a compound that reduces at least one activity of PAD4, i.e., a PAD4 antagonist. In other cases, a “PAD4 modulator” is a compound that increases the activity of PAD4, i.e., a PAD4 agonist. The modulation of the activity of PAD4 may be by any mechanism and may be observed in vitro and/or in vivo. In some cases, a PAD4 modulator may modulate the activity of additional proteins in addition to PAD4, such as, for example, additional PAD enzymes, such as PAD2; while in other cases, the activity modulation may be specific to PAD4 and the PAD4 modulator does not modulate the activity of other proteins such as, for example, other PAD enzymes such as PAD2.

The term “agonist” as used herein refers to a compound that causes an increase in at least one activity or function of a molecule to which it binds, or otherwise activates or helps to activate the molecule. The term “antagonist” or “inhibitor” as used herein refers to a compound that causes a decrease in at least one activity or function of a molecule to which it binds, or that otherwise blocks or inhibits at least one activity or function of the molecule.

The terms “inhibition” or “inhibit” more generally refer to a decrease or cessation of any event (such as protein ligand binding) or to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and/or amount as compared to a reference. It is not necessary that the inhibition or reduction be complete. For example, in certain embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20% or greater. In another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater. In yet another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.

In some cases, a PAD2 modulator may be a small molecule compound, or it may be an antibody. An “anti-PAD2 antibody” or a “PAD2-antibody” or an “antibody that specifically binds to PAD2” or an “antibody that binds to PAD2” and similar phrases refer to an antibody that specifically binds to PAD2 as defined herein. The term “antibody” herein refers to a molecule comprising at least complementarity-determining region (CDR) 1, CDR2, and CDR3 of a heavy chain and at least CDR1, CDR2, and CDR3 of a light chain, wherein the molecule is capable of binding to antigen. The term is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, diabodies, etc.), full length antibodies, single-chain antibodies, antibody conjugates, and antibody fragments, so long as they exhibit the desired PAD2-specific binding activity.

An “antigen” refers to the target of an antibody, i.e., the molecule to which the antibody specifically binds. The term “epitope” denotes the site on an antigen, either proteinaceous or non-proteinaceous, to which an antibody binds. Epitopes on a protein can be formed both from contiguous amino acid stretches (linear epitope) or comprise noncontiguous amino acids (conformational epitope), e.g., coming in spatial proximity due to the folding of the antigen, i.e., by the tertiary folding of a proteinaceous antigen. Linear epitopes are typically still bound by an antibody after exposure of the proteinaceous antigen to denaturing agents, whereas conformational epitopes are typically destroyed upon treatment with denaturing agents.

The term “heavy chain” refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term “full-length heavy chain” refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

The term “light chain” refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term “full-length light chain” refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

The term “complementarity determining regions” (“CDRs”) as used herein refers to each of the regions of an antibody variable region which are hypervariable in sequence and which determine antigen binding specificity. Generally, antibodies comprise six CDRs: three in the VH (CDR-H1 or heavy chain CDR1, CDR-H2, CDR-H3), and three in the VL (CDR- Ll, CDR-L2, CDR-L3). Unless otherwise indicated, the CDRs are determined according to the sequence table herein.

“Framework” or “FR” refers to the residues of the variable region residues that are not part of the complementary determining regions (CDRs). The FR of a variable region generally consists of four FRs: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR- L1)-FR2- CDR-H2(CDR-L2)-FR3- CDR-H3(CDR-L3)-FR4. The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). See, e.g., Kindt et al. Kuby Immunology, 6^th ed., W.H. Freeman and Co., page 91 (2007). A variable domain may comprise heavy chain (HC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and/or FR4; and light chain (LC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and/or FR4. That is, a variable domain may lack a portion of FR1 and/or FR4 so long as it retains antigen-binding activity. A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150 :880-887 (1993) ; Clarkson et al., Nature 352 :624-628 (1991).

The light chain and heavy chain “constant regions” of an antibody refer to additional sequence portions outside of the FRs and CDRs and variable regions. Certain antibody fragments may lack all or some of the constant regions. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant heavy domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain.

The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen (i.e., PAD2) to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005).

A “multispecific” antibody is one that binds specifically to more than one target antigen, while a “bispecific” antibody is one that binds specifically to two antigens. An “antibody conjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a therapeutic agent or a label.

“Percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The term “signal sequence” or “leader sequence” refers to a sequence of amino acid residues located at the N terminus of a polypeptide that facilitates secretion of a polypeptide from a mammalian cell. A leader sequence may be cleaved upon export of the polypeptide from the mammalian cell, forming a mature protein. Leader sequences may be natural or synthetic, and they may be heterologous or homologous to the protein to which they are attached. Nonlimiting exemplary leader sequences also include leader sequences from heterologous proteins. In some embodiments, an antibody lacks a leader sequence. In some embodiments, an antibody comprises at least one leader sequence, which may be selected from native antibody leader sequences and heterologous leader sequences.

In this disclosure, “binds” or “binding” or “specific binding” and similar terms, when referring to a protein and its ligand or an antibody and its antigen target for example, or some other binding pair, means that the binding affinity between the members of the binding pair is sufficiently strong that the interaction cannot be due to random molecular associations (i.e. “nonspecific binding”). Such binding typically requires a dissociation constant (KD) of IpM or less, and may often involve a KD of 100 nM or less.

“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). Affinity can generally be represented by the dissociation constant (KD). Affinity of an antibody for an antigen can be measured by common methods known in the art, such as surface plasmon resonance (SPR), for instance. “Treatment” as used herein, covers any administration or application of a therapeutic for disease in a human, and includes inhibiting the disease or progression of the disease or one or more disease symptoms, inhibiting or slowing the disease or its progression or one or more of its symptoms, arresting its development, partially or fully relieving the disease or one or more of its symptoms, or preventing a recurrence of one or more symptoms of the disease.

An “autoimmune disease” or “autoimmune disorder,” as used herein, encompasses a disease characterized by the subject’s immune system attacking its own normal cells and tissues, and also encompasses immune-mediated diseases which may or may not be characterized by presence of auto-antibodies. The disclosure provides many nonlimiting examples of autoimmune diseases throughout. Some nonlimiting examples of autoimmune diseases include rheumatoid arthritis (RA), lupus (e.g., systemic lupus erythematosus (SLE)), lupus nephritis, vasculitis (e.g., ANCA-associated vasculitis), thrombosis (e.g, venous thrombosis), and inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn’s disease). The terms “disease” and “disorder” are used interchangeably herein. In some embodiments, the autoimmune disease is characterized by the presence of auto-antibodies. The term “effective amount” or “therapeutically effective amount” refers to an amount of a drug effective for treatment of a disease or disorder in a subject, such as to partially or fully relieve one or more symptoms. In some embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

Additional definitions of terms or phrases used herein are included in the sections that follow.

II. Methods of Assessing Citrullination

The present disclosure encompasses several methods of assessing the citrullination by PAD2 and also of assessing the citrullination modifying activity of a PAD2 modulator or another therapeutic agent, and also of assessing citrullination of a biological sample from a subject, inter alia. The citrullination site or sites can be on proteins or peptides. In some cases, the peptides are peptide fragments of proteins, for instance, digested peptides that arise from enzymatic digestion of proteins. Thus, the expression “assessing citrullination of a citrullination site” encompasses assessing one citrullination site on one protein or peptide, as well as assessing more than one citrullination site on one protein or peptide, as well as assessing multiple citrullination sites. And accordingly, while the disclosure herein may recite a citrullination site on a protein or peptide, the “a” in this expression indicates “one or more” citrullination sites, and citrullination of more than one protein or peptide may also be assessed. Assessing multiple citrullination sites can encompass assessing citrullination sites on a plurality of proteins or on a plurality of peptides. For instance, the plurality of citrullination sites may be a plurality of citrullination sites on one protein or on peptide fragments of such protein, and/or the plurality of citrullination sites may be a plurality of citrullination sites on a plurality of different proteins or peptides. Although the methods of assessing citrullination disclosed herein may be used to assess citrullination at any citrullination site, in some embodiments, the citrullination site (e.g., the plurality of citrullination sites) is a citrullination site disclosed herein. Any and all such combinations are contemplated herein.

Among the methods herein include, inter alia, a method of assessing citrullination at a citrullination site by mass spectrometry (MS). In some cases, the methods comprise assessment by liquid chromatography followed by MS (LC-MS). For example, liquid chromatography (e.g., HPLC, RPLC, nLC, pLC, or the like) may be used to separate peptides or proteins for analysis by MS. In some cases, the method comprises measuring, in a biological sample: (i) a first concentration of a citrullinated protein or a citrullinated peptide (which may be from a protein, e.g., a protein that has been enzymatically digested), wherein the citrullinated protein or citrullinated peptide is citrullinated at a citrullination site, and (ii) a second concentration of the corresponding total protein (which includes modified and unmodified forms of the protein) in the sample. In some embodiments, the concentration of corresponding total protein is measured by measuring a concentration of a signature peptide from the protein. The signature peptide is a peptide that is present both in modified and unmodified forms of the protein, and the signature peptide is a peptide that is not itself modified. Accordingly, when the concentration of signature peptide present in a sample within a well is measured by mass spectrometry, for example, the concentration of the signature peptide represents the well total concentration of the protein. In some embodiments, the method of measuring concentrations (i) and (ii) is by MS, such as LC-MS. In some embodiments, the method further comprises calculating a citrullination ratio. A “citrullination ratio,” as used herein, refers to a ratio of the first concentration (i) to the second concentration (ii). A citrullination ratio may be expressed in a variety of ways, for instance, as a percentage, or as any other fraction that is proportional to the aforementioned ratio of the first concentration to the second concentration.

In some cases, the method comprises assessing citrullination of more than one citrullination site. Accordingly, the method can comprise measuring the first concentration and the second concentration for each of a plurality of (meaning two or more) different proteins or peptides that each contain a citrullination site. In some such cases, the method comprises measuring the first concentration and the second concentration for each of the plurality of proteins or peptides. In some such cases, at least two different peptides of the plurality are nonoverlapping fragments of the same protein and contain different citrullination sites; in those cases, the second concentration is the same for the peptides that are fragments of the same protein. In some cases, the method comprises calculating a ratio of the first concentration to the second concentration for each of the plurality of different proteins or peptides.

Exemplary citrullination sites, proteins, and peptide fragments thereof are discussed below and provided in Table 1 and Table 11 herein. For example, Table 1 and Table 11 provide several citrullination sites and associated peptide fragments that were found to be significantly citrullinated by PAD2 at physiological concentrations. In some cases, the peptides were also significantly citrullinated by PAD4; in other cases they were not significantly citrullinated by PAD4. Table 1 and Table 11 also provide certain peptides that were found to be citrullinated significantly by PAD4 but not by PAD2. Thus, in some embodiments, a citrullination site or its associated peptide fragment is a site or peptide listed in Table 1 and/or Table 11 as citrullinated by PAD2 or by both PAD2 and PAD4 (marked either “PAD2” or “common” in the Target column in Tables 1 and 11). In some embodiments, the citrullination site or its associated peptide fragment is listed in Table 1 and/or Table 11 as citrullinated by PAD2 but not by PAD4 (marked “PAD2” in the Target column in Tables 1 and 11). In some cases, the biological sample is obtained from a subject. In some cases, the biological sample is a whole blood, plasma, serum, blood supernatant, or synovial fluid sample. In other cases, the biological sample is a tissue sample. In other cases, the biological sample is a cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), urine, or sputum sample. In some cases, the biological sample is a serum, plasma, or blood supernatant sample. In some cases, the method further comprises comparing the first and second concentrations, or a ratio of the first and second concentrations to first and second concentrations or a ratio of first and second concentrations obtained from a control.

In some cases where citrullination of a biological sample from a subject is assessed herein, the method further comprises determining one or more of the following based on the first and second concentrations or a ratio of the first and second concentrations: probability of a clinical outcome, risk for developing a citrullination-related disease, diagnosis of a citrullinati on-related disease, and selecting the subject from which the biological sample was derived for a treatment of a citrullination-related disease. For instance, in some cases, the subject either has a citrullination-related disease or may be at risk of developing a citrullination-related disease, and an assessment of citrullination in a biological sample, according to methods herein, may be used to confirm a diagnosis of such a disease, or to determine whether or not the subject is at risk of developing such a disease. In some cases, the method may allow for monitoring of citrullination in a subject’s biological samples, in order to determine a probability of clinical outcome, for example, based on comparison to reference subjects whose clinical outcomes are known, or to determine whether a PAD2 modulator or another therapeutic agent would be useful for treating the subject, or to determine if a PAD2 modulator or other therapeutic agent being currently administered to the subject is operating on the subject to reduce citrullination. Thus, for instance, in one example, the method further comprises determining, based on the first and second concentrations or a ratio of the first and second concentrations, whether the subject should receive treatment, for instance, in some cases, for a citrullination-related disease. In some cases, the method further comprises determining, based on the first and second concentrations or a ratio of the first and second concentrations, whether the subject should receive treatment with a therapeutic agent. Nonlimiting examples of such an agent include those that may be used for treatment of citrullination-related diseases, and those that target a biological pathway, such as, for example, NETosis or METosis, which may also involve PAD2, such as, for example, an anti-histone antibody (e.g., CIT-013, for example), as well as PAD2 modulators, such as PAD2 inhibitors, including PAD2 and PAD4 modulators or PAD2 and PAD4 inhibitors, which target both proteins as well as PAD2 modulators or inhibitors that target specifically PAD2. In other cases, the method comprises determining whether a subject already receiving treatment with a therapeutic agent or other therapy should have the treatment adjusted, such as to increase or decrease a dose of a therapeutic agent being administered, or to start treatment with an agent such as a PAD2 modulator or an agent that targets a molecule involved in a biological pathway that involves PAD2. In some cases, the method further comprises determining whether a subject should receive treatment with a PAD2 modulator, or whether a subject already receiving treatment with a PAD2 modulator should have treatment adjusted, such as to increase or reduce the dose of the PAD2 modulator or to discontinue its use. In some cases, the PAD2 modulator is a PAD2 inhibitor. Exemplary PAD2 modulators are provided in this disclosure, and include, for example, therapeutic agents that can impact the expression or activity of PAD2, including the citrullination activity of PAD2. Such molecules include antibodies such as anti-PAD2 antibodies, as well as small molecules, such as small molecule PAD2 inhibitors, among others described in this disclosure.

The present disclosure also encompasses a method of assessing the activity of a therapeutic agent, including, without limitation, an agent that may be used for treatment of a citrullination-related disease, an agent that targets a biological pathway involving PAD2, an agent that targets NETosis and/or METosis, an anti-histone antibody, and a PAD2 modulator such as a PAD2 inhibitor. Such therapeutic agents may, in some cases, comprise large molecules such as antibodies or fusion proteins, or may comprise small molecules. In some embodiments, a method of assessing the activity of a therapeutic agent, such as a PAD2 modulator, comprises assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample from a subject. In such methods, the biological sample has been exposed to a PAD2 modulator or other therapeutic agent, either directly by adding it to the sample prior to assessment, or indirectly, by the subject having received at least one dose of the PAD2 modulator or other therapeutic agent prior to the sample being obtained. In some cases, the therapeutic agent being assessed is a PAD2 modulator. In some cases, the PAD2 modulator is a PAD2 inhibitor.

In any of the methods described herein, in some embodiments, at least two different citrullination sites are assessed. In some cases, at least three different sites are assessed. In some cases, at least four different sites are assessed. In some cases, citrullination of at least two different proteins is assessed. In some cases, citrullination of at least three different proteins is assessed. In some cases, citrullination of at least four different proteins is assessed. In some cases, citrullination of 2-4 citrullination sites and/or 2-4 different proteins is assessed.

In any of the methods herein, in some cases the protein comprising the citrullination site for assessment is selected from one or more of the proteins listed in Table 1 and/or in Table 11. In some cases, the citrullinated protein comprises one or more of: CPB2 (carboxypeptidase B2), AP0A1 (apolipoprotein A-I), APOE (apolipoprotein E), AHSG (alpha-2-HS-glycoprotein), F2 (prothrombin), VTN (vitronectin), AP0A4 (apolipoprotein A- IV), PROS1 (vitamin K-dependent plasma glycoprotein), ALB (albumin), C3 (complement C3), CLU (clusterin),. In some cases, the protein comprises one or more of: CPB2 (carboxypeptidase B2), AP0A1 (apolipoprotein A-I), APOE (apolipoprotein E), AHSG (alpha-2-HS-glycoprotein), F2 (prothrombin), VTN (vitronectin), AP0A4 (apolipoprotein A- IV), PROS1 (vitamin K-dependent plasma glycoprotein), ALB (albumin), C3 (complement C3), CLU (clusterin), ITIH4 (inter-alpha-trypsin inhibitor heavy chain H4), KNG1 (kininogen 1), FGA (fibrinogen alpha chain), AMBP (alpha- 1-microglobulin/bikunin precursor), VTN (vitronectin), CFH (complement factor H), PLG (plasminogen), APOB (apolipoprotein B-100), FN1 (fibronectin- 1), LGALS3BP (galectin-3 binding protein; lectin galactoside-binding soluble 3 binding protein), F5 (factor V), or A2M (alpha-2- macroglobulin). Thus, in some embodiments, a citrullination site from a protein above, or a peptide fragment thereof, is assessed. For example, in some embodiments, the method herein comprises a method of assessing the activity of a PAD2 modulator or other therapeutic agent, the method comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample from a subject, wherein the biological sample has been exposed to a PAD2 modulator or other therapeutic agent, and optionally wherein the protein is selected from one or more of those listed above, or wherein the protein is selected from one or more of those listed in Table 1 and/or in Table 11 below, such as those indicated as citrullinated by PAD2 in Table 1 and/or in Table 11 below.

In some cases, the citrullination site to be assessed is selected from one or more citrullination sites shown in Table 1 and/or in Table 11 below. In these tables below, the amino acid residue corresponding to a citrullination site is noted in the left column, with a reference accession number that may be used to locate the citrullination site in the publicly available UniProt database is listed in the right column. The UniProt accession number for each protein listed is provided in the table. The public database provides an amino acid and gene sequence corresponding to the accession number (e.g., P01023 for alpha-2 - macroglobulin). It is understood that each of the proteins listed in the table below may have more than one naturally occurring amino acid sequence. Different isoforms of a protein can arise, for instance, due to the natural occurrence of different alleles reflecting substitutions, insertions, and/or deletions in the amino acid sequence, as well as due to alternative promoters, splicing and/or translation initiation sites. Accordingly, the accession number and its associated sequence listed for a given protein in Table 1 and/or Table 11, and also in further tables herein that also provide citrullination sites and reference protein accession numbers or sequences, is merely a reference, which may be used to identify the citrullination site in a protein having the same sequence as the reference sequence as well as to identify the citrullination site in a naturally occurring variant protein, for example, by alignment of the variant sequence against the reference sequence. In some embodiments only one of the citrullination sites of Table 1 and/or Table 11 is assessed, while in other cases, at least two, at least three, at least five, at least ten, at least twenty, or at least fifty of the sites are assessed, such as 2-5, 2-10, 5-10, 5-15, 10-20, 2-20, or 5-20 of the sites are assessed. In some cases, citrullination sites of only one protein are assessed, while in other cases, citrullination sites of two or more proteins, such as at least 2, 3, 5, 6, 7, or 8 proteins are assessed. In some cases, citrullination sites of 2-5, 2-10, 5-10, 5-15, 10-20, 2-20, or 5-20 different proteins are assessed. In some cases, the method comprises assessing citrullination of at least two, at least three, at least five, at least ten, at least twenty, at least fifty, or at least one-hundred citrullination sites of Table 1 and/or 11 herein, such as assessing the citrullinome of a sample. In some cases, the method comprises assessing citrullination of two or more, three or more, four or more, 2-150, 2-100, 2-50, 5-150, 5-100, 5-50, 10-150, 10-100, 10-50, 10-25, 25-50, 50-100, 50-150, 100-150, or 2-25 proteins or peptide fragments thereof.

In some cases, the method comprises assessing citrullination of a peptide fragment of a protein, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 1 and/or in Table 11. In some cases at least two peptide sequences as shown in Table 1 and/or Table 11 are assessed, such as 2, 3, 4, 5 or 10 peptides citrullinated by PAD2 listed in Table 1 and/or Table 11. Proteins or peptides that are assessed for citrullination may, in some cases, contain chemical modifications other than citrullination, such as carbamidomethylation of cysteines, oxidation of methionines, and modifications of the N- terminus, among others. Such chemical modifications can be detected, for instance, by mass spectrometry.

In some cases, the citrullination site is selected from one or more sites that are citrullinated significantly by PAD2 listed in Table 1 and/or Table 11, such as significantly by either PAD2 or by both PAD2 and PALM. In some cases, the citrullination site is selected from one or more sites that is listed in Table 1 and/or Table 11 as citrullinated significantly by PAD2 but not by PALM.

In some cases, the citrullination site is selected from one or more of the following: R342 of Uniprot sequence Q961Y4 (i.e., the amino acid sequence Q961Y4 available at www.uniprot.org) for CPB2 (carboxypeptidase B2), R239 of apolipoprotein A-l (APOA1; corresponding to R239 of P02647), R212 of apolipoprotein A-l (APOA1; corresponding to R212 of P02647), R173 of apolipoprotein A-l (APOA1; corresponding to R173 of P02647), R108 of apolipoprotein E (APOE; corresponding to R108 of P02649), R198 of apolipoprotein E (APOE; corresponding to R198 of P02649), R337 of alpha-2-HS- glycoprotein (AHSG; corresponding to R337 of P02765), R383 of prothrombin (F2; corresponding to R383 of P00734), R362 of vitronectin (VTN; corresponding to R362 of P04004), R155 of apolipoprotein A-IV (APOA4; corresponding to R155 of P06727), R306 of apolipoprotein A-IV (APOA4; corresponding to R306 of P06727), R101 of vitamin K- dependent plasma glycoprotein (PROS1; corresponding to RIO 1 of P07225), R496 of albumin (ALB; corresponding to R496 of P02768), R315 of complement C3 (C3; corresponding to R315 of P01024), R194 of clusterin (CLU; corresponding to R194 and/or R198 of Pl 0909), or R198 of clusterin (CLU; corresponding to R194 and/or R198 of Pl 0909), wherein all of the code names beginning with Q or P followed by a number are UniProt accession numbers for the proteins.

In some cases, these citrullination sites may be assessed from one or more peptide fragments of a protein, wherein the sequences of the peptide fragments comprise (where the citrullination site is bold and underlined and other amino acid modifications at non-arginine residues are underlined): DHEELSLVASEAVRAIEK; AKPALEDLRQGLLPVLESFK; ERLGPLVEQGR; HTFMGVVSLGSPSGEVSHPRKT; ENGGARLAEYHAK;

LSPLGEEMRDR; SELEEQLTPVAEETRAR; TPVSDRVTK; IVEGSDAEIGMSPWQVMLFRK; IYISGMAPRPSLAK; RQLTPYAQR; RVEPYGENFNK; SFQTGLFTAARQSTNAYPDLR;

VLLDGVQNPRAEDLVGKSLYVSATVILHSGSDMVQAER; or ASSIIDELFQDRFFTREPQDTYHYLPFSLPHR (corresponding to SEQ ID NOs: 14, 6, 10, 11, 7, 8, 9, 12, 15, 16, 17, 18, 19, 20, or 13, respectively). In some such cases, the biological sample is a whole blood, serum, plasma, blood supernatant, or synovial fluid sample. In some such cases, the biological sample is a whole blood, serum, plasma, or blood supernatant sample.

In some cases, the citrullination site is selected from one or more of the following: R342 of Uniprot sequence Q961Y4 for CPB2 (carboxypeptidase B2), R239 of Uniprot sequence P02647 for APOA1 (apolipoprotein A-I), R198 of Uniprot sequence P02649 for APOE (apolipoprotein E), or R337 of Uniprot sequence P026765 for AHSG (alpha-2 -HS- gly coprotein). In some such cases, the sample is a whole blood, serum, plasma, or blood supernatant sample. In some such cases, the sample is a synovial fluid sample.

The “assessing” in methods herein encompasses a variety of ways of examining citrullination, such as, without limitation, qualitatively determining whether citrullination has occurred at a particular citrullination site on a protein or peptide, and quantitatively determining citrullination of a protein or a peptide. In some embodiments, the assessing comprises quantitatively determining citrullination at a particular citrullination site (e.g., a citrullination site disclosed herein). In some embodiments, quantitatively determining citrullination at a particular citrullination site comprises determining a “citrullination ratio” as defined above. In some cases, mass spectrometry (MS) is used to assess citrullination at a citrullination site. MS can detect differences between the molecular weights of a protein or peptide that is citrullinated and the corresponding protein or peptide that is not citrullinated. As described herein in the examples, intensity measurements obtained with MS can be used to measure the concentration of a citrullinated peptide. MS can also be used to measure the total concentration of corresponding protein from which a citrullinated peptide is derived. For instance, such total concentration of protein can be measured by measuring the concentration of a signature peptide. As used herein, such a “signature peptide” is a peptide that is not modified and is present in both modified and unmodified forms of the protein; accordingly, the concentration of the signature peptide represents the concentration of the corresponding total protein, including modified and unmodified forms of the protein. Selection of signature peptides is known in the art and is described, for instance, in Qiu, X L et al. Signature peptide selection workflow for biomarker quantification using LC-MS based targeted proteomics. Bioanalysis (11 April 2023) 10.4155/bio-2022-0241 C In some cases, assessing citrullination of the citrullination site comprises performing liquid chromatography to assist in separating protein or peptide species, followed by mass spectrometry (LC-MS). For example, liquid chromatography (e.g., HPLC, RPLC, nLC, pLC, or the like) may be used to separate peptides or proteins for analysis by MS.

In methods herein, a sample may be assessed directly after being obtained, or alternatively, a sample may be first prepared in some fashion prior to assessment of citrullination, for example, in order to improve the detection of the proteins or peptides of interest in the sample. For instance, a sample may be diluted with a buffer, a sample comprising cells may be treated to lyse the cells or to separate the cells from the sample fluid such as by centrifugation or filtration. For example, a protein of interest or peptide fragments thereof may first be enriched by separating them from other components of the starting sample prior to assessment of citrullination. For instance, one method of enriching a protein or peptide of interest is to isolate it from the sample. Thus, such a protein or peptide could be exposed to an affinity reagent such as an antibody and thus targeted for purification or removal from the sample. In some cases, such an affinity reagent may be immobilized on a solid surface such as a bead, chip, plate, or well of a plate, in order to facilitate such enrichment. In some cases, one may enrich a protein or peptide of interest in a sample by removing unwanted material from the sample, such as unwanted abundant protein materials or cellular debris. In other cases, a protein or peptide of interest may be enriched by contacting the sample with a binding substance such as an antibody, or a chromatography medium, for example, that binds to the protein or peptide of interest, and then eluting the protein or peptide of interest from the binding substance or medium. In some cases, proteins or peptides of interest may be enriched by a strong anion exchange chromatography (SAX) medium, which, in some embodiments, may be used in a biological sample to bind to proteins or peptides of interest and therefore enrich them with respect to other components of the sample such as abundant proteins or the like such as albumin. For instance, in some embodiments, an SAX medium may comprise particles such as beads or chips, for example magnetic particles, which, upon contact with a sample may be used to bind to proteins or peptides of interest. In other cases, an SAX medium may comprise a resin or may be comprised within a column. In some cases, a sample treated by SAX is a whole blood, plasma, serum, blood supernatant, synovial fluid, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), urine, or sputum sample. In some cases, the biological sample is a serum, plasma, CSF, BALF or blood supernatant sample. In some cases, the proteins or peptides of interest are contacted with a chromatography medium such as SAX and then eluted, optionally with at least one wash step prior to elution, in order to enrich the proteins or peptides of interest. As used herein, the term “strong anion exchange chromatography” or “SAX” refers to anion exchange chromatography with a medium comprising a quarternary ammonium salt attached to a polymer, such as an alkyl or allyl polymer. SAX media may, for example, retain a positive charge within a relatively wide pH range. In some cases, an SAX medium comprises particles such as magnetic particles, which may be in a variety of forms or shapes such as beads or chips or the like. Magnetic particles comprising an SAX medium, for instance, can be manipulated in the laboratory using an appropriate magnet, for example, to add the particles to a sample, allow them to bind to proteins in the sample, and then to remove the protein-bound particles from the sample and elute the proteins from the particles. An exemplary and nonlimiting SAX medium, for instance, is MagReSyn® SAX (ReSyn Biosciences).

In some cases, the proteins of the sample may also be enzymatically digested to form peptide fragments, may be denatured to remove tertiary structure, and/or a sample may be diluted or treated to remove or separate certain components prior to assessment. In some cases, other components could be degraded or removed from the sample, such as nucleic acids for instance, by enzymatic degradation or by alcohol precipitation.

In some cases, a sample may also be treated so as to promote release of endogenous PAD2 in the sample prior to assessment, or may be contacted with exogenous PAD2 to boost the signal from the sample, among other optional treatments. In some cases, for instance in order to boost the overall PAD2 activity in the biological sample, the sample may be incubated with exogenous PAD2 prior to assessing citrullination. For example, in some cases, a concentration of from 1 nM to 5 nM PAD2 may be added, such as from 2nM to 4 nM, from 2 nM to 3 nM. In some cases, a concentration of 1 nM, 2 nM, 3 nM, or 4 nM, or about 1 nM, about 2 nM, about 3 nM, or about 4 nM exogenous PAD2 is added. In some cases, a concentration of 3 nM exogenous PAD2 is added. In some cases, exogenous calcium ion (Ca2+) is also added to the sample. In some cases, where citrullination from additional PAD proteins is also assessed, those may also be added exogenously, such as PAD4, which could be added, for example, at concentrations of 1-15 nM, such as at 10 nM. In other cases, the sample is not exposed to exogenous PAD2 or is not exposed to any exogenous PAD proteins. Thus, in some cases, all PAD2 activity in a biological sample is from endogenous PAD2 present in the sample. For instance, the biological sample may be incubated for a period, which in some cases may induce the release of endogenous PAD2 within the sample. Such incubation may be for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24- 72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48- 60 hours or 60-72 hours. In some cases, the incubation is for 48-96 hours. In some cases, such incubation is conducted at 34-40 °C, such as at 36-39 °C, 36-38 °C, or 37 °C. In some cases, the incubation is conducted in a TruCulture® null tube (Rules Based Medicine, Austin, TX), or a similar container comprising culture media for preservation or analysis of biological samples. In some cases where the method involves assessing an exogenous PAD2 modulator, the PAD2 modulator may be added before the incubation of the sample. In other cases, a PAD2 modulator is added after the incubation. Where exogenous PAD2 is added to a sample, the sample may also be incubated with the exogenous PAD2, such as for 1-3 hours, or 1-2 hours, or 1, 2, or 3 hours, prior to assessing citrullination. In some cases, such incubation is conducted at 34-40 °C, such as at 36-39 °C, 36-38 °C, or 37 °C. In some cases, both exogenous PAD2 and an exogenous PAD2 modulator may be added together. In other cases, a PAD2 modulator may be added after the incubation with exogenous PAD2. In some cases an incubation is conducted in a CO2 incubator. In some cases, the activity of exogenous PAD2 added to a sample is quenched by adding EDTA to the sample. Thus, for example, in some cases the method comprises adding EDTA to the biological sample after incubation with the exogenous PAD2 in order to quench the activity of the PAD2.

In some cases, prior to analysis, a sample could be treated to expose one or more proteins or peptide fragments thereof comprising citrullination sites for later assessment. For example, proteins in a sample may be denatured or digested with enzymes. For instance, proteins may be denatured to remove tertiary structure, for example, by incubation at above physiological temperatures, such as at 60-90°C, such as 60-80°C, 70-85°C, or 80°C, for a period of 5-30 minutes, such as 10-30, 10-20, 15-25, or 20 minutes. In other cases, a chemical denaturant such as urea could be applied. A proteolytic enzyme such as trypsin, LysC, rLysC, LysN, GluC, AspN, rAspN, or a combination of proteolytic enzymes, such as more than one of trypsin, LysC, rLysC, LysN, GluC, AspN, and rAspN, could be used for enzymatic digestion, for example, to form peptide fragments of proteins of interest for later assessment.

In some cases, prior to analysis, a sample is treated with strong anion exchange chromatography (SAX), for example, by contact with particles such as beads or chips comprising an SAX medium, such as magnetic particles (e.g., magnetic beads or chips). In some such cases, the SAX may enrich the proteins or peptides of interest, for example, by enriching proteins or peptides of interest with respect to abundant proteins from the sample, such as albumin in the case of a whole blood, plasma, or serum sample, or with respect to other unwanted debris. In some cases, SAX may enrich membrane proteins in a sample. In some cases, SAX is performed prior to enzymatic digestion of proteins, such as with trypsin.

In some cases, prior to analysis, and in some cases following SAX and enzymatic digestion, the sample is further treated to separate proteins or peptides of interest from unwanted small molecules, enzymes, and salts or buffer ingredients. In some such cases, further chromatography or filtration steps may be used with media that binds to or retains peptides and proteins, such as enzymatically digested peptides, and allows for separation of small molecules, salts, buffer ingredients and the like.

Accordingly, overall, there are a variety of ways in which a sample may be prepared for assessment of citrullination herein.

In embodiments herein in which assessment of citrullination of a biological sample is used to assess a PAD2 modulator or other therapeutic agent, there are also several ways in which the biological sample may be exposed to the PAD2 modulator or other therapeutic agent herein. In some cases, a sample is directly incubated with a PAD2 modulator or other therapeutic agent (i.e., an exogenous PAD2 modulator), such as by contacting the biological fluid with the PAD2 modulator or other therapeutic agent ex vivo. In other cases, the biological sample has been exposed to a PAD2 modulator or other therapeutic agent in vivo in a subject as a result of the PAD2 modulator or other therapeutic agent being administered to the subject. For example, a PAD2 modulator or a metabolite thereof may travel through blood, serum, plasma, or synovial fluid or other bodily fluids or tissues after administration to a subject, or may contact cells in the body which in turn travel to such fluids or to certain bodily tissues.

A biological sample herein may comprise biological fluid (i.e., a biological fluid sample), such as, without limitation, blood, plasma, serum, blood supernatant, synovial fluid, pleural fluid, interstitial fluid, lymph, sweat, tears, or urine. In some embodiments, the biological sample may comprise a biological fluid such as cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), plasma, serum blood supernatant, urine, or sputum. In other cases, a biological sample may comprise tissue, such as from a joint, a neoplasm, or a tumor. In some methods herein, the biological sample comprises synovial fluid. In some methods, the biological sample comprises blood or is derived from blood; in some cases the biological sample comprises serum, plasma, or blood supernatant (e.g. a supernatant that forms after centrifugation or filtration of blood). In some cases, the biological sample comprises plasma. In some cases, the biological sample comprises serum. In some cases, the biological sample comprises BALF. In some cases, the biological sample is fresh, meaning that it has not been frozen and thawed prior to use. In other cases, the sample has been frozen and thawed before use in the method. Thus, in some cases the method comprises freezing and thawing the biological sample before assessing citrullination of the citrullination site. For example, the sample may be frozen at a temperature of -70 °C or below, or -80 °C or below. In some embodiments, the biological sample is prepared for assessment prior to the assessing, such as according to methods provided above. For instance, in some cases the sample is treated by enzymatically digesting proteins, such as with one or more protease enzymes. In some cases, proteins comprising citrullination sites are enriched, in some cases by being removed from the sample, such as with an antibody or affinity reagent, and/or in some cases other proteins or contaminants are removed from the sample. For example, in some cases an antibody that specifically binds to the protein to be assessed may be used to enrich that protein for later assessment of citrullination. In some cases, the antibody or affinity reagent may be immobilized, such as placed on a matrix such as a bead or chip or well of a plate, other solid surface. In some cases, the biological sample may be diluted before assessment of citrullination. In some cases, proteins in the biological sample may be denatured to remove tertiary structure prior to assessment of citrullination. In some cases, any combination of the above treatments may be conducted on the sample.

In some cases, assessing citrullination of a citrullination site comprises measuring a citrullination ratio, as described above. In some cases, the method comprises comparing the citrullination assessment, such as a citrullination ratio or the concentration of a citrullinated protein or peptide fragment thereof, to that of a reference or control. For example, in some cases, the comparison is to a reference citrullination ratio, which may be obtained, for example, from a reference sample, or which may be a value or range of values that represents those obtained from reference samples. Accordingly, in some cases, the method comprises assessing comprises determining a difference between the citrullination ratio for the biological sample and a reference citrullination ratio.

The terms “reference” and “control,” when referring to a biological sample, are used interchangeably to refer to a biological sample against which a measurement from a sample at interest is compared. In some cases, such a control is a sample that has not been exposed to PAD2 but is otherwise comparable to the sample of interest. In some cases, the control is a “baseline” sample, which is a sample taken from a subject prior to some event of interest, such as prior to treatment with a therapeutic agent, e.g., PAD2 modulator, and which, for instance, may be compared to a sample from the same subject taken after the event, such as after treatment with the therapeutic agent. In some cases, the control is a sample from the subject taken prior to treatment with a PAD2 modulator. In some cases, the control is a sample from the subject taken prior to a change in PAD2 modulator treatment. Accordingly, in some cases, one may determine how treatment with a PAD2 modulator impacts a subject by comparing to such a baseline control sample. In other cases, the control biological sample has been exposed to a different treatment than the biological sample, such as to a different PAD2 modulator or different therapeutic agent than the biological sample. In some cases, the control biological sample has been exposed to a different dose of the PAD2 modulator or other therapeutic agent than the biological sample. In some cases, the control is a sample from a healthy subject, which, for instance, could be compared to a sample from a subject with a disorder, or a subject following treatment with PAD2. In other cases, a control biological sample is a sample that has been exposed to a different therapeutic agent or PAD2 modulator, or to a different dose of the same therapeutic agent or PAD2 modulator as the sample of interest. Thus, in some embodiments, citrullination assessments are compared to those of one or more control samples (e.g., baseline samples or samples from other points of time from the same subject, or samples from other subjects), or to values associated with one or more control samples (e.g., from a pool of healthy subjects). For example, in some cases, a citrullination assessment from a biological sample herein may be controlled to a group of control samples representing typical values found in a particular type of subject, or representing values found across a range of subjects. In some embodiments, the present disclosure includes methods of assessing citrullination in a biological sample of a subject comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the biological sample. In some cases, the subject has not received treatment with any PAD2 modulator. In other cases, the subject has received treatment with a PAD2 modulator. In some cases, the subject has not received treatment with any therapeutic agent. In other cases, the subject has received treatment with a therapeutic agent. The present disclosure also encompasses methods of assessing the activity of a PAD2 modulator or other therapeutic agent comprising obtaining a biological sample from a subject following treatment of the subject with at least one dose of a PAD2 modulator or other therapeutic agent, and assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the biological sample. In some cases, these methods further comprises comparing the assessment of the citrullination to that of a control biological sample, such as described above. In some cases, where the subject has been treated with a PAD2 modulator or other therapeutic agent, the control biological sample is a baseline sample obtained from the subject prior to treatment with the PAD2 modulator or other therapeutic agent. In some cases, the method comprises obtaining a baseline biological sample from the subject prior to treatment with the PAD2 modulator or other therapeutic agent, and assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the baseline biological sample, and optionally comparing the assessed citrullination (e.g. a citrullination ratio) of the biological sample and the baseline biological sample. In some cases, a citrullination assessment is performed in order to determine if a subject should receive treatment with a PAD2 modulator, or to serve as a baseline for later monitoring of citrullination following treatment with a PAD2 modulator or other therapeutic agent.

In some cases, the method comprises (a) contacting a biological sample from a subject with exogenous PAD2, and (b) assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the sample. In some cases, the sample is a biological fluid sample, such as plasma or serum, blood supernatant, whole blood, or synovial fluid. In some cases, it is plasma or serum. In some cases, the biological sample is prepared as described above prior to the assessing. In some cases, the concentration of exogenous PAD2 may be as provided above (e.g., from 1 nM to 5 nM, such as from 2nM to 4 nM, from 2 nM to 3 nM, or 1 nM, 2 nM, 3 nM, or 4 nM, or about 1 nM, about 2 nM, about 3 nM, or about 4 nM). In some cases, another PAD protein is also added. In some cases, calcium ion (Ca2+) is also added to the sample. For instance, in some cases, the sample is treated to enrich a protein of interest, and/or to denature and/or enzymatically digest proteins of interest for later assessment, and/or to deplete proteins such as serum albumin. In some cases, the protein of interest is enriched by immunoenrichment techniques such as described below, for example, which may comprise using affinity reagents to isolate or remove the protein of interest.

In some embodiments, the methods comprise in vitro methods of assessing the activity of a PAD2 modulator or other therapeutic agent, comprising (a) treating a biological sample from a subject with a PAD2 modulator or other therapeutic agent to form a treated biological sample, and after the treating, (b) assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the biological sample. In some cases, such methods further comprise assessing citrullination of the citrullination site on the protein or a peptide fragment in a control biological sample, such as described above. Methods herein also comprise, for example, an in vitro method of assessing the activity of a PAD2 modulator or other therapeutic agent, comprising (i) dividing a biological sample obtained from a subject into a plurality of biological samples, (ii) contacting each of the plurality of biological samples with a different dose of the PAD2 modulator or other therapeutic agent, and (iii) assessing, for each of the plurality of biological samples, citrullination of a PAD2- dependent citrullination site on a protein or peptide fragment thereof that is present in the biological sample. In some cases, such a method comprises calculating an IC50 for the PAD2 modulator or other therapeutic agent based on the outcome of the assessing. Methods herein can also include, for example, an in vitro method of assessing citrullination by endogenous PAD2, comprising incubating the biological sample (or the plurality of biological samples) from the subject for an incubation period (for example, to trigger release of endogenous PAD2) and assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the sample. Methods herein also include, for example, an in vitro method of assessing citrullination by endogenous PAD2, comprising (a) incubating a whole blood sample from a subject at 34-40°C for an incubation period (for example, to trigger release of endogenous PAD2), (b) after the incubation period, separating plasma or supernatant from the whole blood sample, and (c) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant. In some cases, the incubating is in a TruCulture® null tube. Such incubation may be for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60- 72 hours. In some cases, the incubation period is 48 to 96 hours. In some cases, the incubating is at a temperature of 35 to 40°C, at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C. In some cases, the sample is prepared for assessment of citrullination, such as to enrich the protein or peptide to be assessed. In some cases, the method comprises, for example, separating the plasma or supernatant from the whole blood sample, such as by centrifuging or filtering the whole blood sample. These methods may also comprise (i) incubating the plasma or supernatant with a protein depletion resin, and (ii) recovering depleted plasma or supernatant that has flowed through the resin to obtain depleted flowthrough, optionally wherein the recovering comprises centrifugation. They may also comprise enzymatically digesting polypeptides in the depleted flowthrough before the assessing, and optionally cleaning up enzymatically digested peptides, such as with an iST-BCT kit (PreOmics, Planegg/Martinsried, Germany) or a similar kit, for instance. Such kits may be used in some embodiments to purify a sample prior to MS analysis and/or to minimize artificial protein modifications induced by reagents added to a sample, such as deamidation and oxidation modifications. In some cases, the whole blood sample has not been frozen and thawed. In other cases, the whole blood sample has been frozen and thawed. In some cases, the method comprises freezing the plasma or supernatant after it has been separated from the whole blood and subsequently thawing the plasma or supernatant before the assessing.

The disclosure herein also encompasses an in vitro method of assessing changes in citrullination in a subject, comprising (a) incubating a whole blood sample from a subject at 34-40°C for an incubation period (such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60-72 hours), wherein the sample is obtained from the subject following administration of a PAD2 modulator to the subject, (b) after the incubation period, separating plasma or supernatant from the whole blood sample, and (c) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant. In some cases, the method further comprises (d) incubating a second whole blood sample from the subject at 34-40°C for an incubation period (such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60-72 hours), wherein the second whole blood sample is obtained from the subject before administration of the PAD2 modulator to the subject, (e) after the incubation period, separating plasma or supernatant from the second whole blood sample, and (f) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant from the second whole blood sample. In some cases, an outcome of the assessing in step (f) is compared with an outcome of the assessing in step (c). Methods herein also include, for example, a method of assessing effects of a PAD2 modulator, the method comprising (a) exposing a whole blood sample from a subject to a PAD2 modulator in vitro, (b) incubating the whole blood sample at 34-40°C for an incubation period (such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60-72 hours), (c) after the incubation period, separating plasma or supernatant from the whole blood sample, and (d) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant. In some embodiments, the method further comprises performing a parallel set of steps on at least one control whole blood sample. In some embodiments, the method further comprises (e) incubating a control whole blood sample at 34-40°C for an incubation period (such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60-72 hours), (f) after the incubation period, separating control plasma or supernatant from the control whole blood sample, and (g) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the control plasma or supernatant. In some cases, an outcome of the assessing in step (g) is compared with an outcome of the assessing in step (d). The disclosure herein also encompasses, for example, an in vitro method of assessing PAD2-dependent citrullination, the method comprising assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in plasma or supernatant that has been separated from a whole blood sample that has been incubated at 34-40°C for an incubation period (such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60-72 hours). In any of these methods herein, the sample can be a plasma or supernatant from a whole blood sample. In some cases the plasma or supernatant sample has not been frozen before assessment, while in other cases the sample has been frozen and thawed, and in some instances the method further comprises freezing and thawing the plasma or supernatant after separation from whole blood but prior to the assessing.

In some cases, a sample may be contacted with a reagent, such as an affinity reagent, to enrich or purify a protein harboring a citrullination site to be analyzed or a peptide fragment thereof. For example, in some cases the method comprises “immunoenriching” a sample for a protein of interest or a peptide fragment thereof, the protein or peptide harboring a citrullination site for assessment. Such “immunoenriching” may comprise contacting the sample with an antibody reagent that specifically binds to the protein of interest and/or to a peptide fragment thereof (e.g., comprising an epitope of the protein of interest). In some cases, the antibody reagent binds to the protein or peptide in both the non-citrullinated and citrullinated form. Accordingly, the protein or peptide fragment thereof may be enriched prior to assessment of citrullination. In some cases, an antibody or other affinity reagent may be immobilized, such as by attachment to a solid surface, such as a bead, chip, plate or well of a plate. Such immobilization, for instance, may assist in separating a protein of interest for assessment of citrullination from other proteins in the sample. In some cases, the reagent used for immunoenriching is an antibody attached to a solid surface, such as a bead, such as a magnetic bead, or a chip, plate, or well of a plate. In some such cases, the sample may be a sample that comprises or is whole blood, serum, plasma, synovial fluid, pleural fluid, interstitial fluid, sputum, urine, blood supernatant, CSF, BALF, or another biological sample.

Accordingly, methods of the disclosure herein also include, for example, a method comprising: (a) immunoenriching a sample for a protein of interest or a peptide fragment thereof to form an immunoenriched sample, wherein the immunoenriching comprises contacting the serum or plasma sample with an antibody that specifically binds to both citrullinated and noncitrullinated forms of the protein of interest or protein fragment thereof, the protein of interest or peptide comprising a citrullination site, and (b) assessing citrullination in the immunoenriched sample at the citrullination site. In some cases, a method disclosed herein comprises: (a) immunoenriching a sample for a protein of interest to form an immunoenriched sample, wherein the immunoenriching comprises contacting the serum or plasma sample with an antibody that binds to both citrullinated and noncitrullinated forms of the protein of interest or peptide fragment thereof, the protein of interest or peptide fragment thereof comprising a citrullination site, (b) enzymatically digesting the immunoenriched sample to form digested peptides from the protein of interest, wherein steps (a) and (b) can be in any order, and (c) assessing citrullination of the digested peptides at the citrullination site. In other cases, the method comprises (a) immunoenriching a sample by contacting the sample with an immobilized antibody that binds to both citrullinated and noncitrullinated forms of a protein of interest, the protein of interest comprising a citrullination site, (b) eluting protein bound to the immobilized antibody, (d) enzymatically digesting the eluted protein to form digested peptides, and (e) assessing citrullination of the digested peptides at the citrullination site. In any of the above methods, the sample may be diluted prior to the immunoenriching step, such as by 2-1000 fold. In any of the above methods, the sample may be a whole blood, plasma, serum, or synovial fluid sample. In some embodiments, it is a plasma or serum sample. In some cases, the immunoenrichment is conducted prior to enzymatic digestion of the protein of interest. In other cases, the protein of interest is first enzymatically digested, and then one or more peptide fragments thereof are immunoenriched. In some cases, the sample may be diluted to a volume of at least 5 pl, e.g., to a volume of 5 pl- 1 OOpl, 5 pl-50pl, 5 pl-25 pl, 5 pl- 15 pl, e.g., to a volume of lOpl. In any of the above methods, the immunoenriching may comprise incubating the sample with the immobilized antibody for at least 30 minutes, such as for a period of 30-90 minutes. In some cases, the incubation is for 30-60 minutes, 40-80 minutes, 50 to 70 minutes, 55 to 65 minutes, or for 60 minutes. The incubating may be at a temperature of 22-28°C, or may be at room temperature. In some cases, the immunoenriching comprises shaking during the incubating (e.g., at 800-1200 rpm or at 1000 rpm). In some cases, the immunoenriching comprises removing the immobilized antibody from the sample and washing the immobilized antibody prior to the eluting, for example with a wash buffer, e.g., a wash buffer comprising PBS (phosphate buffered saline). In some cases, the eluting comprises washing the immobilized antibody with an elution composition. A variety of elution compositions may be used, including, for example elution compositions that elute a protein of interest based on a change of pH, such as elution compositions that are acidic, and/or that comprise a detergent such as a zwitterionic detergent. In some cases, the elution buffer may comprise an acidic solution, optionally further comprising a buffer or ion, such as a zwitterion. In some cases, the buffer is exchanged prior to assessing citrullination, and optionally prior to intermediate steps such as enzymatic digestion, if such steps are performed. For example, if an acidic elution composition is used, then the buffer may be altered to a neutral pH range for subsequent steps, for example. In some cases, proteins that have been immunoenriched according to methods herein are denatured and/or enzymatically digested prior to assessment of citrullination. For example, proteins may be denatured to remove tertiary structure, for example, by incubation at above physiological temperatures, such as at 60-90°C, such as 60- 80°C, 70-85°C, or 80°C, for a period of 5-30 minutes, such as 10-30, 10-20, 15-25, or 20 minutes. In other cases, a chemical denaturant such as urea could be applied. A proteolytic enzyme such as trypsin, LysC, rLysC, LysN, GluC, AspN, rAspN, or a combination of proteolytic enzymes, such as more than one of trypsin, LysC, rLysC, LysN, GluC, AspN, and rAspN, could be used for enzymatic digestion.

In some cases, as noted earlier, a strong anion exchange (SAX) chromatography medium may be used to enrich proteins or peptides of interest. In some cases, the SAX chromatography medium may comprise particles such as beads, chips, or the like, optionally magnetic particles (e.g., magnetic beads or chips or the like). In other cases, the SAX medium may comprise a resin or may be in the form of a column to which a sample is applied. In some cases, a sample may be contacted with the SAX medium. In some cases, SAX treatment may enrich proteins or peptides of interest, for example, with respect to abundant proteins such as albumin in plasma, serum, or blood, or other unwanted proteins or debris. In some such cases, the sample may be a sample that comprises or is whole blood, serum, plasma, synovial fluid, pleural fluid, interstitial fluid, sputum, urine, blood supernatant, CSF, BALF, or another biological sample. In some cases, the SAX treatment comprises contacting the sample with the SAX medium, and optionally incubating the sample with the SAX medium, in order to allow proteins or peptides of interest to bind to the SAX medium, and optionally separating the protein or peptide-bound SAX medium from the rest of the sample, and optionally eluting the proteins or peptides of interest bound to the SAX medium from the medium. One or more wash steps may also be performed prior to the elution. In some cases, the SAX may be performed before enzymatic digestion of the sample. In some cases, proteins that have been enriched by SAX according to methods herein are denatured and/or enzymatically digested prior to assessment of citrullination. For example, proteins may be denatured to remove tertiary structure, for example, by incubation at above physiological temperatures, such as at 60-90°C, such as 60-80°C, 70-85°C, or 80°C, for a period of 5-30 minutes, such as 10-30, 10-20, 15-25, or 20 minutes. In other cases, a chemical denaturant such as urea could be applied. As with other methods herein, a proteolytic enzyme such as trypsin, LysC, rLysC, LysN, GluC, AspN, rAspN, or a combination of proteolytic enzymes, such as more than one of trypsin, LysC, rLysC, LysN, GluC, AspN, and rAspN, could be used for enzymatic digestion.

In methods above, before assessment of citrullination, the treated samples, such as those that have been denatured and/or enzymatically digested, and optionally also treated by immunoenrichment or SAX or other procedures, may be further treated to remove unwanted materials such as salts, buffer components, enzymes, denaturants, and other small molecules. For example, one or more chromatography or filtration steps may be performed to separate peptides and proteins of interest from such materials, for example, by using chromatography or filtration media that bind or retain peptides or proteins to be assessed but that allow unwanted materials to flow through. Such clean-up steps may improve the throughput of analysis methods by allowing cleaner samples to be assessed in MS or LC/MS instrumentation, and reducing the need for cleaning up of the instrumentation between runs.

In these above methods, the protein of interest may comprise any of the proteins listed herein, such as those listed in Table 1 and Table 11, and the citrullination site to be assessed may comprise any one or more of those listed in Table 1 herein, and/or in Table 11 herein, for example. In some cases, the protein of interest comprises CPB2, AP0A1, APOE, or AHSG. In such cases, the citrullination site for assessment may comprise R342 of Uniprot sequence Q961Y4 for CPB2 (carboxypeptidase B2), R239 of Uniprot sequence P02647 for AP0A1 (apolipoprotein A-I), R198 of Uniprot sequence P02649 for APOE (apolipoprotein E), and/or R337 of Uniprot sequence P026765 for AHSG (alpha-2-HS-glycoprotein). Those citrullination sites, in some cases, may be assessed via a peptide of sequence, wherein the underlined and bold R is the citrullination site. In such cases, the citrullination site for assessment may comprise the gelsolin citrullination site located at R32 of P06396, which in some cases may be assessed via a peptide of sequence DHEELSLVASEAVRAIEK; AKPALEDLRQGLLPVLESFK; ERLGPLVEQGR; or HTFMGVVSLGSPSGEVSHPRKT (SEQ ID NOs: 14, 6, 10, or 11, respectively), wherein the underlined and bold R is the citrullination site. In some such embodiments, immunoenrichment of the sample is conducted prior to enzymatic digestion.

In any of the methods above comprising immunoenrichment and enzymatic digestion, assessment of citrullination may be conducted by mass spectroscopy (MS), for example, by liquid chromatography followed by mass spectroscopy such as tandem mass spectroscopy (LC-MS or LC-MS/MS).

The disclosure herein also includes methods of assessing citrullination in a sample at one or more citrullination sites, wherein the sample is contacted with a strong anion exchange (SAX) chromatography medium in order to enrich proteins and peptide fragments thereof within the sample for analysis. In some embodiments, such methods may expand the citrullinome within the sample and allow analysis of citrullination of lower abundance citrullination sites, or analysis of a larger number of citrullination sites, for example. For instance, in some embodiments, SAX chromatography may remove debris and unwanted higher abundance proteins from the sample to allow for assessing of desired citrullination sites. In some embodiments, SAX chromatography may be used to enrich membrane-bound proteins within the sample, including proteins bound to extracellular vesicles or other sub- cellular particles. In some embodiments, methods of determining citrullination of a protein or peptide fragment thereof may comprise assessing citrullination of a citrullination site on a protein or a peptide fragment thereof in a biological sample from a subject,, comprising: (a) contacting the biological sample with a strong anion exchange (SAX) chromatography medium, (b) enzymatically digesting proteins in the sample either before or after the contacting to form the peptide fragment, and (c) assessing citrullination of the peptide fragment at the citrullination site. In some cases, the enzymatic digestion is performed after the contacting of the biological sample with the SAX chromatography medium. In some cases, the SAX chromatography medium comprises particles such as beads, chips, or the like, which may be magnetic particles. In other cases, the SAX chromatography medium is a resin or is comprised within a column. In some cases, the SAX chromatography medium enriches membrane-bound proteins and peptide fragments thereof in the sample. In some cases, the method comprises incubating the sample with the SAX chromatography medium for at least 30 minutes. In some cases, proteins or peptide fragments thereof that are of interest for assessing citrullination are bound to the SAX chromatography medium as a result of the contacting step, and, for example, during incubation. In some cases, the bound proteins or peptides thereof are then eluted from the SAX chromatography medium, for example, with an elution buffer. In some cases, the method further comprises washing the proteins or peptide fragments thereof bound to the SAX chromatography medium at least once prior to eluting the proteins or peptide fragments thereof from the SAX chromatography medium. In such cases, assessing citrullination may then be performed following elution. In some cases, enzymatically digesting is conducted after the elution of the proteins or peptide fragments thereof from the SAX medium. In some cases, the method comprises at least one further chromatography or a filtration step after the enzymatically digesting and prior to the assessing citrullination to separate the proteins or peptide fragments thereof from one or more salts, buffers, or small molecules. In some cases, assessing citrullination comprises measuring a first concentration of citrullinated peptide in the digested peptides and a second concentration of signature peptide in the digested peptides, and optionally determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration. In some cases, proteins in the sample are denatured prior to the assessing. In some cases, the method comprises denaturing the proteins prior to the enzymatically digesting. In some embodiments, such a method of determining citrullination of a protein or peptide fragment thereof comprises (a) contacting the biological sample with a strong anion exchange (SAX) chromatography medium, and optionally incubating the sample with the SAX chromatography medium, (b) eluting proteins bound to the SAX chromatography medium, optionally wherein at least one wash step is performed prior to elution of the proteins from the SAX chromatography medium, (c) enzymatically digesting proteins bound to the SAX chromatography medium to form peptide fragments thereof, optionally wherein the bound proteins are denatured prior to the enzymatically digesting, (d) conducting chromatography or filtration on the enzymatically digested proteins to separate the enzymatically digested peptide fragments of the proteins from one or more salts, buffers, or small molecules, and (e) assessing citrullination of one or more peptide fragments at one or more citrullination sites.

In any of the above methods utilizing SAX chromatography, the biological sample in some embodiments may have been exposed to a PAD2 modulator such as a PAD2 inhibitor, a PAD4 modulator such as a PAD4 inhibitor, or a PAD2 and PAD4 modulator such as a PAD2 and PAD4 inhibitor. Exampleary PAD2 and/or PAD4 modulators are disclosed, for example, in International Patent Publications WO2024/4133161 and US2024/0199761, as well as in publications described in Section III below. In any of these methods, the citrullination site may be a site listed in Table 1 and/or Table 11 herein. In some cases, the sequence of the peptide fragment comprises one of the following sequences, wherein the citrullination site is designated by underlining of R residues and wherein modifications of non-arginine residues are designated by underlining of the modified residues: DHEELSLVASEAVRAIEK; AKPALEDLRQGLLPVLESFK; ERLGPLVEQGR; HTFMGVVSLGSPSGEVSHPRKT; ENGGARLAEYHAK; LSPLGEEMRDR; SELEEQLTPVAEETRAR; TPVSDRVTK; IVEGSDAEIGMSPWQVMLFRK; IYISGMAPRPSLAK; RQLTPYAQR;

RVEPYGENFNK; SFQTGLFTAARQSTNAYPDLR;

VLLDGVQNPRAEDLVGKSLYVSATVILHSGSDMVQAER; or ASSIIDELFQDRFFTREPQDTYHYLPFSLPHR (corresponding to SEQ ID NOs: 14, 6, 10, 11, 7, 8, 9, 12, 15, 16, 17, 18, 19, 20, or 13, respectively). In some cases, the biological sample comprises whole blood, plasma, serum, or blood supernatant. In some cases, the biological sample comprises synovial fluid. In some cases, the peptide fragment comprises the sequence of: DHEELSLVASEAVRAIEK; AKPALEDLRQGLLPVLESFK;

ERLGPLVEQGR; or HTFMGVVSLGSPSGEVSHPRKT (SEQ ID NOs: 14, 6, 10, or 11, respectively). In some cases, the method comprises assessing citrullination of two or more proteins or peptide fragments thereof.

In any of the methods above comprising SAX chromatography and enzymatic digestion, assessment of citrullination may be conducted by mass spectroscopy (MS), for example, by liquid chromatography followed by mass spectroscopy such as tandem mass spectroscopy (LC-MS or LC-MS/MS).

The above methods may be used in a wide variety of contexts, and with a wide range of PAD2 modulators, such as PAD2 inhibitors and PAD2 agonists, and with other therapeutic agents such as those used for treatment of citrullination-related diseases, such as PAD4 modulators and PAD4 inhibitors, as well as those that target biological pathways that involve PAD2, such as NETosis and/or METosis, and those that otherwise can impact citrullination activity by PAD2, or PAD2 expression. For example, they may be used for testing new PAD2 modulators or other agents to determine their effect on citrullination of certain sites. Or they may be used to check the activity of known PAD2 modulators or other agents. Or they may be used to monitor the effect of PAD2 modulators or other therapeutic agents, for example, on citrullination levels, after administration to a patient. Thus, in some cases, a biological sample may be used for testing the effect of a PAD2 modulator or other therapeutic agent that has not been administered to a subject, and the PAD2 modulator or other therapeutic agent may be exposed to the biological sample by incubating the sample with the modulator. In other cases, the biological sample may be taken from a subject who has been previously administered the PAD2 modulator or other therapeutic agent, and thus the sample may have been exposed to the PAD2 modulator through administration of the PAD2 modulator or other therapeutic agent to the subject from whom the sample is obtained. In some cases, the PAD2 modulator or other therapeutic agent is an anti-histone antibody. In some cases, the PAD2 modulator is an anti-PAD2 antibody or small molecule PAD2 inhibitor. In some cases, the other therapeutic agent is a PALM modulator. In some cases, it is a PALM inhibitor, such as an anti-PALM antibody or small molecule PALM inhibitor. In some cases, a PAD2 modulator does not significantly affect the activity of any protein species other than PAD2. In some such cases, the PAD2 modulator does not significantly affect the activity of PALM, for example. In other cases, the PAD2 modulator modulates the activity of PAD2 as well as at least one other protein, such as PALM. Certain exemplary PAD2 modulators compatible with methods herein are described below.

PAD2 modulators may be administered, for example, to subjects who have a citrullination-related disease such as an autoimmune disease or infectious disease or cancer, for example, or who are at risk for developing such a disease. Such diseases and related subjects are discussed in more detail in following sections herein. In some cases, the subject has been diagnosed with an autoimmune disorder or is at risk for developing an autoimmune disorder. In some cases, the citrullination-related disease is the citrullination-related disease is rheumatoid arthritis, lupus (e.g., systemic lupus erythematosus (SLE)), lupus nephritis, vasculitis (e.g., ANCA-associated vasculitis), thrombosis (e.g, venous thrombosis), or inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn’s disease). In some cases the subject has been diagnosed with rheumatoid arthritis (RA) or is at risk for developing rheumatoid arthritis. In other cases, however, the subject has not been diagnosed with an autoimmune disease such as RA, or the subject is not at considered to be at risk of developing such a disease. Thus, in some cases, is a normal, healthy subject. In some cases, the subject is positive for anti-citrullinated protein antibodies (ACPA positive). In some cases, the subject is positive for anti-PAD2 activating antibodies. In cases where a subject is ACPA positive or positive for anti-PAD2 activating antibodies, the subject may also be considered at risk for developing an autoimmune disorder such as RA.

Methods may also be used, for example, to assess citrullination levels in subjects not currently on therapeutic treatment, or who are not receiving a PAD2 modulator. Thus, in some cases, is a normal, healthy subject. In some cases, the subject is positive for anti- citrullinated protein antibodies (ACPA positive). In some cases, the subject is considered at risk for developing an autoimmune disorder or citrullination-related disease.

III. Exemplary PAD2 Modulators

Examples of PAD2 modulators include various small molecule compounds that may increase or reduce PAD2 activity, such as citrullination of amino acid residues. Examples of small molecule PAD2 modulators include benzimidazole-derived compounds and heteroaryl compounds, as well as peptides, such as peptide macrocycles. Exemplary PAD2 modulators, such as small molecule PAD2 modulators and PAD2 inhibitors, for example, include YW3- 56 described, for example, in Wang S, et al. Mol Cancer Ther. 2015 Apr;14(4):877-88, modified and unmodified Streptonigrin described in Dreyton et al. (2012) Probe Reports from the NIH Molecular Libraries Program and Dreyton et al. (2014) Bioorg and Med Chem 22(4): 1362-9, Cl-amidine (Luo et al. (2006) Biochemistry 45(39): 11727-11736), including BB-Cl-amidine (Knight et al (2015) Ann Rhuem Dis , 74(12): 2199-2206), AFM-30a disclosed in Muth et al. (2017) JMedChem 60(7): 3198-3211 and WO2018/102262, NSC95397, ruthenium red, and sanguinarine, described in Lewallen et al. (2014) ACS Chem Biol 9(4): 913-921, compounds 16-20 of Mondal et al. (2019) Acc Chem Res 52(3): 818-832, and ML325 (optimized streptonigrin) described in compounds disclosed in Dreyton et al. (2012) Probe Reports from the NIH Molecular Libraries Program, for example.

In other cases, antibodies may be used as PAD2 modulators. For example, Sims et al, doi 10.1136/annrheumdis-2024-eular.3510 (2024) and Chen et al., doi 10.1136/annrheumdis- 2023-eular.2894 (2023) describe PAD2/PAD4 bispecific antibodies, and WO2016/155745 describes a cross-reactive antibody capable of binding both PAD2 and PAD4. Further anti- PAD2 antibodies are described in WO2014/086365, US2018/0280503 Al, US2018/0284118A1, and WO2019/244934, and in Aosasa et a., J. Immunol. Res. doi:10.1155/2021/6659960 (2021).

Other therapeutic agents in connection with which the methods herein are useful include PAD4 modulators, such as PAD4 inhibitors. For example, in some cases it may be helpful to determine if treatment with a PAD4 modulator impacts citrullination at sites citrullinated by PAD2 or by both PAD2 and PAD4 in a biological sample from a subject. For instance, this may be helpful in determining if further treatment with a PAD2 modulator such as a PAD2 inhibitor is warranted. Examples of small molecule PAD4 modulators include benzimidazole-derived compounds and heteroaryl compounds, as well as peptides, such as peptide macrocycles. Exemplary small molecule PAD4 modulators, for example, include compounds disclosed in International Patent Publication No. W02014/015905, which describes benzimidazole-derived PAD4 modulators such as PAD4 inhibitors, for instance 2- (azaindole-2-yl)benzimidazoles. Additional small molecule PAD4 modulators such as PAD4 inhibitors, are also described in International Patent Publication Nos. WO2016/185279, WO20 17/007405, WO2017/100601, W02017/100594, W02017/147102, WO2018/022897, WO20 18/049296, WO2019/058393, W02020/033490, W02020/033514, W02020/033520, W02020/033488, WO2021/158840, WO2021/163254, WO2022/173722, WO2022/140428, WO2022/221642, and WO2023/083365, for example. Others include compounds disclosed in United States Patent application or publication numbers US11198681, US20220402950, US11524959, US20230203039, US20220348562, US11878965, US20220227787, US 11976083. Others include, for example, GSK121, GSK199, AFM-30a, and GSK 484, described in Lewis et al. (2015) Nat Chem Biol 11(3): 189-191 and Chen et al., doi 10.1136/annrheumdis-2023-eular.2894 (2023; BMS-P5, described in Li et al. (2020) Mol Cancer Ther 19(7): 1530-1538; JBI-589, described in Deng et al. (2022) Cancer Res 82(19): 3561-3572; JBL1044, described in US20200237771, and US2020276206; YW3-56, described in Wang et al. (2012) J Biol Chem 287(31): 25941-25953; ZD-E-1M, described in Zhu et al. (2022) Acta Pharm Sin B 12(5): 2592-2608; modified and unmodified Streptonigrin described in Dreyton et al. (2012) Probe Reports from the NIH Molecular Libraries Program and Dreyton et al. (2014) Bioorg and Med Chem 22(4): 1362-9; Cl- amidine, described in Luo et al. (2006) Biochemistry 45(39): 11727-11736, and its modifications including BB-Cl-amidine (Knight et al (2015) Ann Rhuem Dis , 74(12): 2199- 2206), and o-Cl-amidine (Causey et al. (2011) J. Med Chem 54(19): 6919-6935); F-amidine, Luo et al. (2006) J Am Chem Soc 128(4): 1092-1093, and its modifications including BB-F- amidine (Muth et al. (2017) J Med Chem 60(7): 3198-3211), and o-F-amidine (Causey et al. (2011) J. Med Chem 54(19): 6919-6935); TDFA, described in Jones et al. (2012) ACS Chem Biol 7(1): 160-165; SC97362 and others, described in Aliko et al. (2019) IntJMol Sci 20(9): 2174; NSC95397, ruthenium red, and sanguinarine, described in Lewallen et al. (2014) ACS Chem Biol 9(4): 913-921; chlorotetracycline, minocycline, and streptomycin, described in Knuckley et al. (2007) Bioorg Med Chem 16(2): 739-745; and Lucid-21-302, described in WO-2017/027967. Other examples include the inhibitors described in WO2017/027967; WO20 14/019092; Zhu et al. (2023) Eur JMed Chem 258: 115619; Zhu et al. (2024) J Med Chem 67(10):7973-7994; Jia et al. (2023) Biomed Pharmacother 168: 115826; Muth et al. (2017) J Med Chem 60(7): 3198-3211; Sarswat et al. 2656; Wei et al. (2014) J Med Chem 56(4) 1715-1722; Teo et al. (2017) Chem Biol Drug Des 90(6): 1134-1146; Nadzirin (2021) Comput Biol Chem 92: 107487; and Mondal et al. (2019) Acc Chem Res 52(3): 818-832. Examples of anti-PAD4 antibodies include those described in W02024/020579, WO2016/143753, US2018/0044434, WO2019/131769, WO2022/176970, Zhou et al. (2024) Nat Chem Biol 20:742-750, and Wang et al. (2022) Biomedicine and Pharmacotherapy 153: 113289, as well as PAD2/PAD4 specific antibodies described above.

Each of the above-referenced publications is incorporated herein by reference in its entirety.

Additional therapeutic agents with which the methods herein are useful also include current standard of care treatments for citrullination-related diseases, such as antiinflammatory treatments, for instance, in some cases to determine if further treatment with a PAD modulator such as a PAD2 modulator is warranted.

IV. Therapeutic Compositions and Methods

The methods herein may in some embodiments be conducted in connection with a method of treating a subject with a PAD2 modulator or another therapeutic agent, such as those described in the preceding sections. For example, in some embodiments, the subject from which the biological sample is taken may be a subject that is undergoing treatment with a PAD2 modulator or PAD2 inhibitor, or other therapeutic agent such as a PAD4 modulator or PAD4 inhibitor or other treatment for a citrullination-related disease. In some such cases, the methods may be used to evaluate such treatment, for example, by assessing the activity of the PAD2 modulator in a biological sample from the subject at one or more time points during treatment with the PAD2 modulator. The terms “disease” and “disorder” are used interchangeably herein in the context of an indication to be treated.

In other cases, the subject may have a biological sample assessed for citrullination in the absence of being treated with any PAD2 modulator, or in some cases prior to any treatment. For instance, a sample might be assessed for a purpose such as determining a likely clinical outcome, determining risk for developing a citrullination-related disease, diagnosing a citrullination-related disease, and selecting the subject from which the biological sample was derived for a treatment of a citrullination-related disease. As used herein, a “citrullination-related disease” refers to a disease characterized by presence of increased citrullination of polypeptides in biological samples from disease subjects, and/or by one or more of presence of NETosis, presence of METosis, presence of anti-citrullinated protein antibodies (ACPA), and increased PAD expression such as increased PAD2 and/or PAD4 expression.

A. Exemplary Citrullination-Related Diseases

In some embodiments, the subject may have a citrullination-related disease or be at risk of developing a citrullinati on-associated disease, or a subject’s sample may be tested to determine if they have a citrullination-related disease or are at risk of developing a citrullination-related disease. For example, in some embodiments a citrullination-related disease is associated with one or more of NETosis, METosis, presence of anti-citrullinated protein antibodies (ACPA), increased PAD expression such as PAD2 expression, and increased citrullination of polypeptides in a biological sample. In some embodiments, the citrullination-related disease is an autoimmune disorder or an infectious disease or cancer.

For example, citrullination by PAD enzymes is a stress response and may serve as a signal for removal of stressed cells. (Brentville et al., Oncoimmunology 8: el576490 (2019).) Proteins citrullinated by PAD enzymes become antigenic substrates and are targets for both cellular (i.e., T cell) and humoral (i.e., B cell-derived antibody) adaptive immune responses. (See, e.g., Curran et al. Nat. Rev. Rheumatol. 16: 301-15 (2020); Brentville et al.) Thus, PAD2 activity may lead to generation of anti-citrullinated protein antibodies (ACPA). In neutrophils, PAD4 plays a role in a process called NETosis, by which neutrophils extrude a complex of decondensed chromatin structures containing a DNA scaffold, citrullinated histones, and anti-bacterial neutrophilic granules. (Li et al. J. Exp. Med. 207: 1853-62 (2010).) These extruded complexes are called neutrophil extracellular traps (NET) and, during NETosis, these NETs trap and kill invading microbes as part of the innate immune response. (Chamardani et al., Mol. Cell. Biochem. 477: 673-88 (2022).) PALM has been primarily implicated as essential for NETosis (Li et al. J Exp Med 207: 1853-62 (2010)), however recent studies indicate that PAD2 may also play a role in NET formation (Wu et al. Inflammation 43(4): 1436-1445 (2020), Tian et al. JCI Insight 5(20): el38873 (2020)), while others indicate that PAD2 is not required for NET formation (Bawadekar et al. J Autoimmun 80:39-47 (2017); Holmes et al. J Immunol Res 2019: 2160192 (2019)). A similar process involving monocytes and macrophages is called METosis and involves formation of monocyte extracellular traps (MET). METosis may be mediated by histone citrullination by PAD2 (Mohanan et al. Front Immunol 18(4):67 (2013)).

In some embodiments, the disorder is an autoimmune disorder. In some embodiments, the autoimmune disorder comprises or is rheumatoid arthritis (RA). In some embodiments, the RA is juvenile-onset RA, juvenile idiopathic arthritis (JIA), or juvenile rheumatoid arthritis (JRA). In some embodiments, the subject has rheumatoid synovitis or significant systemic involvement secondary to RA (including but not limited to vasculitis, pulmonary fibrosis or Felty's syndrome). In some embodiments, the subject is positive for ACPA. In some embodiments, the subject is positive for anti -PAD autoantibodies.

A variety of data suggest that PAD citrullination plays a role in autoimmune diseases such as rheumatoid arthritis (RA), RA-associated interstitial lung disease and idiopathic pulmonary fibrosis (IPF), lupus (including systemic lupus erythematosus (SLE), lupus nephritis, multiple sclerosis (MS), , and cystic fibrosis, for example. (See, e.g., Curran et al.; Fresneda Alarcon et al. Frong. Immunol. 12: 649693 (2021); Wu et al. Front Immunol 12: 761946 (2021); Boon et al. Matrix Biology 95: 68-83 (2021); Liu et al. JCI Insight 3(23): el24729 (2018); Tsoyi et al. Scientific Reports 12: 2847 (2022); Li et al. Sci Translational Med 13 eaba2927 (2021); Calabrese et al. Mult Scler 18(3):299-304 (2012); Wood et al. Laboratory Investigation 88(4): 354-364 (2008); Yang et al. Neurochem Res 41(8): 1845-56 (2016))

In some embodiments, the autoimmune disorder comprises or is rheumatoid arthritis (RA). In some embodiments, the disorder is RA, or the subject to be treated has been diagnosed with RA. In some embodiments, the subject is considered at risk of developing RA. In some embodiments, the RA is juvenile-onset RA, juvenile idiopathic arthritis (JIA), or juvenile rheumatoid arthritis (JRA). In some embodiments, the subject has rheumatoid synovitis or significant systemic involvement secondary to RA (including but not limited to vasculitis, pulmonary fibrosis or Felty's syndrome). In some embodiments, the subject is positive for anti-citrullinated protein antibodies (ACPA). In some embodiments, the subject is positive for anti-PAD autoantibodies.

Rheumatoid arthritis (RA) is a major autoimmune disease the pathobiology of which commonly includes the presence of auto-antibodies including anti-citrullinated protein antibodies (ACPA). Citrullinated proteins, for example, may serve as neo-auto-antigens. These neo-auto-antigens, when presented, result in the production of ACPAs and are recognized by ACPAs to form immune complexes, thus leading to the initiation and progression of the disease. On a mechanistic level, PAD2 citrullinates various proteins known to be targets of ACPA, which antibodies are used as part of the classification and diagnosis of RA in subjects. However, anti-PAD2 antibodies have also been found in ACPA-negative patients, though their function in disease progression remains unknown (Darrah et al. Front Immunol 9 2696 (2018)) and PAD2 may contribute to pathogenesis in ACPA-independent fashion. (Reviewed in Curran AM, Naik P, Giles JT, Darrah E. Nat Rev Rheumatol. 2020 Jun;16(6):301-315)

In some embodiments, a subject whose biological sample is assessed for citrullination herein is at risk for developing RA or is found to be at risk for developing RA at least in part on the basis of the assessment results herein. In some embodiments, the subject at risk for developing RA has a first-degree relative with RA (i.e., a parent or sibling) and/or presence of anti-citrullinated protein antibodies (ACPA) in serum and/or presence of rheumatoid factor (RF) in serum. For example, presence of anti-citrullinated protein antibodies may be determined in some cases using an anti-CCP test, such as an ELISA test. For example, anti- CCP antibody test positivity (i.e., presence of ACPA) was found in 46% of 340 individuals who did not meet the classification criteria for RA but nonetheless had anti-CCP testing performed, such as due to joint pain or lung disease. Those 46% went on to meet the classification for RA within the subsequent 5 years. (Ford et al., Rheum. Dis. Clin North Am 45: 101-112 (2019). In some cases, such anti-CCP test results may be positive up to 10 years prior to onset of RA symptoms. (See, e.g., Jones et al., Curr. Op. Drug Discov. Dev., 12(5): 616-627 (2009).) PAD2 has been found in synovial fluid and synovial biopsies of RA subjects, along with citrullinated proteins, and it has also been found in NETs generated from neutrophils of RA subjects (Foulquier et al. Athritis & Rheumatism 56(11): 3541-3553 (2007); Damgaard et al. Arthritis Res Ther 16(6): 498 (2014); Spengler et al. Arthritis Rheumatol 67(12): 3135-45 (2015)). It is thought that citrullination of these target proteins such as fibrinogen, vimentin, and histones, is promoted in the subclinical phase of RA development, and may be triggered by factors such as cigarette smoking (which is known to increase PAD2 expression in lung tissue, Makrygiannakis et al. Ann Rheum Dis 67(10): 1488-92 (2008)) and periodontal disease (via PAD activity of the oral microbe P. gingivalis (Engstrom et al. 2018 J Transl Med 16:214 (2018)). (Curran et al (2020).; ) Accordingly, in some cases, a subject at risk for developing RA has a history of smoking (e.g., cigarettes, cigars) or of using tobacco products (e.g., chewing tobacco), and/or has periodontal disease.

In some such cases, a subject at risk for developing RA does not have clinical symptoms of arthritis. In some embodiments, however, the subject shows subclinical symptoms of arthritis, such as joint inflammation visible by imaging, such as ultrasound or magnetic resonance imaging (MRI), presence of ACPA via an anti-CCP test, presence of rheumatoid factor (RF), or an SNP or other genetic alteration in the PADI2 gene characteristic of subjects with RA (Chang et al. PLOS One 8(12): e81259 (2013); Guzman- Guzman et al. Front Immunol 12: 718246 (2021)) . In some cases, the subject has such subclinical symptoms along with one or both of a first-degree relative with RA and serum ACPA or serum rheumatoid factor (RF). In other cases, the subject has been diagnosed with arthralgia or undifferentiated arthritis. For example, “arthralgia” herein refers to symptoms of pain or aching in at least one joint, such as an ankle, toe, shoulder, elbow, wrist, knee, hip, or one or more joints of the hand, fingers, or spine. A subject with arthralgia may also have tenderness, redness, warmth, loss of mobility, stiffness, weakness, numbness and/or tingling in one or more joints. “Undifferentiated arthritis” refers to diagnosis of arthritis in a subject for which the type of arthritis, such as RA or osteoarthritis, is not specified or cannot be determined. In the case of a subject with arthralgia or undifferentiated arthritis, the subject may also have one or more of a first-degree relative with RA, ACPA in serum, RF in serum, and subclinical joint inflammation (e.g., by ultrasound or MRI). For example, in such subjects at risk of developing RA, the treating may comprise, for example, lessening effects of one or more present clinical symptoms and/or one or more present sub-clinical symptoms.

In some embodiments, an RA subject or a subject at risk of developing RA has a comorbidity. In some embodiments, the comorbidity is a lung disorder such as interstitial lung disease (ILD), pleural effusion, cricoarytenoiditis, constrictive or follicular bronchiolitis bronchiectasis, pulmonary vasculitis, or pulmonary hypertension. (See, e.g., S. Kadura & G. Raghu, Eur. Respiratory Rev. 30: 210011 (2021).) In some cases, the lung disorder is a parenchymal lung disease (e.g., pneumonia), an airway disease (e.g., cricoarytenoiditis), or a pleural disease (e.g., pleural effusion). (S. Kadura & G. Raghu.) In some embodiments, the comorbidity is a lung disorder characterized by inflammation and/or scarring (fibrosis) of the lung, such as interstitial lung disease (ILD), also known as pulmonary fibrosis.

In other embodiments, the subject has not been diagnosed with RA, but has a lung disorder, such as a disorder characterized by inflammation and/or scarring of the lung, such as interstitial lung disease (ILD), also known as pulmonary fibrosis, or has a parenchymal lung disease (e.g., pneumonia), an airway disease (e.g., cricoarytenoiditis), or a pleural disease (e.g., pleural effusion), or has interstitial lung disease (ILD), pleural effusion, cricoarytenoiditis, constrictive or follicular bronchiolitis bronchiectasis, pulmonary vasculitis, or pulmonary hypertension. For example, ILD can also be a comorbidity with other autoimmune diseases such as scleroderma, dermatomyositis and polymyositis, mixed connective tissue disease, Sjogren’s syndrome, and sarcoidosis, as well as result from certain infectious diseases such as pneumonia, or exposure to certain drugs or harmful substances such as asbestos, or can result from uncontrolled gastroesophageal reflux.

In some embodiments, the autoimmune disorder comprises or is a rheumatic autoimmune disease other than RA. NETosis is associated with the pathophysiology of lupus and other autoimmune and renal diseases, including, for instance systemic lupus erythematosus, vasculitis (e.g., ANCA-associated vasculitis), antiphospholipid antibody syndrome, type 1 diabetes mellitus, and renal inflammatory diseases (gomerulophritides, e.g., proliferative glomerulonephritis and non-proliferative gromerulonephritis), and is also associated with the pathophysiology of cancer. (See, e.g., Li et al., Molecular Cancer Therapeutics, 19: 1530-38 (2020), Teijeira et al. Immunity: 56, 856-871 (2020), Gupta, S. and Kaplan, M.J. Nat Rev Nephrol 12(7):402-413 (2016).) NETs are extracellular web-like structures composed of chromatin backbone and various peptides and proteins that are formed by neutrophils in response to various stimuli in a process called NETosis. NETosis has been found to involve the citrullination of histones, such as histone H3, which requires PAD4 activity. PAD2 may also play a role in NET formation in some disease models (Wu et al. Inflammation 43(4): 1436-1445 (2020), Tian et al. JCI Insight 5(20): el38873 (2020)), however see also Bawadekar et al. J Autoimmun 80:39-47 (2017); Holmes et al. J Immunol Res 2019: 2160192 (2019). In vasculitis, for instance, NETosis is a key driver of disease. (See, for example, B. Arneth et al., Int. J. Med. Sci. 18: 1532-40 (2021); JM Berthelot et al., Joint Bone Spine 84(3): 255-262 (2017); ZL Wang et al., Beijing Da Xue Xue Bao Yi Xue Ban 46(2): 200-6 (2014).) In some embodiments, the disease is cancer (e.g., a cancer disclosed herein), or an autoimmune disease, such as, e.g., lupus (e.g., systemic lupus erythematosus), vasculitis (e.g., ANCA-associated vasculitis), antiphospholipid antibody syndrome, type 1 diabetes mellitus, inflammatory bowel disease (IBD) (e.g., ulcerative colitis and Crohn’s disease), cystic fibrosis, or a renal disease such as renal inflammatory disease (e.g., proliferative glomerulonephritis and non-proliferative gromerulonephritis).

Furthermore, PAD2 plays a role in thrombosis via citrullination of fibrinogen and antithrombin. Citrullination of antithrombin by PAD2 Citrullination of fibrinogen by PAD2 has been shown to slow fibrin polymerization, reduce its hemostasis potential, and lead to increased fibrinolysis (Damiana et al. Clinica Chimica Acta 501 : 6-11 (2020)). Citrullination of antithrombin by PAD2 in the presence of heparin impairs complexing with thrombin (Tilvawala et al. Cell Chem Biol 25(6): 691-704)).

In addition, abnormal PAD activity is known to be associated with autoimmune disorders in addition to RA, such as multiple sclerosis (MS), autoimmune encephalomyelitis, obstructive nepropathy, Alzheimer’s disease (AD), and inflammatory bowel disease (IBD) (e.g., ulcerative colitis and Crohn’s disease), as well as ankylosing spondylitis, osteoarthritis, glaucoma, Scrapie, and HIV/AIDS. For example, elevated levels of PAD enzymes and/or citrullinated proteins have been found in all of those conditions. (See, e.g., Chumanevich et al., Am. J. Physiol. Gastrointest. Liver Physiol. 300(6): G929-G938 (2011); Jones et al., Curr. Op. Drug Discov. Dev., 12(5): 616-627 (2009).) For example, PAD2 overexpression in transgenic mice led to nerve demyelination and development of MS-like symptoms (Musse et al. Dis Model Meeh 1(4-5): 229-40 (2008). Moreover, deamination of PAD substrates has been suggested to occur in response to TNF-alpha signaling, while anti -TNF -alpha antibodies have been used as treatments for various autoimmune conditions such as RA and IBD, suggesting that the elevated PAD activity may result from uncontrolled TNF-alpha signaling. See Chumanevich et al., supra.) Chumanevich and colleagues, for example, found elevated levels of both PAD2 and PAD4 in a colitis model and showed that a small molecule pan-PAD inhibitor could be used to treat colitis in a dextran sulfate sodium (DSS)-induced murine colitis model. (Id.) Thus, in some embodiments, the autoimmune disorder comprises IBD. In some embodiments, the autoimmune disorder comprises colitis, such as ulcerative colitis, Crohn's disease, gluten-sensitive enteropathy, or Whipple’s disease.

In some embodiments, the autoimmune disorder comprises lupus, such as systemic lupus erythematosus, cutaneous lupus erythematosus, or lupus nephritis. In some embodiments, the autoimmune disorder comprises vasculitis. Exemplary types of vasculitis include Bechet’s Disease, Buerger’s Disease (Thromboangiitis Obliterans), eosinophilic granulomatosis with polyangiitis (EGPA; formerly known as Churg Strauss), cryoglobulinemia, giant cell arteritis (temporal arteritis), Henoch-Schbnlein purpura (HSP; IgA vasculitis), microscopic polyangiitis, polyarteritis nodosa, polymyalgia rheumatica, rheumatoid vasculitis, Takayasu’s arteritis, granulomatosis with polyangiitis (GPA; formerly known as Wegener’s), ANCA-associated vasculitis (such as PR3-ANCA associated vasculitis or MPO-ANCA associated vasculitis), hypersensitivity vasculitis, isolated aortitis, central nervous system vasculitis, primary angiitis of the central nervous system (PACNS), Kawasaki Disease, urticarial vasculitis, drug-induced vasculitis, relapsing polychondritis (RP). In some embodiments, the autoimmune disorder comprises thrombosis. In some cases, the autoimmune disorder comprises arthritis, such as, e.g., acute arthritis, chronic arthritis, gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, septic arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, menopausal arthritis, estrogen-depletion arthritis, ankylosing spondylitis, or rheumatoid spondylitis. In some cases, the autoimmune disorder comprises multiple sclerosis (MS), such may include: primary progressive multiple sclerosis (PPMS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), and progressive relapsing multiple sclerosis (PRMS). In some cases, the autoimmune disorder comprises systemic sclerosis (scleroderma), idiopathic inflammatory myopathy (such as, e.g., dermatomyositis, polymyositis, necrotizing autoimmune myopathy, or sporadic inclusion body myositis), Sjogren’s syndrome, sarcoidosis, autoimmune hemolytic anemia, immune pancytopenia, paroxysmal nocturnal hemoglobinuria, autoimmune thrombocytopenia (such as, e.g., idiopathic thrombocytopenic purpura, immune- mediated thrombocytopenia, acute thrombocytopenic purpura, chronic thrombocytopenic purpura), thyroiditis (such as, e.g., Grave's disease, Hashimoto’s thyroiditisjuvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephritis, tubulointerstitial nephritis), a demyelinating disease of the central and/or peripheral nervous system (such as, e.g., multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, or chronic inflammatory demyelinating polyneuropathy), a hepatobiliary disease (such as, e.g., infectious hepatitis (e.g., hepatitis A, B, C, D, E or other non-hepatotropic virus), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, or sclerosing cholangitis), inflammatory bowel disease (IBD)(such as, e.g., ulcerative colitis, Crohn's disease, glutensensitive enteropathy, or Whipple’s disease), an autoimmune or immune-mediated skin disease (such as, e.g., a bullous skin disease, erythema multiforme, contact dermatitis, or psoriasis), an allergic disease (such as, e.g., asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity, or urticaria), an immunologic disease of the lung (such as, e.g., eosinophilic pneumonia, idiopathic pulmonary fibrosis or hypersensitivity pneumonitis), a transplantation associated disease (such as, e.g., graft rejection or graft-versus-host-disease), fibrosis (such as, e.g., kidney fibrosis or hepatic fibrosis), cardiovascular disease, including atherosclerosis and coronary artery disease, cardiovascular events associated with chronic kidney disease, myocardial infarction, and congestive heart failure, diabetes, including type II diabetes, Bronchiolitis obliterans with organizing pneumonia (BOOP), or hemophagocytic syndrome, macrophage activation syndrome, sarcoidosis, or periodontitis). In some cases, the autoimmune disorder comprises a methotrexate-resistant autoimmune disorder, such as methotrexate-resistant RA, lupus, vasculitis, thrombosis, MS, or the like. In some cases, the autoimmune disorder comprises a renal disease, such as a renal inflammatory disease such as, e.g., kidney fibrosis, chronic kidney disease, proliferative glomerulonephritis, or nonproliferative gromerulonephritis.

In some cases, the subject has a disorder comprising: acid-induced lung injury, acne (PAPA), acute lymphocytic leukemia, acute respiratory distress syndrome, Addison’s disease, adrenal hyperplasia, adrenocortical insufficiency, ageing, AIDS, alcoholic hepatitis, alcoholic liver disease, allergen induced asthma, allergic bronchopulmonary, aspergillosis, allergic conjunctivitis, alopecia, Alzheimer’s disease, amyloidosis, amyotrophic lateral sclerosis, weight loss, angina pectoris, angioedema, anhidrotic ecodermal dysplasia-ID, ankylosing spondylitis, anterior segment, inflammation, antiphospholipid syndrome, aphthous stomatitis, appendicitis, arthritis, asthma, atherosclerosis, atopic dermatitis, autoimmune diseases, autoimmune hepatitis, bee sting-induced inflammation, Bechet’s disease, Bechet’s syndrome, Bells Palsy, berylliosis, Blau syndrome, bone pain, bronchitis, bronchiolitis, bums, bursitis, cardiac hypertrophy, carpal tunnel syndrome, catabolic disorders, cataracts, cerebral aneurysm, chemical irritant-induced inflammation, chorioretinitis, chronic heart failure, chronic lung disease of prematurity, chronic lymphocytic leukemia, chronic obstructive pulmonary disease, colitis, complex regional pain syndrome, connective tissue disease, COPD, corneal ulcer, Crohn’s disease, cryopyrin- associated periodic syndromes, cryptococcosis, cystic fibrosis, deficiency of the interleukin- 1-receptor antagonist (DIRA), dermatitis, dermatitis endotoxemia, dermatomyositis, diffuse intrinsic pontine glioma, dry eye disease, endometriosis, endotoxemia, epicondylitis, erythroblastopenia, familial amyloidotic polyneuropathy, familial cold urticarial, familial Mediterranean fever, fetal growth retardation, glaucoma, glomerular disease, glomerular nephritis, gout, gouty arthritis, graft-versus-host disease, gut diseases, head injury, headache, hearing loss, heart disease, hemolytic anemia, Henoch-Scholein purpura, hepatitis, hereditary periodic fever syndrome, herpes zoster and simplex, HIV-1, Hodgkin’s disease, Huntington’s disease, hyaline membrane disease, hyperammonemia, hypercalcemia, hypercholesterolemia, hyperimmunoglobulinemia D with recurrent fever (HIDS), hypoplastic and other anemias, hypoplastic anemia, idiopathic thrombocytopenic purpura, incontinentia pigmenti, infectious mononucleosis, inflammatory bowel disease, inflammatory lung disease, inflammatory neuropathy, inflammatory pain, insect bite-induced inflammation, iritis, irritant-induced inflammation, ischemia/reperfusion, juvenile rheumatoid arthritis, keratitis, kidney disease, kidney injury caused by parasitic infections, kidney injury caused by parasitic infections, kidney transplant rejection prophylaxis, leptospirosis, Lewy body dementia, Loeffler’s syndrome, lung injury, lupus, lupus nephritis, meningitis, mesothelioma, mixed connective tissue disease, Muckle-Wells syndrome (urticaria deafness amyloidosis), multiple sclerosis, multiple system atrophy, muscle wasting, muscular dystrophy, myasthenia gravis, myocarditis, mycosis fungoides, myelodysplastic syndrome, myositis, nasal sinusitis, necrotizing enterocolitis, neonatal onset multisystem inflammatory disease (NOMID), nephrotic syndrome, neuritis, neuropathological diseases, non-allergen induced asthma, obesity, ocular allergy, optic neuritis, organ transplant, osteoarthritis, otitis media, Paget’s disease, pain, pancreatitis, Parkinson’s disease, pemphigus, pericarditis, periodic fever, periodontitis, peritoneal endometriosis, pertussis, pharyngitis and adenitis (PFAPA syndrome), plant irritant-induced inflammation, pneumonia, pneumonitis, pneumocystis infection, poison ivy or urushiol oil-induced inflammation, polyarteritis nodosa, polychondritis, polycystic kidney disease, polymyositis, psoriasis, psychosocial stress diseases, pulmonary disease, pulmonary hypertension, pulmonary fibrosis, pyoderma gangrenosum, pyogenic sterile arthritis, renal disease, retinal disease, rheumatic carditis, rheumatic disease, rheumatoid arthritis, sarcoidosis, seborrhea, sepsis, severe pain, sickle cell, sickle cell anemia, silica-induced disease, Sjogren’s syndrome, skin diseases, sleep apnea, spinal cord injury, spondylitis, spondyloarthropathy, Stevens- Johnson syndrome, stroke, subarachnoid hemorrhage, sunburn, temporal arteritis, tenosynovitis, thrombocytopenia, thyroiditis, tissue transplant, TNF receptor associated periodic syndrome (TRAPS), toxoplasmosis, transplant, traumatic brain injury, tuberculosis, type 1 diabetes, type 2 diabetes, ulcerative colitis, urticarial, uveitis (including nongranulamotous uveitis and granulomatous uveitis), wound healing, Wegener’s granulomatosis, interstitial lung disease, psoriatic arthritis, juvenile idiopathic arthritis, antineutrophil cytoplasmic antibody (ANCA)- associated vasculitis, antiphospholipid antibody syndrome, deep vein thrombosis, fibrosis, Alzheimer’s, scleroderma or CREST syndrome.

In some embodiments, the disorder is cancer. For example, neutrophil inflammation, neutrophil extracellular traps (NET), and/or monocyte extracellular traps (MET) have been identified in cancers, and are associated with poorer prognosis. (See, e.g., Li et al., Molecular Cancer Therapeutics, 19: 1530-38 (2020).) PAD2 has been demonstrated to play a role in various tumor types, further reviewed in Wu et al. Front Immunol 12: 761946 (2021). PAD2 may play a role in the uncontrolled proliferation of tumor cells via citrullination of RNA polymerase II (RNAP2) (Sharma et al. Molecular Cell 73(1): 84-96 (2019). Inhibition of PAD2 has also been demonstrated to synergize with other chemotherapies, for example combination treatment with a PAD inhibitor and docetaxel inhibited proliferation of breast cancer cells in vitro Li et al. J Exp Clin Cancer Res 38: 414 (2019). In some embodiments, the cancer is a cancer that is typically responsive to immunotherapy. In some embodiments, the cancer is a cancer that is not typically responsive to immunotherapy. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a blood malignancy (liquid tumor).

In some embodiments, the cancer is carcinoma, lymphoma, blastoma, sarcoma, or leukemia. In some embodiments, the cancer is squamous cell cancer, small-cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma squamous cell carcinoma, small-cell lung cancer (SCLC), non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell renal carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer (including triple-negative breast cancer, ER positive breast cancer, ER negative breast cancer, node positive breast cancer, and node negative breast cancer), colon carcinoma, and head and neck cancer (or carcinoma), gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer/T-cell lymphoma, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain cancer, brain stem glioma, pituitary adenoma, Kaposi’s sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, an environmentally-induced cancer (e.g., a cancer induced by asbestos, a virus-related cancer or a cancer of viral origin (e.g., human papilloma virus (HPV-related or -originating tumors)), a hematologic malignancy derived from either of the two major blood cell lineages (i.e., the myeloid cell line (which produces granulocytes, erythrocytes, thrombocytes, macrophages and mast cells) or lymphoid cell line (which produces B, T, NK and plasma cells), such as, e.g., a leukemia, lymphoma, or myeloma (of any type), e.g., acute, chronic, lymphocytic and/or myelogenous leukemia, such as acute leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), undifferentiated AML (MO), myeloblastic leukemia (ML), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3 V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma; a lymphoma, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B cell hematologic malignancy, e.g., B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large-cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angio-immunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma/leukemia (T-Lbly/T- ALL), peripheral T- cell lymphoma, lymphoblastic lymphoma, post-transplantation lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, B cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B- cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmocytoma, and multiple myelomas, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; a hematopoietic tumor of myeloid lineage, a tumor of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; seminoma, teratocarcinoma, a tumor of the central or peripheral nervous system, such as astrocytoma, schwannoma; a tumors of mesenchymal origin, such as fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; or another tumor, such as melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer, teratocarcinoma, a hematopoietic tumor of lymphoid lineage, for example a T-cell or B-cell tumor, such as a T-cell disorder such as T-prolymphocytic leukemia (T-PLL), such as of the small cell or cerebriform cell type; a large granular lymphocyte leukemia (LGL) of the T-cell type; a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma (pleomorphic or immunoblastic subtype); angiocentric (nasal) T-cell lymphoma; cancer of the head or neck, renal cancer, rectal cancer, cancer of the thyroid gland; acute myeloid lymphoma, or any combinations of said cancers. The methods described herein can also be used for treatment of metastatic cancers, unresectable cancers, refractory cancers (e.g., cancers refractory to previous immunotherapy, e.g., with an anti-CTLA-4 or anti-PD-1 antibody), and/or recurrent cancers.

In some embodiments, the disease is an infectious disease. For example, occurrence of NETosis has also been found in various infections. (See, e.g., Li et al., Molecular Cancer Therapeutics, 19: 1530-38 (2020).) Infectious diseases may include, for example, viral diseases (including AIDS (HIV infection), hepatitis (A, B, C, D, and E), and herpes), bacterial infections, fungal infections, protozoal infections and parasitic infections. Examples of pathogenic infections include, but are not limited to, HIV, Hepatitis (A, B, & C), Influenza, Herpes (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, Epstein Barr virus), Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa, adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, Lyme disease bacteria, Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absihlorambzopus), Sporothrix schenkii. Blastomyces dermalilidis. Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum, Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis. In some cases, the infectious disease is caused by a viral pathogen. In other cases, it is caused by a bacterial pathogen.

B. Combination Therapy

A PAD2 modulator may be administered alone or with other modes of treatment. Such other modes of treatment may be provided before, substantially contemporaneous with, or after administration of a PAD2 modulator. Thus, some subjects herein receiving treatment with a PAD2 modulator, such as a PAD2 inhibitor, or being considered for treatment with a PAD2 modulator or PAD2 inhibitor, may also be receiving treatment with another therapy.

In some embodiments, the additional treatment may be an additional PAD modulator such as a PAD inhibitor, e.g., a PAD4 modulator such as a PAD4 inhibitor.

For treatment of rheumatoid arthritis, for example, a subject may be treated with therapeutic agents, for example, such as a disease-modifying anti-rheumatic drug (DMARD) such as methotrexate (Trexall® or Otrexup®), adalimumab (Humira®), etanercept (Enbrel®), infliximab (Remicade®), hydroxychloroquine (Plaquenil®), sulfasalazine (Azulfidine®), leflunomide (Arava®), abatacept (Orencia®), anakinra (Kineret®), Certolizumab (Cimzia®), golimumab (Simponi®), rituximab (Rituxan®), sarilumab (Kevzara®), tocilizumab (Actemra®), baricitinib (Olumiant®), tofacitinib (Xeljanz®), upadacitinib (Rinvoq®), and Orencia® (abatacept); an non-steroidal anti-inflammatory drug (NS AID) such as ibuprofen (Advil, Motrin, and diclofenac) and naproxen sodium; a COX-2 inhibitor (celecoxib or etoricoxib); a steroid such as prednisolone or prednisone. In some cases, a PAD2 modulator such as a PAD2 inhibitor may be administered with one or more of: anti-TNF agents (e.g., anti-TNF antibodies) such as infliximab (Remicade®), adalimumab (Humira®), golimumab (Simponi®), certolizumab (Cimzia®), and etanercept (Enbrel®); glucocorticoids such as prednisone or methylprednisolone; leflunomide (Arava®); azathioprine (Imuran® or Azasan®); JAK inhibitors such as CP 590690; SYK inhibitors such as R788; TYK2 inhibitors such as deucravacitinib (Sotyktu®), anti-IL-6 antibodies; anti-IL-6R antibodies; anti-CD-20 antibodies; anti-CD19 antibodies; anti-GM-CSF antibodies; and anti-GM-CSF-R antibodies. For treatment of autoimmune conditions, a PAD2 modulator such as a PAD2 inhibitor may be administered with other therapeutic agents, for example, interferon alpha; interferon beta; anti-Type I interferon receptor antibodies such as anifrolumab (Saphnelo®); prednisone; anti-alpha4 integrin antibodies such as Tysabri®; anti-BAFF/BLyS antibodies such as belimumab (Benlysta®); anti-CD20 antibodies such as Rituxan® (rituximab); calcineurin inhibitors such as cyclosporin or voclosporin (Lupkynis®); complement inhibitors such as eculizumab (Soliris®) or avacopan (Tavneos®); mycophenolate mofetil (CellCept®) or mycophenolate sodium (MyFortic®); cyclophosphamide (Cytoxan®); FTY720 (fmgolimod, e.g., Gilenya®); and Cladribine® (Leustatin). In some cases, a PAD2 modulator such as a PAD2 inhibitor may be administered with methotrexate.

For the treatment of lupus, for example, a subject may be treated with one or more therapeutic agents such as cyclosporine, tacrolimus, cyclophosphamide, azathioprine (Imuran®), mycophenolate (CellCept®), rituximab (Rituxan®), and Belimumab (Benlysta®), steroids (e.g., prednisone or prednisolone), blood pressure medication (e.g., antiotensin- convertin enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs)).

For the treatment of vasculitis, for example, includes administration of therapeutic agents such as steroids (e.g., prednisone, prednisolone, methylprednisolone, or dexamethasone), methotrexate (Trexall®), azathioprine (Imuran®, Azasan®), mycophenolate (CellCept®), cyclophosphamide, tocilizumab (Actemra®), rituximab (Rituxan®), Avacopan, plasma exchange, mycophenolate mofetil (MMF), azathioprine (AZA), leflunomide (LEF), belimumab, meprolizumab, and omalizumab. For treatment of cancer, a subject may be treated with anti-cancer agents, such as an immune checkpoint inhibitor, a chemotherapeutic agent, growth inhibitory agent, radiotoxic agent, immunosuppressive agent, anti-cancer vaccine such as a gene therapy vaccine, anti-angiogenesis agent and/or anti-neoplastic composition.

Examples of immune checkpoint inhibitors include molecules that inhibit particular signaling pathways that regulate the immune system. See e.g., Weber (2010) Semin. Oncol. 37:430; Pardoll (2012) Nat. Rev. Cancer 12:252. Immune checkpoint inhibitors, in some embodiments, comprise an antagonist of PD-1, PD-L1, CTLA4, LAG-3, Galectin 1, Galectin 9, CEACAM-1, BTLA, CD25, CD69, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM1, TIM3, TIM4, ILT4, IL-6, IL-10, TGFp, VEGF, KIR, LAG-3, adenosine A2A receptor, PI3Kdelta, or IDO. In some embodiments, an immune checkpoint inhibitor comprises an agonist of B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, 0X40, OX40L, GITR, GITRL, CD27, CD40, CD40L, DR3, CD28H, IL-2, IL-7, IL-12, IL-15, IL-21, IFNa, STING, or a Toll-like receptor agonist such as a TLR2/4 agonist. In some embodiments, an immune checkpoint inhibitor comprises an agent that binds to a member of the B7 family of membrane-bound proteins such as B7-1, B7-2, B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. In some embodiments, an immune checkpoint inhibitor binds to a member of the TNF receptor family or a co-stimulatory or co- inhibitory molecule binding to a member of the TNF receptor family such as CD40, CD40L, 0X40, OX40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL/ Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fnl4, TWEAK, BAFFR, ED AR, XEDAR, EDAI, EDA2, TACI, APRIL, BCMA, LTPR, LIGHT, DeR3, HVEM, VEGL/TL1A, TRAMP/DR3, TNFR1, TNFP, TNFR2, TNFa, ip2, FAS, FASL, RELT, DR6, TROY, or NGFp. In some embodiments, an immune checkpoint inhibitor antagonizes or inhibits a cytokine that inhibits T cell activation such as IL-6, IL- 10, TGFP, VEGF. In some embodiments, an immune checkpoint inhibitor comprises an agonist of a cytokine that stimulates T cell activation such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNa. In some embodiments, the at least one immune stimulating agent comprises an antagonist of a chemokine, such as CXCR2, CXCR4, CCR2, or CCR4. In some embodiments, an immune checkpoint inhibitor comprises an antibody. In some embodiments, an immune checkpoint inhibitor comprises a vaccine, such as a mesothelin-targeting vaccine or attenuated listeria cancer vaccine such as CRS-207.

Exemplary non-limiting example targets of immune checkpoint inhibitors are CTLA- 4, PD-1, and PD-L1. Non-limiting examples of such immune checkpoint inhibitors include anti-CTLA4, anti-PD-1, and anti-PD-Ll antibodies, such as, e.g., pembrolizumab (Keytruda®), ipilimumab (Yervoy®), nivolumab (Opdivo®), atezolizumab (Tecentriq®), avelumab (Bavencio®), dostarlimab (Jemperli®), cemiplimab (Libtayo®), and durvalumab (Imfinzi®).

Non-limiting examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and Cytoxan®/Neosar® cyclosphosphamide; lenalidomide (Revlimid®); bortezomib (Velcade®); bendamustine (Treanda®); rituximab (Rituxan®); alemtuzumab (Campath®); ofatumumab (Kesimpta®); everolimus (Afinitor®, Zortress®); carfilzomib (Kyprolis™); ifosamade; dexamethasone; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophy cin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil (Leukeran®), chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Inti. Ed. EngL, 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine (Fludara®), 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as gemcitabine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; chlorambucaxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2- ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone’ 2”2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxanes, e.g., Taxol® paclitaxel (Bristol- Myers Squibb Oncology, Princeton, N. J.), Abraxane® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and Taxotere® doxetaxel (Rhone- Poulenc Rorer, Antony, Fran75hlorambucilbucil; Gemzar® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine (Oncovin®); thalidomide (Thalomid®); Navelbine® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluorometlhyl ornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Further nonlimiting exemplary chemotherapeutic agents include anti-hormonal agents that act to regulate or inhibit hormone action on cancers such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and Fareston® toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, Megase® megestrol acetate, Aromasin® exemestane, formestanie, fadrozole, Rivisor® vorozole, Femara® letrozole, and Arimidex® anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGF expression inhibitor (e.g., Angiozyme® ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapy vaccines, for example, Allovectin® vaccine, Leuvectin® vaccine, and Vaxid® vaccine; Proleukin® rIL-2; Lurtotecan® topoisomerase 1 inhibitor; Abarelix® rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In some embodiments, an anti-angiogenesis agent may be administered. Non-limiting examples of an anti-angiogenesis agent can include an antibody or other antagonist to an angiogenic agent, e.g., antibodies to VEGF-A (e.g., bevacizumab (Avastin®)) or to the VEGF-A receptor (e.g., KDR receptor or Fit- 1 receptor), anti-PDGFR inhibitors such as Gleevec® (Imatinib Mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668, Sutent®/SUl 1248 (sunitinib malate), AMG706, or those described in, e.g., international patent application WO 2004/113304). Anti-angiogensis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D’Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 (e.g., Table 3 listing anti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12): 1359-1364; Tonini etal. (2003) Oncogene 22:6549-6556 (e.g., Table 2 listing known anti-angiogenic factors); and Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., Table 1 listing anti-angiogenic agents used in clinical trials).

In some embodiments, a tumor growth inhibitory agent may be administered. Nonlimiting examples of growth inhibitory agents include, but are not limited to, agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S- phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (W.B. Saunders, Philadelphia, 1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (Taxotere®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (Taxol®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.

For treatment of inflammatory or autoimmune or infectious disease conditions or cancer, in some embodiments, an anti-inflammatory drug may be administered. The antiinflammatory drug can be, e.g., a steroid or a non-steroidal anti-inflammatory drug (NSAID). In cases where it is desirable to render aberrantly proliferative cells quiescent, hormones and steroids (including synthetic analogs) could be administered, such as 17a-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone, Mege str ol acetate, Methylprednisolone, Methyl-testosterone, Prednisolone, Triamcinolsone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, or goserelin (ZOLADEX®), can also be administered to the patient.

In some cases, a subject with a disease may receive a vaccination protocol. Many experimental strategies for vaccination against infectious diseases and tumors have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). Examples are cell-based therapies such as dendritic cells, or vaccine-like particles (VLPs).

In other cases, a subject may be administered other therapies, such as radiation therapy in the case of a tumor, surgical interventions, or the like.

Any of the above agents may, in some cases, be administered with a PAD2 modulator, such as a PAD2 inhibitor, to a subject, or alternatively, a subject already receiving one of the above therapies or agents may have a biological sample assessed by methods herein in order to determine if the subject should receive a PAD2 modulator or PAD2 inhibitor either in combination with or as an alternative to a current treatment for the subject.

EXAMPLES

Example 1: Citrullination in human serum induced in vitro by exogenous PAD4 and/or exogenous PAD2

Global proteomics experiments were carried out using serum samples from normal healthy volunteers to identify sites on human proteins that are citrullinated by PAD4 and/or PAD2.

A. Materials and Methods

Induction of citrullination in vitro by exogenous PAD2 or exogenous PAD4 in human serum. A starting enzyme reaction mixture containing a concentration of 1 mM of the recombinant human (rh) enzyme (1 mM of rhPAD4 or ImM of rhPAD2), 1 M Tris, 20 mM DDT, and 10 mM CaCh was used. The starting enzyme reaction mixture was diluted as needed with a 10-fold reaction buffer that contained 1 M Tris, 20 mM DDT, and 10 mM CaCh to achieve a mixture with lOx the final desired enzyme concentration. Then, 4 pl of the relevant lOx enzyme reaction mixture was added to 36 pl of serum in plates (BD 353263) to provide a final concentration of lOnM PAD4 or 3nM of PAD2. The wells without added PAD enzyme served as non-induction controls. Plates were incubated at 37°C in CO2 incubator for 2 hours. Reactions were stopped with 2 pl of 0.5 M EDTA. Plates were sealed and stored at -80°C.

Global proteomics. The serum mixture (serum + enzyme + antibody, prepared as described above) was thawed and 5 pL was passed over lOOul Thermo Top 14 Depletion Igg agarose beads (Cat. No. A36372). This was performed on an Agilent microfilter plate (Cat. No. 200989-100) after shaking for 15min and eluting with 1200xG for 2min. The eluate was combined with 50 pl 2X iST-BCT LYSE buffer (PreOmics, cat# P.O.00120) and heated for 10 minutes at 95°C. Samples were enzymatically digested with addition of trypsin and LysC, and resulting peptides cleaned up according to manufacturer’s protocol using iST-BCT kit (PreOmics, cat# P.O.00116).

Peptides were analyzed by nanoLC-MS/MS on a Bruker timsTOF mass spectrometer using a 13min diaPASEF method with a 200ng load. diaPASEF data was searched against a previously generated Data dependent acquisition (DDA) spectral library generated from NHV serum. Data independent acquisition (DIA) runs were subsequently analyzed using the Fragpipe and DIANN workflow (Demichev, V., Szyrwiel, L., Yu, F. et al. dia-PASEF data analysis using FragPipe and DIA-NN for deep proteomics of low sample amounts. Nat Commun 13, 3944 (2022). doi. org/ 10.1038/s41467-022-31492-0).

For differential citrullinome analysis across different treatment groups, the precursor values were log2 -normalized and precursors carrying arginine residues with (citrullination) modification were annotated as citrullinated (see bold R residues in peptide sequences of table below). Precursors showing citrullination on the last arginine residue were removed from further analysis as they were deemed misassigned. Protein level intensity values were also log2 -transformed before downstream analysis. Both citrullinated precursor and protein data were modeled using linear regression (“limma” package in R) where donor-to-donor variation was accounted for. For assessing which citrullination sites were citrullinated by which PAD enzymes (PAD2, PAD4, or both), citrullinated precursors at 3nM PAD2 and lOnM PAD4 treatment groups were compared to no-treatment control group separately. Precursors that were significantly citrullinated by PAD2 at adjusted p-value cut-off of 0.01, but showed no statistically significant effect by PAD4 treatment, are designed in the results below as "PAD2-citrullinated". Similarly, precursors that were significantly citrullinated by PAD4, but showed no statistically significant effect by PAD2 treatment, are designated in the results below as PAD4-citrullinated. Precursors that were significantly citrullinated by both PAD2 and PAD4 are designated in the results below as “common” targets.

A. Global Proteomics Results

PAD2 or PAD4 enzyme concentration levels considered to be physiologically relevant were used in this assay, and many citrullinated proteins were induced at these concentrations. A list of selected peptides that were citrullinated under these conditions, i.e., with addition of 10 nM PAD4 or 3 nM PAD2 or both are shown in Table 1 (below). Table 1 shows particular arginine (R) residues in each peptide or protein that were citrullinated, using bold face and underlining. The peptides in the tables below also in some cases were found to also include chemical modifications at other residues, such as oxidation of methionine (M), carbamidomethylation at certain cysteine (C) residues, or modifications at the N-terminus of the peptide, which are noted by underlining those residues, or in the case of a modification at the N-terminus, underlining the first residue in the peptide sequence. Each row of the tables provides the protein of which the peptide is a fragment, by name and abbreviation, as well as a UniProt accession number for the UniProt database (www.uniprot.org) providing the complete protein sequence. The amino acid residue of the UniProt complete protein sequence corresponding to the citrullination site shown in the peptide fragment of the table is also provided.

Peptides citrullinated by PAD4 alone are indicated as “PAD4 citrullinated” in the “Target” column below, while those citrullinated by both PAD4 and PAD2 are indicated by the term “common” in the “Target” column of the table below.

Example 2: Citrullination in human serum induced in vitro by exogenous PAD4 and inhibited by an anti-PAD4 antibody

This Example and subsequent Examples describe experiments assessing citrullination by PAD4 and in some cases the impact of a PAD4 inhibitor. These experiments can also be applied to assess citrullination by PAD2 and the impact of a PAD2 inhibitor, for example by replacing PAD4 and/or a PAD4 inhibitor with PAD2 and/or a PAD2 inhibitor in the protocol, and using a PAD2-citrullinated peptide or protein.

In this example, global proteomics experiments were carried out to identify sites on human proteins that are citrullinated by PAD4 and where citrullination is inhibited by PAD4 inhibitors. This Example shows how citrullination of particular peptides may be used to monitor activity of a PAD inhibitor, for example. The anti-PAD4 antibody hzl3-5 D3 IE, described in International Patent Publication No. W02024/020579, was used as an exemplary PAD4 inhibitor in this and subsequent examples.

B. Methods

Induction of citrullination in vitro by exogenous PAD4 in human serum. In this series of experiments, 20 pl of enzyme reaction mix was added to 80 pl of serum in plates (100 nM PAD4, 100 mM Tris, 2 mM DDT, and 1 mM CaCh final concentrations). Plates were incubated at 37°C in CO2 incubator for 2 hours. Reactions were stopped with 10 pl of 0.5 M EDTA. Plates were sealed and stored at -80°C.

Inhibition curves. To produce inhibition curves, 7.5 pl of Ab hzl3-5 D31E serial dilutions in PBS (final concentrations ranging from 0.0038 nM to 100 nM) or isotype control (final concentration 100 nM) were added to 80 pl of serum in plates, mixed and stored overnight at -80°C. Serum plates were thawed and 12.2 pl of PAD4 enzyme reaction mix with or without 2 mM CaCh (7 nM or 13.5 nM PAD4, 100 mM Tris, 2 mM DDT final) was added and incubated at 37°C in CO2 incubator for 2 hours. Reactions were stopped with 10 pl of 0.5 M EDTA. Plates were sealed and stored at -80°C.

Global proteomics. The serum mixture (serum + enzyme + antibody, prepared as described above) was thawed and diluted (1 part serum mixture to 10 parts water). Then 5 pl of the resulting diluted serum mixture was combined with 45 pl iST-BCT LYSE buffer (PreOmics, cat# P.O.00116) and heated for 10 minutes at 95°C. Samples were enzymatically digested with addition of trypsin and LysC, and resulting peptides cleaned up according to manufacturer’s protocol using iST-BCT kit (PreOmics, cat# P.O.00116). Peptides were analyzed by nanoLC-MS on Bruker timsTOF mass spectrometer using a 13 minute diaPASEF method. Data dependent acquisition (DDA) data was collected on samples with 100 nM PAD4 without compound to generate a spectra library. Data independent acquisition (DIA) runs were subsequently analyzed using the Fragpipe and DIANN workflow (Demichev, V., Szyrwiel, L., Yu, F. et al. dia-PASEF data analysis using FragPipe and DIA-NN for deep proteomics of low sample amounts. Nat Commun 13, 3944 (2022). doi . org/ 10.1038/s41467-022-31492-0).

To identify citrullination sites that showed statistically significant dose response to Ab hzl3-5 D3 IE, a four-parameter non-linear model was applied to a data matrix containing citrullination ratio (CitRatio) of each citrullinated peptide calculated by dividing the intensity of the citrullinated peptide by the total protein intensity (summarized based maxLFQ algorithm). For each citrullinated site, CitRatio was fitted against doses in a four-parameters (upper bound, lower bound, ec50 and hill) non-linear model. The non-linear model was then compared against a null linear regression model using chi-square statistics. The null model fits the mean (CitRatio) against doses. This process generates a p. value (DRC.p.value) that represents goodness of fit and effect size of the dose dependency for each individual citrullination site.

C. Global Proteomics Results

The addition of 100 nM exogenous PAD4 induced more than 500 unique citrullinated peptide precursors which originated from more than 100 serum proteins, many of which were inhibited by 100 ug/ml Ab hzl3-5 D3 IE. When PAD4 enzyme concentrations at more physiologically relevant levels were used in the same assay, many citrullinated proteins were induced even at these lower concentrations and were inhibited by Ab hzl3-5 D3 IE in a dose dependent fashion. Citrullinated peptides from a variety of proteins were detected in at least 2/3 of all the samples, and about a p value (for goodness of fit) < 0.01 with addition of 13.5 nM PAD4 or 7 nM PAD4, and with or without added calcium in enzyme reaction mix. About 50 such peptides were identified with 13.5 nM PAD4 with and without calcium, whereas 8- 15 such peptides were identified with 7 nM PAD4 with and without calcium. In addition to citrullination of arginine (R) residues, in some cases peptides also were found to also include chemical modifications at other residues, such as oxidation of methionine (M), carbamidomethylation at certain cysteine (C) residues, or modifications at the N-terminus of the peptide.

Example 3: Baseline citrullination in human plasma

Citrullination in human plasma was assessed. A. Methods

Sample collection. Plasma was separated by centrifugation from blood drawn on heparin from healthy volunteers and the plasma was frozen for further use.

Global proteomics. 5 pl plasma was thawed and incubated with 100 pl High-Select™ Top 14 Abundant Protein depletion resin (Thermo Fisher, cat# A36372) at room temperature for 15 minutes. Depleted plasma samples were recovered in flowthrough by centrifugation, and subsequently enzymatically digested and cleaned up using iST-BCT kit (PreOmics, cat# P.O.00116) following the manufacturer’s protocol.

Peptides were analyzed by nanoLC-MS on Bruker timsTOF mass spectrometer using a 13 -minute diaPASEF (DIA) method. A pooled sample generated by combining peptides from all samples were fractionated into 24 fractions using high PH reversed phase HPLC. Data dependent acquisition (DDA) data were collected on the fractions to generate a spectra library. DIA runs were analyzed using the Fragpipe and DIANN workflow (Demichev, V., Szyrwiel, L., Yu, F. et al. dia-PASEF data analysis using FragPipe and DIA-NN for deep proteomics of low sample amounts. Nat Commun 13, 3944 (2022). doi.org/10.1038/s41467- 022-31492-0).

B. Global Proteomics Results

Citrullinated peptides were detected by unbiased proteomics in plasma from 10 healthy donors (Table 2). Table 2 shows particular arginine positions of each peptide that were found to be citrullinated. The citrullination of the peptides at the sites shown in Table 2 (in bold and underlining) was also found in Example 2 to be induced by exogenously added PAD4 and inhibited by the anti-PAD4 antibody hzl3-5 D31E.

Example 4: Induction of citrullination ex vivo in incubated blood and inhibition of citrullination by a PAD4 inhibitor

This Example provides another experimental system for assessing citrullination in blood samples, and the impact of a PAD inhibitor. The experimental protocol was carried out in the presence of a PAD4 inhibitor, but can be also carried out in the presence of a PAD2 inhibitor.

Samples obtained from normal healthy volunteers were analyzed for citrullination of specific peptides by endogenous PAD4. The blood samples were incubated at 37°C to induce release of endogenous PAD4 from dying cells. The samples were also treated with a PAD4 inhibitor (the anti-PAD4 antibody hzl3-5 D31E) and analyzed for inhibition of citrullination by Ab hzl3-5 D31E.

A. Methods

Sample generation. PAD4 inhibitor (hzl3-5 D3 IE), isotype control, or PBS were added to whole blood collected in Sodium Heparin tubes or TruCulture® null tubes. Blood was then incubated at 37°C and 5% CO2 for 72 hours. Plasma was collected from Sodium Heparin tubes; supernatant from the blood was collected from TruCulture® tubes, and the resulting samples (plasma or TruCulture® supernatants) were stored at -80°C until analysis for citrullinated peptides identification and quantification. Reproducibility between donors, and within donors, between 2 timepoints that were 1 week apart were evaluated (N=6). Analysis method for identification and measure of citrullinated peptides: Global proteomics. 5 pl of thawed sample (plasma or supernatant) was incubated with 100 pl High- Select™ Top 14 Abundant Protein depletion resin (Thermo Fisher, cat# A36372) at room temperature for 15 minutes. Depleted samples were recovered in flowthrough with centrifugation, and subsequently enzymatically digested and cleaned up using iST-BCT kit (PreOmics, cat# P.O.00116) following manufacture protocol. The LC-MS method was as discussed above in Example 3, with one exception: spectra library was created using HPLC fractions from pooled samples of the 72-hour incubation group without PAD4 antibody treatment.

B. Results

After 48 hours or 72 hours of incubation at 37°C, induction of specific citrullinated peptides was observed. Citrullination was inhibited by Ab hzl3-5 D31E in a dose dependent manner (Table 3 and FIG. 1 A to FIG. ID). The expectation was that PAD4 would be released following cell death during the incubation. Consistent with this expectation, MPO levels as well as PAD4 levels showed increases between 0 hours and 48 hours (results not shown). Table 3 also shows the particular arginine positions of each peptide that were citrullinated sites in bold text and underlining.

Example 5: Identification of citrullinated peptides in RA synovial fluid (SF)

A. Methods

This example illustrates a method for assessment of citrullination in synovial fluid. 5 pl synovial fluid (SF) (instead of serum) was used as starting material. LC-MS sample preparation and analysis methods were as discussed in Example 3 above. Spectra libraries were made for SF from pooled samples generated by combining a portion of each sample. B. Results

Spearman correlation analysis was performed to assess the correlation between percent citrullination (calculated as citrullinated peptide intensity divided by total protein intensity) and MPO LC-MS intensity. MPO was used as a surrogate for NETosis. MPO and PAD4 levels measured by unbiased proteomics in this SF sample set are correlated (Spearman p=0.56 p=2.9xl0'³). The correlation of MPO with PAD4 was confirmed by measuring PAD4 concentrations using an affinity capture LC-MS assay on the same SF samples (Spearman p=0.69, p=2.8xl0'⁷).

The expectation is that citrullinated peptides that are positively correlated with MPO levels are relevant to NETosis and PAD4 activity in the joint. A number of citrullinated peptide precursors were detected in at least 35% of patients with Spearman p >0.3, and p- value <0.05. The charged form of the peptide that correlates the best with MPO was selected to provide correlation coefficient and p-value. Based on the global proteomics analysis, a subset of those peptides were selected for targeted LC-MS assay of rheumatoid arthritis synovial fluid (RA SF) for several reasons: (1) they showed in vivo inhibition in mouse models with Ab hzl3-5 D3 IE (PRG4 peptide is also present in mice); (2) they showed a good prevalence across donors (especially PRG4, FGA, AMBP and C3 peptides); (3) they were citrullinated by exogenous PAD4 in serum and inhibited with Ab hzl3-5 D3 IE (AMBP, FGA and C3 peptides); and (4) they were also detected in plasma (ITH44). These selected peptides are listed in Table 4 below. The level of citrullinated ITH44 is lower than other citrullinated peptides listed in Table 4; this observation might be due to a natural variant in the amino acid sequence of ITH44 (669Q to L), which may have resulted in a variant peptide was not detected by mass spectrometry and/or levels of citrullination below the limit of detection in some donor samples.

The levels of the citrullinated peptides listed in Table 4 above were compared between anti-citrullinated protein antibodies positive (ACPA+) and ACPA negative (ACPA-) patients and no differences were observed.

Example 6: Analysis of serum or plasma for citrullination by exogenous PAD4 and inhibition of citrullination by a PAD4 inhibitor

This example relates to an ex vivo assay for the measurement of citrullination inhibition by a PALM inhibitor (such as Ab hzl3-5 D3 IE) in serum or plasma. The assay may be used as a pharmacodynamic assay, and may also be used for measurement of citrullination inhibition of PAD2 citrullinated peptides by a PAD2 inhibitor.

A. Method

Plasma or serum samples from human subjects were collected and analyzed to assess citrullination of particular citrullination sites.

Sample collection. Serum and plasma samples from normal healthy volunteers were used in this example. To obtain serum, blood was collected in serum tubes (Vacutainer SST blood collection tubes) to separate serum. To obtain plasma, whole blood samples were collected in sodium heparin vacutainer tubes and plasma was isolated from the whole blood samples using standard methods.

Ex Vivo Treatment with PAD 4 Inhibitor for Generating Inhibition curves. Serum or plasma aliquots of 80 pl were pipetted into 96 well microplates (plate cat# 351190, Falcon). Various concentrations of PALM inhibitor (anti-PALM antibody hzl3-5 D31E) were titrated into the samples in the wells and the microplates were incubated for 6 h at 37degrees C. Then the samples were frozen.

In vitro citrullination by exogenous PAD4. Exogenous P AD4 was added to thawed serum or plasma samples to induce citrullination of proteins. 12.2 pl of PALM enzyme mix (13.5 nM PALM, lOOmM Tris, 2 mM DDT) was added to each sample. The reactions were incubated at 37°C in a CO2 incubator for 2 hours. Each reaction was stopped with 10 pl of 0.5 M EDTA and frozen prior to subsequent treatment and proteomics analysis.

Preparations Prior to Proteomic Analysis. Ten (10) pl of serum or plasma was thawed and diluted 15-fold with PBS. Sixty (60) pl of 15-fold diluted serum or plasma was used for depletion of human serum albumin using CUSTOM PhyTip 200 pL CaptureSelect™ Human Albumin tips (Biotage, San Jose, CA) containing 20 pL CaptureSelect™ human albumin affinity matrix. Five (5) pl of albumin-depleted serum or plasma was combined with 45 pl ist-BCT LYSE buffer and heated for 20 minutes at 80°C. Samples were enzymatically digested with addition of trypsin and LysC according to manufacturer’s protocol. Peptides were cleaned up using iST-BCT kit (PreOmics, cat# P.O.00116).

Targeted proteomic analysis. Peptides listed in Table 5 were analyzed by nanoLC-MS on Bruker timsTOF of mass spectrometer using a 30-minute prm-PASEF method. Data dependent acquisition (DDA) data was acquired for samples with high PAD4 concentrations with spiked internal standards (heavy peptides) for each peptide of interest. Skyline software was used to generate a list of cit-peptide and unmodified peptide sequences from cit-proteins previously identified by global proteomics experiments. Next, the library in Skyline was created using MaxQuant generated msms.txt; mqpar.xml, and modifications.xml files. Ion mobility library was created after importing DDA data and using the imported data to generate a spectra library. Three parameters for each analyte were exported from Skyline to build a prm-PASEF method in TimsTOF Control 3.0 software: retention time (RT); Ion Mobility (IM), and precursor m/z. Data was analyzed using Skyline workflow.

Citrullination of specific peptides, listed in Table 5, was quantified on nanoLC- TimsTOF platform using Skyline data processing software. In the peptides of Table 5, the cysteine at position 3 of the AMBP peptide and the cysteine at position 10 of the TF peptide were also found to be modified by carb amidomethylation, as noted by underlining of those residues.

Analyses on TripleQuad® platform were also performed. TripleQuad® platform is much higher throughput and can be used to achieve faster turnaround times. Peptides that were analyzed on TripleQuad® are shown in Table 6.

B. Results

The results showed dose-dependent inhibition of citrullination by the PAD4 inhibitor hzl3-5 D3 IE. Inhibition curves were generated using the citrullination ratios (ratio of citrullinated peptide to total corresponding protein) for each peptide and IC50s were calculated. The IC50 results (reported as mean ± standard deviation) for representative peptides, as measured in plasma and serum samples, are shown in Table 7 and inhibition curves for three donors are shown in FIG. 2 A to FIG. 2C. These results (shown in Table 7 and FIG. 2A to FIG. 2C) were generated from measurements on nanoLC-TimsTOF platform. Measurements from the TripleQuad® platform yielded similar results (results not shown).

These results show that the assay can be used as an in vitro pharmacodynamic assay to track the citrullination inhibition by a PAD4 inhibitor. In clinical applications, baseline-samples (prior to treatment with a PAD4 inhibitor) can be collected, processed and analyzed as described in this Example. PAD4 inhibition by a PAD4 inhibitor can be assessed using samples obtained from a subject at one or more timepoints after treatment with the PAD4 inhibitor. For instance, PAD4 inhibition (% decrease in each of one or more citrullinated peptides) in post-treatment samples may be calculated using inhibition curves generated with baseline (pre-treatment) samples to which titrated concentrations of PAD4 inhibitor have been added.

Example 7: Analysis of whole blood for ex vivo citrullination by endogenous PAD4 and inhibition of citrullination by a PAD4 inhibitor

This example relates to an ex vivo assay for the measurement of citrullination by endogenous PAD4 and its inhibition by a PAD4 inhibitor using whole blood samples. This assay design may also be used to measure citrullination by endogenous PAD2 and its inhibition by a PAD2 inhibitor.

A. Method i. Sample Collection and Preparation

Sample collection. Whole blood samples were collected from healthy subjects in sodium heparin vacutainer tubes or TruCulture® tubes. Various concentrations of PAD4 inhibitor (anti-PAD4 antibody hzl3-5 D31E) were titrated whole blood aliquots from each donor to generate titration curves.

Ex vivo citrullination in whole blood by endogenous PAD4. Fresh whole blood samples that had been treated with PAD4 inhibitor or not treated with PAD4 inhibitor (samples not treated with PAD4 inhibitor were treated with PBS or isotype control) were incubated (in the vacutainer tubes or TruCulture® tubes) on heat blocks at 37°C for 72 hours to trigger release of endogenous PAD4. Plasma was collected from sodium heparin tubes, supernatant was collected from TruCulture® tubes. Shorter incubation times (<72 h) were also tested (data not shown). With the 72 hour incubation, the supernatant separated without centrifugation; if short incubation times (e.g., 18 hours or less) are used or if non-incubated samples are tested, centrifugation can be used to separate supernatant. Centrifugation was used to obtain supernatant for control samples that were treated with PBS and not incubated. The plasma or supernatant samples were frozen prior to further preparation and proteomic analysis.

Preparation for Proteomic analysis. Frozen samples were thawed and further prepared for analysis by LC-MS to quantify citrullination at the sites specified in Table 7 above. Ten (10) pl of sample was thawed and diluted 15-fold with PBS. Sixty (60) pl of 15- fold diluted supernatant was used for depletion of human serum albumin using CUSTOM PhyTip 200 pL CaptureSelect™ Human Albumin tips (Biotage, San Jose, CA) containing 20 pL CaptureSelect™ human albumin affinity matrix. Twenty five (25) pl of the albumin- depleted sample was combined with 25 pl ist-BCT LYSE 2-fold buffer and heated for 20 minutes at 80°C. Samples were enzymatically digested with addition of trypsin and LysC according to manufacturer’s protocol. Peptides were cleaned up using iST-BCT kit (PreOmics, cat# P.O.00116). ii . Targeted Proteomic Analysis

Samples were analyzed by LC-MS and citrullination of specific peptides was analyzed and quantified as described in Example 6 above. Peptides assessed included those listed in Table 8. Changes in citrullination at specific citrullination sites were measured as in the Example 6.

B. Results

IC50 results (mean ± standard deviation) for two citrullinated peptides measured in ex vivo endogenous PAD4 assay on TimsTOF platform are listed in Table 8. These results illustrate detection of inhibition of citrullination by a PAD4 inhibitor.

Induction and inhibition of citrullination of GSN and FGA peptides is also shown in FIG. 3A and FIG. 3B, respectively. All results (Table 8 and Fig. 3A-B) were generated from measurements on nanoLC-TimsTOF platform.

The results show that this assay can be used to assess inhibition by a PAD4 inhibitor (e.g., Ab hzl3-5 D31E) using whole blood samples. The assay can be used, for instance, to assess citrullination in samples obtained from subjects before and/or after treatment with a PAD4 inhibitor or PAD2 inhibitor. This and other assays disclosed herein can be used to assess citrullination in subjects that are treated with an agent or considered for a treatment (e.g., treatment with a PAD2 and/or PAD4 inhibitor or another agent that affects citrullination and/or related biological mechanisms such as NETosis or METosis). Example 8: Targeted proteomic analysis of citrullinated peptides after immunocapture enrichment

This example describes a method for performing proteomic analysis of two particular citrullinated proteins, fibrinogen (FGA) and gelsolin (GSN). It further illustrates an immunocapture method that may be employed with other citrullinated proteins and citrullination targets of PAD2 as well as PAD4.

Immunocapture enrichment and trypsin digestion

Samples were collected in TruCulture® null tubes and treated as described in the Methods subsections of Example 7 titled “ Sample collection” and “Ex vivo citrullination in whole blood by endogenous PAD 4" . Before quantifying citrullinated and total FGA and GSN, the samples were immunoenriched using anti-FGA and anti-GELS monoclonal antibodies (mAb) immobilized on tosylated magnetic beads. The antibodies used for the immunoenrichment have high affinity to both citrullinated and non-citrullinated FGA and GSN.

TruCulture® supernatants were thawed and diluted 200- fold for FGA and 10-fold for GSN in a 96-well KingFisher® plate. 40 pL of beads containing 0.10 pg/pL of anti-FGA mAb (ab244636, Abeam) and 40 pL of beads containing 0.05 pg/pL anti-GSN mAb (ab247406, Abeam) were added to 200 pL of diluted sample. Samples were incubated for 60 min at 25 °C with shaking at 1000 rpm. After incubation, 96-well plate was placed on the KingFisher® robotic system. Beads were washed twice with 500 pL of PBST buffer, followed by one wash with 500 pL of PBS buffer. Proteins bound to the beads were eluted using 110 pL of 12 mM HC1 with 0.05% Zwittergent. Samples were neutralized with 10 pL of 500 mM NH4HCO3 buffer and transferred into a clean LoBind™ 96-well plate. Samples were incubated at 80 °C for 20 min to denature proteins. After cooling the plate down to room temperature, 2.0 pg of Promega trypsin (20 pL of 100 pg/mL trypsin solution in 100 mM NH4HCO3 buffer) was added to each sample. Samples were incubated at 50°C for 60 min with shaking at 500 rpms. After incubation, the digestion was stopped by the addition of 10 pL of the internal standard working solution in 10% formic acid in 80/20 water/acetonitrile.

HPLC-MS/MS conditions

The digested peptides were monitored with selective reaction monitoring on Sciex® 6500;/ mass spectrometer using a 15 min LC gradient. Chromatographic separation was performed on an ACQUITY UPLC HSS T3 100A, 1.8 pm, 2.1 mm x 50 mm column (Waters Corporation, MA, USA) with the column temperature set to 60 °C. Mobile phase A contained 0.1% formic acid (FA) in water and mobile phase B contained 0.1% FA in acetonitrile.

A gradient separation program was used as following: 0-0.5 min 2% B; 0.5-13.0 min 2-30% B; 13.0-13.1 min 30-95% B; 13.1-14.0 min hold at 95% B; 14.0-14.1 min 95-2% B; and the run was stopped at 15.0 min. The flow rate was set at 0.6 mL/min and the injection volume was 40 pL. The mass spectrometer was operated using positive ion electrospray ionization. The following optimized MS conditions were used: curtain gas and collision gas were set as 20 and 10; the turbo spray voltage was set at 5500 V and ion source gas 1 and gas 2 were both set at 50 psi. The probe temperature was set at 500°C. FGA and GSN peptides were quantified using SRM transitions of m/z 682.03 > 870.89 (CE: 29.7 eV), m/z 571.11 > 610.68 (CE: 29.4 eV), m/z 496.53 > 585.64 (CE 24.6 eV), and m/z 920.00 > 1135.21 (CE 42.0 eV) for QFTSSTSYNRGDSTFESK (SEQ ID NO: 81) (Cit-FGA signature), GSESGIFTNTK (SEQ ID NO: 86) (total FGA signature), ATASRGASQAGAPQGR (SEQ ID NO: 54) (Cit-GSN signature), and TPSAAYLWVGTGASEAEK(SEQ ID NO: 87) (total GSN signature), respectively.

Results

Representative results for FGA peptide QFTSSTSYNRGDSTFESK (SEQ ID NO: 81) measured in TruCulture® samples after the immunocapture enrichment described above are shown in FIG. 4. Percent (%) citrullination was calculated using the concentration ratio of citrullinated FGA to total FGA (total FGA includes modified and unmodified versions) that was multiplied by 100%.

The results showed that the assay detects citrullination and its inhibition by hzl3-5 D31E.

Example 9: Targeted proteomic analysis of peptides

To quantify citrullinated peptide concentrations and their related total protein concentrations in plasma samples from patients (e.g., rheumatoid arthritis (RA) patients), samples are immunoenriched using monoclonal antibodies (mAb) specific to the proteins to be analyzed, wherein the antibodies are immobilized on tosylated magnetic beads. The selected antibodies have high affinity to both citrullinated and non-citrullinated forms of the proteins.

The patient plasma samples are collected in sodium heparin tubes using standard blood collection procedures. Samples may be collected before and/or after patients are treated with a PAD inhibitor, such as a PAD2 and/or PAD4 inhibitor. Baseline samples collected before treatment (e.g., with a PAD inhibitor) may be subjected to ex vivo treatment (e.g., with the PAD inhibitor or isotype control), or one or more control treatments (e.g., PBS) as disclosed herein in other Examples for the purpose of assessing citrullination and determining IC50s.

The patient plasma samples are diluted for assessment of the proteins (such as 20- 1000 fold) in a 96-well KingFisher® plate. 40 pL of beads containing 0.10 pg/pL of a particular antibody specific for a first protein and 40 pL of beads containing 0.05 pg/pL of an antibody specific for a second protein are added to 200 pL of diluted plasma sample. Samples are incubated for 120 min at 25 °C with shaking at 1000 rpm. After incubation samples are processed and analyzed as described in Example 8.

Example 10: Targeted Analysis of Citrullination of EGA in Cultured Human Blood by IC-LC-MS/MS

A targeted analysis of citrullination of FGA in cultured human blood samples by IC- LC-MS/MS was developed for use in evaluating samples from subjects participating in clinical testing of PAD4 inhibitors such as anti-PAD4 antibodies. A similar assay may be developed for evaluation of samples from subjects receiving or intended for administration of a PAD2 inhibitor as well, utilizing a peptide significantly citrullinated by PAD2.

Summary and assay qualification experiments

Percent citrullination of FGA is measured in whole blood from normal, healthy volunteers in TruCulture® null tubes at 37 °C to trigger release of endogenous PAD4. Two samples are collected from each donor, one to be incubated at 37 °C for 72 hours (TruCulture-72h) and the other to be analyzed immediately (TruCulture-Oh), and which is expected to have low levels of endogenous PAD4. Each sample is diluted 200-fold in assay buffer, incubated with magnetic beads conjugated to anti-FGA antibody (Abeam catalog no. ab244636, rabbit monoclonal antibody to fibrinogen), after which beads are washed, and immunocaptured (IC) proteins are eluted with a low pH buffer. Beads are Dynabeads™ M- 280, tosylactivated, at 30 mg/mL (ThermoFisher catalog no. 14204). Trypsin was added to the samples to digest the FGA protein. Internal standards were spiked into all samples, and samples were analyzed by LC-MS/MS on an Acquity® UPL HCC T3 column (100 Angstrom, 1.8 pm, 2.1 mm x 50 mm) for liquid chromatography and a Sciex® TripleQuad™ 7500 system, operated in positive ion electrospray mode, with detection by MRM, for MS/MS analysis.

Citrullination was assessed at FGA peptide QFTSSTSYNRGDSTFESK (SEQ ID NO: 81), with the citrullination site arginine bold and underlined. The total FGA peptide GSESGIFTNT (SEQ ID NO: 88) was also assessed to determine total FGA. Internal standard peptides were QFTSSTSYNCitGDSTFESLys (SEQ ID NO: 89) (13C6; 15N2) for Cit-FGA internal standard, and GSESGIFTNTLys (SEQ ID NO: 86) (13C6,15N2) for total FGA internal standard.

For assay qualification and calibration, synthetic FGA peptides were added to samples over a concentration range of 4-1000 nM for Cit-FGA peptide and 40-10,000 nM for total FGA peptide to obtain a standard curve.

To address donor-to-donor variability, TruCulture® samples from 12 healthy donors at the 0 and 72 hour timepoints were analyzed. The % cit (percentage citrullination) varied from 0.12% to 0.62% in the TruCulture® Oh samples from the donors and varied from 0.51% to 3.65% in the TruCulture® 72h samples from the donors.

Clinical assay protocol

Peripheral blood samples are collected in TruCulture® Null tubes and incubated for 72 hours at 37 °C to boost citrullination. TruCulture® supernatant is separated from blood cells after incubation (TruCulture-72h). Representative samples with low citrullinated FGA concentrations are obtained by processing TruCulture® Null samples immediately after collection (TruCulture-Oh). Samples for method qualification are collected at Discovery Life Sciences (DLS). Only Truculture-72h samples are collected from clinical subjects for testing. Samples are collected pre- and post-dose of PAD4 inhibitor to measure inhibition of FGA citrullination after each dose. Citrullinated FGA (Cit-FGA) concentrations are expected to decrease with increasing doses of PAD4 inhibitor.

Cit-FGA and total FGA are quantified by IC-LC-MS/MS assay as described above. % citrullination is calculated as follows: [Cone. Cit-FGA (nM)/ Cone. Total FGA (nM)] X 100%.

Example 11: Therapeutic PAD4 antibody maintained potency in presence of endogenous PAD4 antibodies from rheumatoid arthritis patients

A. Introduction

PAD4 is released extracellularly in the inflamed joint through neutrophil activation, NETosis and cell death. PAD enzymes catalyze the modification of an arginine into a citrulline residue and drive the formation of citrullinated neoantigens that are recognized by anti-citrullinated peptide antibodies (ACPA), which are a hallmark of rheumatoid arthritis. Formation of immune complexes between ACPAs and citrullinated proteins are believed to drive tissue damage and perpetuate inflammation. Besides ACPA, about 25-35% of RA patients express anti-PAD4 IgG, of which about 20-40% cross-react between PAD3/PAD4 and have the potential to enhance PAD4 activity leading to more erosive disease. In this Example, the impact of the presence of endogenous anti-PAD4 antibodies on the potency of antibody hzl3-5 D3 IE was investigated in serum or with purified immunoglobin G (IgG).

B. Materials and methods a. Serum samples

The samples were serum samples from rheumatoid arthritis (RA) patients with established disease. Serum from healthy controls (NHV) was collected in BD Vacutainer SST Blood collection tubes (Cat#367988). All serum samples were frozen until aliquoting in plates. b. PAD4 antibody ELISA

Serum anti-PAD4 autoantibodies were measured using PAD4 Autoantibody ELISA kit (500930) from Cayman Chemicals following manufacturer's protocol. Serum samples were tested at 1 : 150 dilution in an assay buffer provided in the ELISA kit. c. IgG purification

Human IgG was purified using Melon™ gel IgG Spin Purification Kit (45206) from Thermo Scientific. Briefly, 500 pl purification gel was loaded to spin columns and washed twice with 300 pl of purification buffer. 80 pl of serum was diluted 5-fold with purification buffer and added to purification gel containing spin columns. Columns were mixed endover-end for 5 minutes and centrifuged to collect purified IgG. Concentration of purified IgG was determined using NanoDrop™ One instrument from Thermo Scientific. d. Citrullinated H3 enzymatic assay

All reagents were calculated for 100 pl final reaction. In 42.5 pl volume, recombinant PAD4 (Cayman #10500) was pre-incubated with purified IgG on ice for 45 minutes. To produce inhibition curves, 7.5 pl of hzl3-5 D31E serial dilutions in PBS or isotype control were added to the PAD4 / IgG mix and incubated on ice for an additional 45 minutes. Histone H3 in buffer containing calcium chloride was added, and the reaction (final concentrations: 13.5 nM PAD4, 2 mM CaC12, 10 pg/ml H3, 100 pg/ml purified IgG) was incubated at 37° C in an incubator for 2 hours. The H3 citrullination reaction was stopped with 10 pl of 0.5M EDTA. Citrullinated histone H3 was measured using Citrullinated Histone H3 (Clone 11D3) ELISA kit (501620) from Cayman Chemicals following manufacturer's protocol. Citrullinated samples were tested @ 1 : 100 dilution in an assay buffer provided in the ELISA kit. e. Citrullination induction in serum

To produce inhibition curves, 7.5 pl of hzl3-5 D31E serial dilutions in PBS or isotype control were added to 80 pl of thawed serum in plates, mixed, incubated at 37 °C in CO2 incubator for 6h then stored overnight at -80 °C. Plates containing serum mixed with antibody were thawed and 12.2 pl of PAD4 enzyme reaction mix (13.5 nM PAD4, 100 mM Tris, 2mM DTT final) was added and incubated at 37°C for 2h. Reactions were stopped with 10 pl of 0.5 M EDTA. Plates were sealed and stored at -80 °C. f. Measure of citrullinated peptides by LC-MS

Ten (10) pl of serum was thawed and diluted 15-fold with PBS. Sixty (60) pl of 15- fold diluted plasma was used for depletion of human serum albumin using CUSTOM PhyTip 200 pL CaptureSelect™ Human Albumin tips (Biotage, San Jose, CA) containing 20 pL CaptureSelect™ human albumin affinity matrix. Five (5) pl of albumin-depleted serum was combined with 45 ul LYSE BCT buffer and heated for 20 minutes at 80 °C. Samples were enzymatically digested with addition of trypsin and LysC according to manufacturer’s protocol. Peptides were cleaned up using preOmics® BCT kit (cat# P.O.00116).

Peptides were analyzed by nanoLC-MS on Bruker’s timsTOF of mass spectrometer using a 30-minute prm-PASEF method. Chromatographic separation was done using Bruker’s NanoElute™ LC on AURORA ELITE (AUR-15075C18-CSI) column. Data dependent acquisition (DDA) data was acquired for samples with high PAD4 concentrations with spiked internal standards (heavy peptides) for each peptide of interest. Skyline® software was used to generate a list of cit-peptide (citrullinated peptide) and unmodified peptide sequences from cit-proteins previously identified by global proteomics experiments. Next, the library in Skyline® was created using MaxQuant® generated msms.txt; mqpar.xml, and modifications. xml files. Ion mobility library was created after importing DDA data and using the imported data to generate a spectra library. Three parameters for each analyte were exported from Skyline® to build a prm-PASEF method in TimsTOF Control 3.0 software: retention time (RT); Ion Mobility (IM), and precursor m/z. Data was analyzed using Skyline® workflow. g. Calculation of IC50

IC50 calculations and statistics were performed in GraphPad® Prism software. Citrullinated H3 or % citrullination were plotted against the log of the concentration of the antibody and a non linear fit (Hill Slope = -1) was performed.

C. Results a. PAD4 antibody status

The presence of endogenous PAD4 autoantibodies was evaluated in the serum samples from the 21 RA patients and 10 healthy controls (NHV) by ELISA. On average the OD was significantly higher in RA than in NHV (1.3 in RA vs 0.38 in NHV, p<0.0001, Mann Whitney T test). Using 1 as a cutoff for positivity, 13 out of 21 (62%) RA sera and none of the NHV sera could be considered anti-PAD4 IgG+. FIG. 49A shows serum anti- PAD4 autoantibodies measured by OD450 by ELISA in 21 RA and 10 NHV purified IgG. b. Hzl3-5 D3 IE potency in the presence of purified IgG

Potency of hzl3-5 D31E on inhibition of H3 citrullination in the presence of 100 ug/ml purified IgG from the 21 RA and 6 NHV sera was evaluated (3 plates containing each 7 RA and 2 NHV donors). hzl3-5 D3 IE inhibited in vitro PAD4 driven H3 citrullination in the presence of purified IgG and there was no difference in IC50 whether purified IgG came from RA or NHV sera (FIG. 49B, Table 28), despite the fact that a large proportion of RA IgG had anti-PAD4 autoantibodies. The mean IC50 (nM) for the RA sera (n = 21) was 8.2 (standard deviation of 1.3), while the mean IC50 (nM) for the NHV sera (n = 6) was 9.0 (with standard deviation 2.5). c. Hzl3-5 D3 IE potency in serum from RA patients

Serum from 21 RA and 10 NHV donors was incubated with 13.5 nM PAD4 to induce citrullination, without or with different doses of hzl3-5 D3 IE. Citrullination of 5 selected peptides for proteoglycan 4 (PRG4), fibrinogen A (FGA), Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha- 1-microglobulin/bikunin precursor (AMBP) and gelsolin (GSN) was measured by LC-MS and reported as a percent of citrullinated over total peptide. Citrullination of these peptides was observed in all samples with inter-donor variability. hzl3-5 D3 IE inhibited citrullination of each of these peptides in a dose dependent manner and potency similar between NHV and RA (Table 9, no statistical differences between NHV and RA by Mann Whitney test). In addition, there was no correlation between any of the IC50s and the presence of anti-PAD4 autoantibodies (Spearman, not shown), as measured by ELISA (FIG. 5 A), suggesting that the presence of endogenous PAD4 antibodies do not affect potency of hzl3-5 D31E. Table 9 shows IC50 of hzl3-5 D31E on citrullination of 5 serum peptides from RA or NHV donors. SD refers to the standard deviation.

Table 9

SEQ ID NO Peptide sequence IC50 IC50

Protein (citrullination sites that were (nM) (nM) assessed are bold and underlined) mean mean

(SD) for (SD) for

RA NHV

(n=21) (n=10)

PRG4 80 AITTRSGQTLSK 4.9 (3 5) 4.4 (2 3)

FGA 81 QFTSSTSYNRGDSTFESK 4.5(1 6) 5.5(2 3)

ITIH4 21 QLGLPGPPDVPDHAAYHPFRR 5.2(1 5) 5.7(2 6)

AMBP 28 GPCRAFIQLWAFDAVK 4.3(1 0) 5.1(2 1)

GSN 54 ATASRGASQAGAPQGR 7.1(3 6) 7.7(9 3)

With two independent assays, no shift in potency of hzl3-5 D3 IE in the presence of endogenous anti-PAD4 autoantibodies was observed in this set of 21 RA serum samples.

Example 12: Targeted proteomic analysis of citrullinated PRG4 after immunocapture enrichment of citrullinated signature peptide

This example describes a method for performing targeted analysis of citrullinated proteoglycan 4 (PRG4).

A. Materials and methods

Plasma samples from normal healthy volunteers and rheumatoid arthritis patients were analyzed in this example. To obtain plasma, whole blood samples were collected in sodium heparin vacutainer tubes and plasma was isolated from the whole blood samples using standard methods. All plasma samples were frozen until aliquoting into plates for sample preparation for analysis. Cit-PRG4 concentrations in plasma samples from 30 rheumatoid (RA) patients and 10 normal healthy volunteers (NHV) were analyzed.

1. Sample preparation for HPLC-MS/MS

Before quantifying Cit-PRG4, plasma proteins were denatured, trypsin-digested, and cit-peptide of interest (AITTRSGQTLSK)(SEQ ID NO: 80) was immunocaptured with rabbit anti-human PRG4 (1384-1397) polyclonal antibody (pAb). The antibody used for the immunoenrichment was custom generated by immunizing rabbits with (KLH)-C- TARAITTRSGQTLS, where KLH is a carrier protein keyhole limpet hemocyanin.

Twenty-five pL of each plasma sample was aliquoted into a 96-well KingFisher® plate and 225 pL of 50 mM ammonium bicarbonate in water was added to dilute samples 10- fold. Proteins in the diluted samples were denatured for 20 min at 80 °C and digested with 5 pg of trypsin per sample. After digestion, trypsin was heat inactivated for 10 min at 95 °C. To immunocapture AITTRSGQTLSK (SEQ ID NO: 80), samples were cooled to room temperature and 40 pL of beads containing 0.20 pg/pL of anti-PRG4 pAb immobilized on tosylated magnetic beads were added to each well. After incubation for 60 min at 25 °C with shaking at 1000 rpm, 96-well plate was placed on the KingFisher® robotic system. Beads were washed twice with 500 pL of PBST buffer, followed by one wash with 500 pL of PBS buffer. Peptides bound to the beads were eluted using 115 pL of 12 mM HC1 with 0.05% Zwittergent. Ten pL of the internal standard working solution in 80/20 water/acetonitrile with 10% formic acid was added to each well, samples were mixed, transferred to a clean LoBind™ 96-well plate, and analyzed by LC-MS.

2. HPLC-MS/MS conditions

The digested surrogate peptide AITTKSGQTLSK (SEQ ID NO: 90) was monitored with selective reaction monitoring (SRM) on Sciex® 7500 mass spectrometer using a 7 min LC gradient. Chromatographic separation was performed on an AC QUIT Y UPLC HSS T3 100 A, 1.8 pm, 2.1 mm x 50 mm column (Waters Corporation, MA, USA) with the column temperature set to 60 °C. Mobile phase A contained 0.1% formic acid (FA) in water and mobile phase B contained 0.1% FA in acetonitrile. A gradient separation program was used as following: 0-1.0 min 2% B; 1.0-4.9 min 2-20% B; 4.9-5.0 min 20-95% B; 5.0-6.0 min hold at 95% B; 6.0-6.1 min 95-2% B; and the run was stopped at 7.0 min. The flow rate was set at 0.6 mL/min and the injection volume was 30 pL. The mass spectrometer was operated using positive ion electrospray ionization. The following optimized MS conditions were used: curtain gas and collision gas were set as 40 and 12; the turbo spray voltage was set at 2500 V and ion source gas 1 and gas 2 were both set at 55 psi. The probe temperature was set at 650°C. AITTKSGQTLSK (SEQ ID NO: 90) was quantified using SRM transition of m/z 422.14 > 540.59 (CE: 18.3 eV). B. Results

Comparison of Cit-PRG4 (surrogate peptide AITTKSGQTLSK) (SEQ ID NO: 90) concentrations measured in RA and NHV plasma samples is shown in FIG. 6. These results indicate that citrullination of PRG4 is higher in RA patients than in normal healthy volunteers.

Example 13: Citrullinome Study Performed With Human Plasma Enriched by Strong Anion Exchange (SAX) Chromatography

The following study was the first scaled citrullinome study performed utilizing enriched human plasma.

A. Proteome enrichment of plasma from normal, healthy volunteers, incubated with exogenous PAD2 or PAD4 via strong anion exchange

Strong ion exchange chromatography (SAX) was used to enrich human plasma to enrich the proteome and obtain a deeper citrullinome, and to deplete high-abundance plasma proteins such as albumin. 50 pl of treated plasma from normal, healthy volunteers was transferred to a sterile KingFisher® 96 deep-well plate (Thermo Cat No 95040460) with 50 pl 100 mM Bis-Tris Propane, pH 6.3, 150 mM NaCl. 13 pl SAX beads (MagReSyn® MR- SAXO 10) were washed two times in 1 mL 50 mM Bis-Tris Propane, pH 6.5, 150mM NaCl before being transferred to the plate containing the treated plasma. There, the beads were incubated for 34 minutes alternating slow mixing on and off again. The beads were then washed three times, 5 min each, in 1 mL 50 mM Bis-Tris Propane, pH 6.5, 150mM NaCl mixing slowly. Finally, the beads were released into 100 pl BCT-LYSE buffer from the Preomics® ist-BCT 96 sample kit in conjunction with their Phoenix™ cartridge for peptide cleanup (Preomics P.O.00099).

B. Enriched Plasma Tryptic Digestion

The plates containing the beads in BCT-LYSE buffer were heat sealed and warmed to 60°C shaking at 1000 RPM for lOmin (Eppendorf® Thermomixer EP5382000023). After plates were allowed to cool back to room temperature, BCT-DIGEST solution was added to each well as described by the manufacturer and lysates were digested at 37°C for 18 hours. 200 pl of BCT-STOP solution was added to each well and peptides were transferred to the Preomics® Phoenix™ Cartridge plate. The procedure then followed manufacturer guidelines. Eluate was captured in a white, round bottom polyproylene plate (Thermo Cat. No 267350), and peptide concentration was measured by tryptophan fluorescence (TEC AN MPLEX 200), Excitation 295nm:5nm bandwidth, Emission 350nm:20nm bandwidth. Samples were then dried via SpeedVac™. Before injection, peptides were resuspended to 50 ng/pl in Optima™ Water+0. l%Formic Acid and 1000 ng were loaded onto EVOSEP Evotip® Pure tips (EVOSEP EV-2018).

C. Mass Spectrometry and Data Analysis Peptides were loaded onto a 15cm reverse phase column (Bruker 1893474) (15cm x

150pm ID, 1.5pm C18) joined to a 20pm ZDV captive spray emitter (PR10781883-V2). The column was maintained at 40°C. An EVOSEP One (EV- 1000) System (EVOSEP) was directly coupled online with the mass spectrometer (Bruker timsTOF Pro2) via electrospray source set to 1500V. Peptides were separated with a binary buffer system of Buffer A (0.1% Formic Acid in Water) and Buffer B (0.1% Formic Acid in Acetonitrile) using the EVOSEP 30SPD method. Data was collected in Data Independent Parallel Accumulation Serial Elution Fragmentation mode (DIA-PASEF) with 100ms accumulation. DIA windows are shown in Table 10. Table 10: Data Independent Parallel Accumulation Windows:

Collisional energy was stepped tracking ion mobility: 0.85 V*s/cm² (31eV) to 1.3 V*s/cm² (57eV).

Data was searched against the Human SwissProt™ Proteome using the software DIA- NN 1.8.1 (https://github.com/vdemichev/DiaNN) with the following parameters: Variable modifications were maxed at 1 per peptide and included N-term Met Excision, Cys Carbamidomethylation, and Ox(Met), Citrullination on Arg, and deamination on Asn and Glu. Missed cleavages were set to max 1. Precursors and fragment ion m/z range was 200- 1,700 with a 1.0% FDR. Mass accuracy and MSI Accuracy was set to lOppm, and match between runs and Smart Profiling was enabled. Neural network was set to single-pass mode and protein inferences were turned off. Label free quantification strategy implemented the Robust LC (high precision) algorithm. The spectral library utilized for this search was a bespoke library generated from pooled enriched samples fractionated on a high pH Cl 8 column.

D. Bioinformatics Analysis and Results

Charge State Aggregation: To address peptide identification across multiple charge states, three distinct aggregation strategies were implemented across experimental runs. A summation-based approach was used where intensities from multiple charge states of the same modified sequence were directly summed to create a single peptide-level intensity value.

Feature Filtering and Quality Assessment: Citrullinated peptides were identified using the UniMod:7 modification signature pattern in the modified sequence, excluding peptides with terminal citrullination patterns. Quality control metrics included signal-to-noise ratios with a threshold of 3, and features with >50% missing values across all experimental groups were excluded from downstream analysis.

Data Normalization and Batch Effect Correction:

Missing Value Imputation: A hybrid imputation strategy combining Bayesian Principal Component Analysis (bPCA) and minimum detection (MinDet) methods was implemented. Accurate variance estimation in differential protein expression analysis requires consideration of the inherent dependence of protein variance on the number of peptide spectrum matches used for quantification. Group-specific bPCA imputation was performed with the number of principal components set to min(sample size - 1, 6) to account for experimental design constraints. Missing values not imputed by bPCA were subsequently imputed using MinDet with a quantile threshold of 0.01, representing values below the limit of detection.

Batch Effect Removal: Limma's removeBatchEffect function was employed to correct for donor-specific effects, as donor variability represented the primary source of unwanted variation in the experimental design. Batch-corrected data were used for visualization purposes while preserving the original variance structure for statistical modeling.

Differential Expression Analysis:

Linear Mixed Models: Differential expression analysis was performed using the limma framework with linear mixed-effects models to account for the hierarchical structure of the data. The model design included: ~ 0 + Group + Donor. Where Group represents the treatment conditions (PAD enzyme combinations and inhibitor treatments) and Donor accounts for individual biological variation. The limma method uses an empirical-Bayes approach to shrink protein-wise sample variance toward a common estimate, providing improved statistical power compared to standard t-tests.

Technical Replicate Handling: Duplicate correlation analysis was performed to estimate the correlation between technical replicates within the same biological sample, with consensus correlations ranging from 0.26 to 0.40 across experimental runs. This correlation structure was incorporated into the linear model fitting process.

Contrast Analysis: A comprehensive contrast matrix was constructed to evaluate:

• Enzyme induction effects: PAD2, PAD4, and dual PAD2+PAD4 versus naive conditions

• Inhibitor efficacy: Antibody treatments versus isotype controls within each enzyme condition

• Interaction effects: Unique effects of dual antibody treatment versus individual antibody effects

Statistical significance was assessed using moderated t-statistics with Benjamini-Hochberg false discovery rate correction (adjusted p-value < 0.05).

Downstream Analysis for Marker Selection:

Quantitative Assessment of Citrullination: Relative citrullination levels were quantified using the ratio of citrullinated peptide intensity to total protein intensity (PR2PG ratio), calculated as:

PR2PG ratio = (2^rcilru Hi naled _peptide intensity) / (2^rlolal protein intensity)

This metric provided a normalized measure of site-specific citrullination that accounts for total protein abundance variations.

PAD Enzyme Specificity Analysis: Enzyme preference was determined by calculating fold-change ratios between PAD2 and PAD4 individual treatments:

• PAD2 preference: FC(PAD2)/FC(PAD4) > 2

• PAD4 preference: FC(PAD4)/FC(PAD2) > 2

• Common targets: Similar fold-changes between both enzymes

E. Results

This was the first citrullinome study performed with enriched plasma. This was also the first comprehensive list of citrullination sites identified in plasma and matched to a cognate enzyme.

The increased proteome depth of the magnetic bead enrichment yielded 192 sites of citrullination from human plasma. Of these sites, 116 showed a >2X fold preference by PAD4 while 38 were preferred substrates of PAD2. The rest were deemed as common substrates. The citrullination sites identified in this study can be used to track activity of PAD2 and/or PALM in healthy, diseased, or clinically suspect subjects.

By combining the enrichment protocol with the ist-BCT cleanup as described, the depth of the citrullinome was unparalleled (192 citrullination sites) and the throughput was scaled to analyze hundreds of human plasma samples per day. Furthermore, the cleanup removed residual salts from the biofluid itself as well as those introduced during the tryptic digestion process, which allowed for MS analysis without intermittent instrument cleaning, which is otherwise common with biofluid samples. Citrullination sites identified in this Example are shown in Table 11. Table 11 shows particular arginine (R) residues in each peptide or protein that were citrullinated, using bold face and underlining. The peptides in the tables below also in some cases were found to also include chemical modifications at other residues, such as oxidation of methionine (M), carbamidomethylation at certain cysteine (C) residues, or modifications at the N-terminus of the peptide, which are noted by underlining those residues, or in the case of a modification at the N-terminus, underlining the first residue in the peptide sequence. Each row of the tables provides the protein of which the peptide is a fragment, by name and abbreviation, as well as a UniProt accession number for the UniProt database (www.uniprot.org) providing the complete protein sequence.

I l l

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DESCRIPTION OF THE SEQUENCES

The following table provides a listing of certain sequences referenced herein. In the peptide sequences of the table below, the Description column provides the protein acronym, Uniprot (www.uniprot.org) reference number, and the residue position of the citrullination site shown in the peptide in bold/underlining from the Uniprot sequence of the protein. In some cases, other peptide residues are also modified (e.g., oxidized or the like), and the existence of a modification at another residue is denoted by underlining.

Claims

What is Claimed is:

R342 of carboxypeptidase B2 (CPB2; corresponding to R342 of Q96IY4), R239 of apolipoprotein A-l (APO Al; corresponding to R239 of P02647), R198 of apolipoprotein E (APOE; corresponding to R198 of P02649), R337 of alpha-2-HS-glycoprotein (AHSG; corresponding to R337 of P02765), R212 of apolipoprotein A-l (APO Al; corresponding to R212 of P02647), R173 of apolipoprotein A-l (AP0A1; corresponding to R173 of P02647), R108 of apolipoprotein E (APOE; corresponding to R108 of P02649), R383 of prothrombin (F2; corresponding to R383 of P00734), R362 of vitronectin (VTN; corresponding to R362 of P04004),

R155 of apolipoprotein A-IV (AP0A4; corresponding to R155 of P06727), R306 of apolipoprotein A-IV (AP0A4; corresponding to R306 of P06727), R101 of vitamin K-dependent plasma glycoprotein (PROS1; corresponding to RIO 1 ofP07225),

R496 of albumin (ALB; corresponding to R496 of P02768),

R315 of complement C3 (C3; corresponding to R315 of PO 1024),

R194 of clusterin (CLU; corresponding to R194 and/or R198 of P10909), or

R198 of clusterin (CLU; corresponding to R194 and/or R198 of Pl 0909).

2. The method of claim 1, wherein the biological sample has been exposed to a PAD2 modulator such as a PAD2 inhibitor.

R342 of carboxypeptidase B2 (CPB2; corresponding to R342 of Q96IY4), R239 of apolipoprotein A-l (APO Al; corresponding to R239 of P02647), R198 of apolipoprotein E (APOE; corresponding to R198 of P02649), R337 of alpha-2-HS-glycoprotein (AHSG; corresponding to R337 of P02765), R212 of apolipoprotein A-l (APO Al; corresponding to R212 of P02647), R108 of apolipoprotein E (APOE; corresponding to R108 of P02649),

R383 of prothrombin (F2; corresponding to R383 of P00734),

R362 of vitronectin (VTN; corresponding to R362 of P04004),

R496 of albumin (ALB; corresponding to R496 of P02768),

R315 of complement C3 (C3; corresponding to R315 of PO 1024),

4. The method of claim 3, wherein the PAD2 modulator is a PAD2 inhibitor.

5. The method of claim 3 or 4, wherein the PAD2 modulator reduces citrullination at the citrullination site in a dose-dependent manner.

6. The method of any one of claims 1-5, wherein the method comprises assessing citrullination of a peptide fragment of the protein, wherein the peptide fragment comprises the citrullination site.

7. The method of any one of claims 2-6, wherein the biological sample has been exposed to a PAD2 inhibitor in vivo in a subject as a result of the PAD2 inhibitor being administered to the subject.

8. The method of any one of claims 2-7, wherein the biological sample has been exposed to the PAD2 inhibitor by contacting the biological sample with the PAD2 inhibitor ex vivo.

9. The method of any one of claims 1-8, wherein the assessing citrullination of the citrullination site comprises mass spectrometry (MS).

10. The method of any one of claims 1-8, wherein the assessing citrullination of the citrullination site comprises liquid chromatography and mass spectrometry (LC-MS).

11. The method of any one of claims 1-8, wherein the assessing citrullination comprises selective reaction monitoring and chromatographic separation.

12. The method of any one of claims 1-11, wherein assessing citrullination of the citrullination site comprises measuring, in the biological sample, a first concentration of the citrullinated protein or peptide fragment thereof and a second concentration of corresponding total protein.

13. The method of claim 12, wherein assessing citrullination of the citrullination site comprises determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.

14. The method of claim 13, wherein the method comprises comparing the citrullination ratio to a reference citrullination ratio.

15. The method of claim 14, wherein the reference citrullination ratio is a citrullination ratio determined for a control biological sample.

16. The method of claim 15, wherein the control biological sample is a biological sample that: (a) has not been exposed to a PAD2 modulator or PAD2 inhibitor, (b) is from the same subject, and/or (c) is from the same subject prior to treatment with the PAD2 modulator.

17. The method of any one of claims 14-16, wherein the assessing comprises determining a difference between the citrullination ratio for the biological sample and the reference citrullination ratio.

18. The method of any one of claims 1-17, wherein the method comprises contacting the biological sample with exogenous PAD2.

19. A method comprising:

(i) contacting a biological sample from a subject with exogenous PAD2, and

(ii) assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample; wherein the citrullination site is selected from one or more of the following:

R342 of carboxypeptidase B2 (CPB2; corresponding to R342 of Q96IY4),

R239 of apolipoprotein A-l (APO Al; corresponding to R239 of P02647),

R198 of apolipoprotein E (APOE; corresponding to R198 of P02649),

R337 of alpha-2-HS-glycoprotein (AHSG; corresponding to R337 of P02765),

R212 of apolipoprotein A-l (APO Al; corresponding to R212 of P02647), R173 of apolipoprotein A-l (AP0A1; corresponding to R173 of P02647), R108 of apolipoprotein E (APOE; corresponding to R108 of P02649), R383 of prothrombin (F2; corresponding to R383 of P00734), R362 of vitronectin (VTN; corresponding to R362 of P04004),

R155 of apolipoprotein A-IV (AP0A4; corresponding to R155 of P06727),

R306 of apolipoprotein A-IV (AP0A4; corresponding to R306 of P06727),

R496 of albumin (ALB; corresponding to R496 of P02768), R194 of clusterin (CLU; corresponding to R194 and/or R198 of P10909), or R198 of clusterin (CLU; corresponding to R194 and/or R198 of Pl 0909).

20. The method of claim 18 or 19, wherein a concentration of 1-5 nM, 2-4 nM, 1 nM, 2 nM, 3 nM, 4 nM, or 5 nM exogenous PAD2 is added to the biological sample.

21. The method of claim 18 or 19 or 20, wherein the method comprises incubating the exogenous PAD2 with the biological sample.

22. The method of claim 21, wherein the method comprises incubating the exogenous PAD2 with the biological sample at a temperature of 36-38 °C.

23. The method of claim 21 or 22, wherein the incubating is for 1-3 hours.

24. The method of any one of claims 21-23, wherein the method comprises adding EDTA to the biological sample after the incubation with exogenous PAD2.

25. The method of any one of claims 1-24, wherein the method comprises incubating the biological sample for an incubation period before assessing citrullination.

26. The method of claim 25, wherein the incubating is performed at 35-40°C.

27. The method of claim 25 or 26, wherein the incubation period is 48 to 96 hours.

28. The method of any one of claims 1-27, comprising preparing the biological sample for assessment prior to the assessing.

29. The method of claim 28, wherein the preparing comprises enzymatically digesting proteins in the biological sample.

30. The method of claim 28 or 29, wherein the preparing comprises depleting proteins from the biological sample that are not targeted by the assessing.

31. The method of any one of claims 28-30, wherein the preparing comprises diluting the biological sample.

32. The method of any one of claims 28-31, wherein the preparing comprises denaturing proteins in the biological sample.

33. The method of any one of claims 28-32, wherein the preparing comprises enriching the biological sample for a protein or peptide fragment thereof that comprises the citrullination site to be assessed.

34. The method of claim 33, wherein the preparing comprises contacting the biological sample with an antibody that specifically binds to citrullinated and noncitrullinated forms of the protein or peptide fragment thereof that comprises the citrullination site to be assessed.

35. The method of any one of claims 28-34, wherein the method comprises enriching the biological sample for membrane-bound proteins and peptide fragments thereof prior to the assessing.

36. The method of any one of claims 28-35, wherein the method comprises contacting the biological sample with a strong anion exchange (SAX) chromatography medium.

37. The method of claim 36, wherein the SAX chromatography medium comprises particles.

38. The method of claim 36, wherein the particles comprise magnetic particles.

39. The method of any one of claims 36-38, wherein the SAX chromatography medium enriches membrane-bound proteins and peptide fragments thereof in the sample.

40. The method of any one of claims 36-39, wherein the method further comprises eluting proteins or peptide fragments thereof bound to the SAX chromatography medium and assessing citrullination of the eluted proteins or peptide fragments thereof.

41. The method of any one of claims 35-40, comprising enzymatically digesting proteins in the biological sample following the enriching or the contacting of the biological sample with the SAX chromatography medium or following the eluting of the proteins or peptide fragments thereof from the SAX chromatography medium.

42. The method of any one of claims 1-41, wherein the method comprises freezing and thawing the biological sample before assessing citrullination of the citrullination site.

43. The method of any one of claims 1-42, comprising:

44. The method of claim 43, wherein the immunoenriching comprises contacting the sample with an immobilized antibody that binds to both citrullinated and noncitrullinated forms of the protein and/or to both citrullinated and noncitrullinated forms of the peptide fragment thereof comprising the citrullination site, and eluting protein and/or peptide bound to the immobilized antibody.

45. The method of claim 43 or 44, wherein the enzymatically digesting is conducted before the immunoenriching.

46. The method of claim 43 or 44, wherein the enzymatically digesting is conducted after the immunoenriching.

47. The method of any one of claims 43-46, wherein the immunoenriching comprises incubating the sample with the immobilized antibody for at least 30 minutes.

48. The method of claim 47, wherein the incubating is at a temperature of 22-28°C.

49. The method of claim 47 or 48, wherein the incubating is for a period of 30 to 90 minutes.

50. The method of any one of claims 43-49, wherein the immunoenriching comprises shaking during the incubating.

51. The method of any one of claims 44-50, wherein the immunoenriching comprises removing the immobilized antibody from the sample and washing the immobilized antibody prior to the eluting.

52. The method of any one of claims 44-51, wherein the eluting comprises washing the immobilized antibody with an elution composition under acidic conditions.

53. The method of claim 52, wherein the elution composition comprises a detergent.

54. The method of claim 53, wherein the detergent is a zwitterionic detergent.

55. The method of any one of claims 44-54, the method comprises neutralizing eluted protein in a buffer prior to assessing, and optionally prior to enzymatically digesting.

56. The method of any one of claims 43-55, wherein the antibody is immobilized by attachment to a solid surface.

57. The method of any one of claims 43-56, wherein the assessing citrullination comprises measuring a first concentration of citrullinated peptide in the digested peptides and a second concentration of signature peptide in the digested peptides, and optionally determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.

58. The method of any one of claims 43-57, comprising denaturing proteins in the immunoenriched sample prior to the assessing.

59. The method of any one of claims 44-58, comprising denaturing the eluted protein prior to the assessing.

60. The method of claim 59, comprising denaturing the eluted protein prior to enzymatically digesting the eluted protein.

61. The method of any one of claims 43-60, comprising diluting the plasma or serum sample prior to the immunoenriching and performing the immunoenriching on the sample that has been diluted.

62. The method of claim 61, comprising diluting the plasma or serum sample by 2 to 1000 fold.

63. The method of claim 61 or 62, wherein the sample that has been diluted has a volume of at least 5 pl.

64. The method of any one of claims 1-63, wherein the sequence of the peptide fragment comprises one of the following sequences, wherein the citrullination site is designated by underlining of R residues and wherein modifications of non-arginine residues are designated by underlining of the modified residues: DHEELSLVASEAVRAIEK;

IVEGSDAEIGMSPWQVMLFRK; IYISGMAPRPSLAK; RQLTPYAQR;

RVEPYGENFNK; SFQTGLFTAARQSTNAYPDLR;

65. The method of any one of claims 1-64, wherein the biological sample comprises whole blood, plasma, serum, or blood supernatant.

66. The method of any one of claims 1-64, wherein the biological sample comprises synovial fluid.

67. The method of any one of claims 64-66, wherein the peptide fragment comprises the sequence of: DHEELSLVASEAVRAIEK; AKPALEDLRQGLLPVLESFK;

ERLGPLVEQGR; or HTFMGVVSLGSPSGEVSHPRKT (corresponding to SEQ ID NOs: 14, 6, 10, or 11, respectively).

68. The method of any one of claims 1-67, wherein the method comprises assessing citrullination of two or more proteins or peptide fragments thereof.

69. The method of any one of claims 1-68, wherein the method further comprises assessing citrullination of at least one additional citrullination site listed in Table 1.

70. The method of any one of claims 1-68, wherein the method further comprises assessing citrullination of at least one additional citrullination site listed in Table 11.

71. A method comprising:

72. A method comprising:

73. A method comprising

(b) eluting protein bound to the immobilized antibody,

74. A method comprising

(b) immunoenriching the enzymatically digested biological sample by contacting the sample with an immobilized antibody that specifically binds to both citrullinated and noncitrullinated forms of a peptide fragment of a protein of interest, the peptide fragment comprising a citrullination site, (c) eluting peptides bound to the immobilized antibody, and

75. The method of any one of claims 71-74, wherein the immunoenriching comprises incubating the sample with the immobilized antibody for at least 30 minutes.

76. The method of claim 75, wherein the incubating is at a temperature of 22-28°C.

77. The method of claim 75 or 76, wherein the incubating is for a period of 30 to 90 minutes.

78. The method of any one of claims 71-77, wherein the immunoenriching comprises shaking during the incubating.

79. The method of any one of claims 73-77, wherein the immunoenriching comprises removing the immobilized antibody from the sample and washing the immobilized antibody prior to the eluting.

80. The method of any one of claims 73-79, wherein the eluting comprises washing the immobilized antibody with an elution composition under acidic conditions.

81. The method of claim 80, wherein the elution composition comprises a detergent.

82. The method of claim 81, wherein the detergent is a zwitterionic detergent.

83. The method of any one of claims 80-82, the method comprises neutralizing eluted protein or peptide in a buffer prior to assessing, and optionally prior to enzymatically digesting.

84. The method of any one of claims 71-83, wherein the antibody is immobilized by attachment to a solid surface.

85. The method of any one of claims 71-84, wherein the assessing citrullination comprises measuring a first concentration of a citrullinated peptide and a second concentration of a signature peptide in the enzymatically digested sample, and optionally determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.

86. The method of any one of claims 71-85, comprising denaturing proteins or peptides in the immunoenriched sample prior to the assessing.

87. The method of any one of claims 73-86, comprising denaturing the eluted protein or peptide prior to the assessing.

88. The method of claim 87, comprising denaturing the eluted protein prior to enzymatically digesting the eluted protein.

89. The method of any one of claims 71-88, comprising diluting the sample prior to the immunoenriching and performing the immunoenriching on the sample that has been diluted.

90. The method of claim 89, comprising diluting the sample by 2 to 1000 fold.

91. The method of claim 89 or 90, wherein the sample that has been diluted has a volume of at least 5 pl.

92. The method of any one of claims 71-91, wherein the method comprises assessing citrullination of two or more proteins of interest or peptide fragments thereof and the antibody comprises antibodies that specifically bind citrullinated and noncitrullinated forms of each of the two or more proteins of interest or peptide fragments thereof.

93. The method of any one of claims 71-92, wherein the biological sample is a serum or plasma sample.

94. A method of assessing the activity of a PAD2 modulator, the method comprising

96. The method of claim 94 or 95, further comprising assessing citrullination of the citrullination site on the protein or a peptide fragment thereof in a control biological sample.

97. The method of claim 96, further comprising comparing an outcome of the assessment of citrullination in the biological sample with a corresponding outcome of the assessment of citrullination in the control biological sample.

98. The method of any one of claims 84-97, wherein the assessing comprises (i) measuring a first concentration of citrullinated protein or peptide in the sample and (ii) measuring a second concentration of corresponding total protein in the sample, wherein the citrullinated protein or peptide is citrullinated at the citrullination site and the corresponding total protein encompasses modified and unmodified forms of the protein, and optionally (iii) determining a citrullination ratio, which is a ratio of the first concentration to the second concentration.

99. The method of claim 98, wherein the method comprises comparing the citrullination ratio of the biological sample to the citrullination ratio of a control biological sample.

100. The method of claim 99, wherein the control biological sample is from the same subject as the biological sample.

101. The method of claim 100, wherein the control biological sample is a biological sample obtained from the subject prior to treatment with a PAD2 modulator.

102. The method of any one of claims 99-101, wherein the control biological sample has not been exposed to the PAD2 modulator.

103. The method of any one of claims 99-102, wherein the control biological sample has been exposed to a different treatment than the biological sample.

104. The method of any one of claims 94 or 96-103, further comprising (a) selecting or adjusting a dose of a PAD2 modulator based on the outcome of the assessing; or (b) selecting the subject for treatment with a PAD2 modulator based on the outcome of the assessing.

106. The method of claim 105, further comprising calculating an IC50 for the PAD2 modulator based on the outcome of the assessing.

107. The method of any one of claims 105-106, further comprising selecting or adjusting a dose of a PAD2 modulator to be administered to the subject based on the outcome of the assessing.

108. The method of any one of claims 1-106, further comprising selecting the subject from which the biological sample was derived for treatment of a citrullination-related disease based on the outcome of the assessing.

(b) after the incubation period, separating plasma or supernatant from the whole blood sample, and (c) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant.

110. The method of claim 109, wherein the incubation period is 48 to 96 hours.

111. The method of any one of claims 109 or 110, wherein the incubating is at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C.

112. The method of any one of claims 109-111, comprising enzymatically digesting polypeptides in the plasma or supernatant before the assessing.

113. The method of any one of claims 109-112, further comprising (i) incubating the plasma or supernatant with a protein depletion resin, and (ii) recovering depleted plasma or supernatant that has flowed through the resin to obtain depleted flowthrough, optionally wherein the recovering comprises centrifugation.

114. The method of claim 113, comprising enzymatically digesting polypeptides in the depleted flowthrough before the assessing.

115. The method of claim 113 or 114, further comprising cleaning up enzymatically digested plasma or supernatant before the assessing.

116. The method of any one of claims 109-115, wherein the whole blood sample and the plasma or supernatant separated therefrom have not been frozen and thawed.

117. The method of any one of claims 109-116, wherein the whole blood sample and/or the plasma or supernatant separated therefrom has been frozen and thawed.

118. The method of claim 117, wherein the method comprises freezing the plasma or supernatant after the separating and subsequently thawing the plasma or supernatant before the assessing.

119. The method of any one of claims 109-118, wherein the whole blood sample has been exposed to a PAD2 modulator.

120 The method of claim 119, wherein the whole blood sample has been exposed to a PAD2 modulator in vitro.

121. The method of claim 119, wherein the whole blood sample has been exposed to a PAD2 modulator in vivo in the subject as a result of the PAD2 modulator being administered to the subject.

(a) incubating a whole blood sample from a subject at 35-40°C for an incubation period, wherein the sample is obtained from the subject following administration of a PAD2 modulator to the subject, (b) after the incubation period, separating plasma or supernatant from the whole blood sample, and

123. The method of claim 122, further comprising

124. The method of claim 123, further comprising comparing an outcome of the assessing in step (f) with an outcome of the assessing in step (c).

125. A method of assessing effects of a PAD2 modulator, the method comprising

(a) exposing a whole blood sample from a subject to a PAD2 modulator in vitro,

(b) incubating the whole blood sample at 35-40°C for an incubation period,

126. The method of claim 125, further comprising

127. The method of claim 126, further comprising comparing an outcome of the assessing in step (g) with an outcome of the assessing in step (d).

129. The method of claim 128, wherein the plasma or supernatant has been frozen and the method comprises thawing the plasma or supernatant prior to the assessing.

130. A method comprising

(i) contacting a biological sample from a subject with exogenous PAD2 and

131. The method of claim 130, wherein the biological sample comprises plasma or serum.

132. The method of claim 130 or 131, wherein the method comprises preparing the biological sample for assessment prior to the assessing.

133. The method of claim 123, wherein the preparing comprises enzymatically digesting proteins in the biological sample to produce digested peptides and optionally cleaning up the digested peptides.

134. The method of claim 132 or 133, wherein the preparing comprises depleting proteins from the biological sample that are not targeted by the assessing.

135. The method of any one of claim 134, wherein depleting comprises depletion of human serum albumin.

136. The method of any one of claims 130-135, wherein the preparing comprises immunoenriching the biological sample for the protein or peptide fragment thereof using an antibody that binds to citrullinated and non-citrullinated forms of the protein or peptide.

137. The method of any one of claims 71-136, wherein the assessing comprises analysis with mass spectrometry (MS).

138. The method of any one of claims 71-136, wherein the assessing is performed using liquid chromatography and mass spectrometry (LC-MS).

139. The method of any one of claims 71-136, wherein the assessing comprises selective reaction monitoring and chromatographic separation.

141. The method of claim 140, wherein the method comprises (i) measuring a first concentration of a citrullinated peptide from a target protein in a biological sample by MS, wherein the citrullinated peptide is citrullinated at a citrullination site, and (ii) measuring the second concentration by measuring the concentration of a signature peptide from the target protein in the biological sample by MS.

142. The method of claim 140 or 141, further comprising calculating a ratio of the first concentration to the second concentration

143. The method of any one of claims 140-142, wherein the method comprises measuring the first concentration and the second concentration for each of a plurality of different proteins or peptides.

144. The method of claim 143, wherein the method comprises measuring the first concentration for each of two different peptides, each of which are nonoverlapping fragments of the same protein and contain different citrullination sites.

145. The method of claim 143 or 144, wherein the method comprises calculating a ratio of the first concentration to the second concentration for each of the plurality of different proteins or peptides.

146. The method of any one of claims 71-145, wherein the PAD2 modulator is a PAD2 inhibitor.

147. The method of any one of claims 71-146, wherein the citrullination site is a site listed in Table 1.

148. The method of any one of claims 71-146, wherein the citrullination site is a site listed in Table 11.

149. The method of any one of claims 71-146, wherein the citrullination site is selected from one or more of the following:

R155 of apolipoprotein A-IV (APOA4; corresponding to R155 of P06727), R306 of apolipoprotein A-IV (AP0A4; corresponding to R306 of P06727),

R496 of albumin (ALB; corresponding to R496 of P02768),

R315 of complement C3 (C3; corresponding to R315 of PO 1024),

R194 of clusterin (CLU; corresponding to R194 and/or R198 of P10909), or

R198 of clusterin (CLU; corresponding to R194 and/or R198 of Pl 0909).

150. The method of claim 149, wherein the method comprises assessing citrullination at one of the following peptide sequences, wherein the citrullination site is designated by underlining: DHEELSLVASEAVRAIEK; AKPALEDLRQGLLPVLESFK;

ERLGPLVEQGR; HTFMGVVSLGSPSGEVSHPRKT; ENGGARLAEYHAK;

LSPLGEEMRDR; SELEEQLTPVAEETRAR; TPVSDRVTK;

IVEGSDAEIGMSPWQVMLFRK; IYISGMAPRPSLAK; RQLTPYAQR;

RVEPYGENFNK; SFQTGLFTAARQSTNAYPDLR;

152. The method of claim 151, wherein the enzymatically digesting is conducted after the contacting.

153. The method of claim 151 or 152, wherein the SAX chromatography medium comprises particles.

154. The method of claim 153, wherein the particles comprise magnetic particles.

155. The method of claim 153 or 154, wherein the SAX chromatography medium enriches membrane-bound proteins and peptide fragments thereof in the sample.

156. The method of any one of claims 153-155 wherein the contacting comprises incubating the sample with the SAX chromatography medium for at least 30 minutes.

157. The method of any one of claims 153-156, wherein the method further comprises eluting proteins or peptide fragments thereof bound to the SAX chromatography medium either before or after the enzymatically digesting, and assessing citrullination of the eluted proteins or peptide fragments thereof.

158. The method of claim 157, wherein the method further comprises washing the proteins or peptide fragments thereof bound to the SAX chromatography medium at least once prior to eluting the proteins or peptide fragments thereof from the SAX chromatography medium.

159. The method of claim 157 or 158, wherein the enzymatically digesting is conducted after the eluting.

160. The method of any one of claims 151-159, wherein the method comprises at least one further chromatography or a filtration step after the enzymatically digesting and prior to the assessing citrullination to separate the protein or peptide fragment thereof from one or more salts, buffers, or small molecules.

161. The method of any one of claims 151-160, wherein the assessing citrullination comprises measuring a first concentration of citrullinated peptide in the digested peptides and a second concentration of signature peptide in the digested peptides, and optionally determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.

162. The method of any one of claims 151-161, comprising denaturing proteins in the sample prior to the assessing.

163. The method of claim 162, comprising denaturing the proteins prior to the enzymatically digesting.

165. The method of any one of claims 151-164, wherein the assessing citrullination comprises analysis with mass spectrometry (MS) or performing liquid chromatography and mass spectrometry (LC-MS).

166. The method of any one of claims 151-165, wherein the biological sample has been exposed to a PAD2 modulator such as a PAD2 inhibitor.

167. The method of any one of claims 151-166, wherein the citrullination site is a site listed in Table 1.

168. The method of any one of claims 151-166, wherein the citrullination site is a site listed in Table 11.

169. The method of any one of claims 151-166, wherein the sequence of the peptide fragment comprises one of the following sequences, wherein the citrullination site is designated by underlining of R residues and wherein modifications of non-arginine residues are designated by underlining of the modified residues: DHEELSLVASEAVRAIEK;

IVEGSDAEIGMSPWQVMLFRK; IYISGMAPRPSLAK; RQLTPYAQR;

RVEPYGENFNK; SFQTGLFTAARQSTNAYPDLR;

170. The method of any one of claims 151-169, wherein the biological sample comprises whole blood, plasma, serum, or blood supernatant.

171. The method of any one of claims 151-169, wherein the biological sample comprises synovial fluid.

172. The method of any one of claims 151-171, wherein the peptide fragment comprises the sequence of: DHEELSLVASEAVRAIEK; AKPALEDLRQGLLPVLESFK;

173. The method of any one of claims 151-172, wherein the method comprises assessing citrullination of two or more proteins or peptide fragments thereof.

174. The method of any one of claims 1-173, wherein the biological sample is obtained from a subject that is ACPA positive.

175. The method of any one of claims 1-174, wherein the biological sample is obtained from a subject that is positive for endogenous PAD antibodies.

176. The method of any one of claims 1-175, wherein the biological sample is obtained from a subject that has been diagnosed with a citrullinati on-related disease or is at risk for developing a citrullination-related disease.

177. The method of any one of claims 1-176, wherein the method further comprises selecting the subject from which the biological sample was derived for treatment of a citrullination- related disease.

178. The method of claim 176 or 177, wherein the citrullination-related disease is an autoimmune disorder.

179. The method of claim 176 or 177, wherein the citrullination-related disease is rheumatoid arthritis, lupus (e.g., systemic lupus erythematosus (SLE)), lupus nephritis, vasculitis (e.g., ANCA-associated vasculitis), thrombosis (e.g, venous thrombosis), or inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn’s disease).

180. The method of any one of claims 1-176, wherein the biological sample is obtained from a subject that is a normal, healthy subject.

181. The method of any one of claims 1-179, wherein the biological sample is obtained from a subject following treatment of the subject with at least one dose of a PAD2 modulator such as a PAD2 inhibitor.

182. The method of any one of claims 1-179, wherein the method further comprises administering a PAD2 modulator such as a PAD2 inhibitor to the subject, and optionally assessing citrullination of a citrullination site on a protein or peptide fragment thereof in a sample from the subject collected following administering of the PAD2 modulator or PAD2 inhibitor.