Methods of assessing activity of citrullination and pad4 modulators

Abstract

The present application relates to methods for quantifying citrullination and methods that can be used to assess the activity of agents, including, for example, PAD4 (peptidyl arginine deiminase 4) modulators, such as small molecules or antibodies. In certain embodiments, the methods include assessing citrullination at one or more citrullination sites on a protein or peptide fragment thereof present in a biological sample.

Description

Technical Field

[0001] This application relates to a method for evaluating citrullination sites on proteins or peptide fragments thereof present in biological samples from subjects. The method can be particularly used to evaluate the citrullination modification activity of pharmaceutical agents, including, for example, PAD4 (peptidyl arginine deiminase 4) modulators, such as small molecules or antibodies. Background Technology

[0002] PAD4 (peptidyl arginine deiminase 4) is a major PAD enzyme found in the synovial tissue 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. PAD4 is an enzyme that catalyzes the conversion of arginine to citrulline. Approximately 15% of RA patients have antibodies that activate PAD4, and the presence of such PAD4-activating antibodies is associated with severe joint erosion. Compounds capable of modulating PAD4 activity may be useful in treating a variety of conditions. For example, PAD4 inhibitors may be used to treat autoimmune diseases such as RA and others.

[0003] Therefore, the methods used to assess the activity of PAD4 modulators can be used, for example, to monitor treatment with existing PAD4 modulators, to evaluate novel PAD4 modulators under development, and for other purposes. Summary of the Invention

[0004] This disclosure provides methods for evaluating the citrullination of proteins or peptides, and methods for evaluating the activity of PAD4 modulators or other therapeutic agents that can affect citrullination, by assessing the citrullination of one or more citrullination sites on a protein or peptide fragment present in a biological sample.

[0005] This disclosure specifically relates to embodiments including, for example:

[0006] 1. A method for determining citrullination of a protein or a peptide fragment thereof, the method comprising evaluating citrullination at citrullination sites on a protein or a peptide fragment thereof in a biological sample from a subject, wherein the biological sample has been exposed to a PAD4 inhibitor, wherein the citrullination sites are selected from one or more of the following:

[0007] R1391 of proteoglycan 4 (corresponding to R1391 of Q92954).

[0008] R573 of the fibrinogen α chain (corresponding to R573 of P02671).

[0009] R591 of the fibrinogen α chain (corresponding to R591 of P02671).

[0010] R573 of complement C3 (corresponding to R573 of P01024).

[0011] R748 of complement C3 (corresponding to R748 of P01024).

[0012] R688 of the heavy chain H4, an inhibitor of inter-α-trypsin (corresponding to R688 of Q14624).

[0013] R297 of the α-1-microglobulin / biskunitz inhibitor precursor (corresponding to R297 of P02760).

[0014] R715 of α-2-macroglobulin (corresponding to R715 of P01023).

[0015] R32 of gel sol protein (corresponding to R32 of P06396).

[0016] R59 of haptoglobin (corresponding to R59 of P00738), and

[0017] R651 of serum transferrin (corresponding to R651 of P02787).

[0018] 2. A method for evaluating the activity of a PAD4 inhibitor, the method comprising evaluating citrullination at citrullination sites on a protein or peptide fragment thereof present in a biological sample from a subject, wherein the biological sample has been exposed to the PAD4 inhibitor, wherein the citrullination sites are selected from one or more of the following:

[0019] R1391 of proteoglycan 4 (corresponding to R1391 of Q92954).

[0020] R573 of the fibrinogen α chain (corresponding to R573 of P02671).

[0021] R591 of the fibrinogen α chain (corresponding to R591 of P02671).

[0022] R573 of complement C3 (corresponding to R573 of P01024).

[0023] R748 of complement C3 (corresponding to R748 of P01024).

[0024] R688 of the heavy chain H4, an inhibitor of inter-α-trypsin (corresponding to R688 of Q14624).

[0025] R297 of the α-1-microglobulin / biskunitz inhibitor precursor (corresponding to R297 of P02760).

[0026] R715 of α-2-macroglobulin (corresponding to R715 of P01023).

[0027] R32 of gel sol protein (corresponding to R32 of P06396).

[0028] R59 of haptoglobin (corresponding to R59 of P00738), and

[0029] R651 of serum transferrin (corresponding to R651 of P02787).

[0030] 3. The method according to embodiment 2, wherein the PAD4 inhibitor reduces citrullination at the citrullination site in a dose-dependent manner.

[0031] 4. The method according to embodiment 1, 2 or 3, wherein the method includes evaluating citrullination of a peptide fragment of the protein, wherein the peptide fragment contains the citrullination site.

[0032] 5. The method according to any one of embodiments 1-4, wherein the biological sample has been exposed to the PAD inhibitor in the subject due to administration of the PAD inhibitor to the subject.

[0033] 6. The method according to any one of embodiments 1-4, wherein the biological sample has been exposed to the PAD inhibitor by ex vivo contacting the biological sample with the PAD inhibitor.

[0034] 7. The method according to any one of embodiments 1-6, wherein the evaluation of citrullination at the citrullination site includes mass spectrometry (MS).

[0035] 8. The method according to any one of embodiments 1-6, wherein the evaluation of citrullination of the citrullination site includes liquid chromatography and mass spectrometry (LC-MS).

[0036] 9. The method according to any one of embodiments 1-6, wherein the evaluation of citrullination includes selective reaction monitoring and chromatographic separation.

[0037] 10. The method according to any one of embodiments 1-9, wherein evaluating the citrullination of the citrullination site includes measuring a first concentration of the citrullinated protein or a peptide fragment thereof and a second concentration of the corresponding total protein in the biological sample.

[0038] 11. The method according to embodiment 10, wherein evaluating the citrullination of the citrullination site includes determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.

[0039] 12. The method according to embodiment 11, wherein the method includes comparing the citrullination ratio with a reference citrullination ratio.

[0040] 13. The method according to embodiment 12, wherein the reference citrullination ratio is a citrullination ratio determined for a control biological sample.

[0041] 14. The method according to embodiment 13, wherein the control biological sample is a biological sample that is (a) not yet exposed to a PAD4 modulator or a PAD4 inhibitor, (b) from the same subject, and / or (c) from the same subject prior to treatment with the PAD4 inhibitor.

[0042] 15. The method according to any one of embodiments 12-14, wherein the evaluation includes determining the difference between the citrullination ratio of the biological sample and the reference citrullination ratio.

[0043] 16. The method according to any one of embodiments 1-15, wherein the method includes contacting the biological sample with exogenous PAD4.

[0044] 17. A method, the method comprising:

[0045] (i) Expose biological samples from the subject to exogenous PAD4, and

[0046] (ii) Assess citrullination at citrullination sites on proteins or their peptide fragments present in biological samples;

[0047] The citrullination site is selected from one or more of the following:

[0048] R1391 of proteoglycan 4 (corresponding to R1391 of Q92954).

[0049] R573 of the fibrinogen α chain (corresponding to R573 of P02671).

[0050] R591 of the fibrinogen α chain (corresponding to R591 of P02671).

[0051] R573 of complement C3 (corresponding to R573 of P01024).

[0052] R748 of complement C3 (corresponding to R748 of P01024).

[0053] R688 of the heavy chain H4, an inhibitor of inter-α-trypsin (corresponding to R688 of Q14624).

[0054] R297 of the α-1-microglobulin / biskunitz inhibitor precursor (corresponding to R297 of P02760).

[0055] R715 of α-2-macroglobulin (corresponding to R715 of P01023).

[0056] R32 of gel sol protein (corresponding to R32 of P06396).

[0057] R59 of haptoglobin (corresponding to R59 of P00738), and

[0058] R651 of serum transferrin (corresponding to R651 of P02787).

[0059] 18. The method according to embodiment 16 or 17, wherein the method includes incubating the exogenous PAD4 together with the biological sample.

[0060] 19. The method according to embodiment 18, wherein the method includes incubating the exogenous PAD4 with the biological sample at a temperature of 36ºC-38ºC.

[0061] 20. The method according to embodiment 18 or 19, wherein the incubation lasts for 1-3 hours.

[0062] 21. The method according to any one of embodiments 18-20, wherein the method comprises adding EDTA to the biological sample after incubation with exogenous PAD4.

[0063] 22. The method according to any one of embodiments 1-21, wherein the method includes incubating the biological sample for an incubation period prior to evaluating citrullination.

[0064] 23. The method according to embodiment 22, wherein the incubation is carried out at 35ºC-40ºC.

[0065] 24. The method according to embodiment 22 or 23, wherein the incubation period is 48 to 96 hours.

[0066] 25. The method according to any one of embodiments 1-24, wherein the method includes preparing the biological sample for evaluation prior to the evaluation.

[0067] 26. The method according to embodiment 25, wherein the preparation includes enzymatic digestion of proteins in the biological sample to form peptide fragments thereof.

[0068] 27. The method according to embodiment 25 or 26, wherein the preparation includes depleting the biological sample of proteins or peptide fragments thereof that are not the target of the evaluation.

[0069] 28. The method according to any one of embodiments 25-27, wherein the preparation includes diluting the biological sample.

[0070] 29. The method according to any one of embodiments 25-28, wherein the preparation includes denaturing a protein or a peptide fragment thereof in the biological sample.

[0071] 30. The method according to any one of embodiments 25-29, wherein the preparation comprises enriching the biological sample with a protein or peptide fragment thereof containing the citrullination site to be evaluated.

[0072] 31. The method according to embodiment 30, wherein the preparation includes contacting the biological sample with an antibody that specifically binds to both citrullinated and non-citrullinated forms of the protein or a peptide fragment thereof containing the citrullinated site to be evaluated.

[0073] 32. The method according to any one of embodiments 1-31, wherein the method comprises freezing the biological sample and thawing the biological sample prior to evaluating the citrullination of the citrullination site.

[0074] 33. The method according to any one of embodiments 1-32, the method comprising:

[0075] (a) Immunoenriching the sample with the protein or a peptide fragment thereof to form an immunoenriched sample, wherein the immunoenrichment comprises contacting the sample with an antibody that binds to both the citrullinated and non-citrullinated forms of the protein containing the citrullinated site, and / or to both the citrullinated and non-citrullinated forms of the peptide fragment of the protein containing the citrullinated site.

[0076] (b) Enzymatically digesting the proteins in the sample before or after the immunoenrichment to form the peptide fragments, and

[0077] (c) Evaluate the citrullination of the peptide fragment at the citrullination site.

[0078] 34. The method according to embodiment 33, wherein the immunoenrichment comprises contacting the sample with a fixed antibody, the fixed antibody binding to both the citrullinated and non-citrullinated forms of the protein containing the citrullinated site, and / or binding to both the citrullinated and non-citrullinated forms of a peptide fragment of the protein containing the citrullinated site, and eluting the protein and / or peptide bound to the fixed antibody.

[0079] 35. The method according to embodiment 33 or 34, wherein the enzymatic digestion is performed prior to the immunoenrichment.

[0080] 36. The method according to embodiment 33 or 34, wherein the enzymatic digestion is performed after the immunoenrichment.

[0081] 37. The method according to any one of embodiments 33-36, wherein the immunoenrichment comprises incubating the sample with the immobilized antibody for at least 30 minutes.

[0082] 38. The method according to embodiment 37, wherein the incubation is carried out at a temperature of 22ºC-28ºC.

[0083] 39. The method according to embodiment 37 or 38, wherein the incubation lasts for a period of 30 to 90 minutes.

[0084] 40. The method according to any one of embodiments 33-38, wherein the immunoenrichment includes oscillation during the incubation.

[0085] 41. The method according to any one of embodiments 34-40, wherein the immunoenrichment comprises removing the fixed antibody from the sample and washing the fixed antibody prior to the elution.

[0086] 42. The method according to any one of embodiments 34-41, wherein the elution comprises washing the fixed antibody with an elution composition under acidic conditions.

[0087] 43. The method according to embodiment 42, wherein the elution composition comprises a detergent.

[0088] 44. The method according to embodiment 43, wherein the detergent is an amphoteric detergent.

[0089] 45. The method according to any one of embodiments 34-44, wherein the method includes neutralizing the eluted protein in a buffer before evaluation and optionally before enzymatic digestion.

[0090] 46. ​​The method according to any one of embodiments 33-45, wherein the antibody is immobilized by attaching to a solid surface.

[0091] 47. The method according to any one of embodiments 33-46, wherein the evaluation of citrullination includes measuring a first concentration of citrullinated peptides in the digested peptide and a second concentration of characteristic peptides in the digested peptide, and optionally determining a citrullination ratio, wherein the citrullination ratio is the ratio of the first concentration to the second concentration.

[0092] 48. The method according to any one of embodiments 33-47, wherein the method includes denaturing the proteins in the immunoenriched sample prior to the evaluation.

[0093] 49. The method according to any one of embodiments 34-48, wherein the method includes denaturing the eluted protein prior to the evaluation.

[0094] 50. The method according to embodiment 49, wherein the method includes denaturing the eluted protein prior to enzymatic digestion of the eluted protein.

[0095] 51. The method according to any one of embodiments 33-50, the method comprising diluting a plasma or serum sample prior to the immunoenrichment and performing the immunoenrichment on the diluted sample.

[0096] 52. The method according to embodiment 51, wherein the method comprises diluting the plasma or serum sample by 2 to 1000 times.

[0097] 53. The method according to embodiment 51 or 52, wherein the volume of the diluted sample is at least 5 μl.

[0098] 54. The method according to any one of embodiments 1-53, wherein the sequence of said peptide fragment comprises one of the following sequences, wherein said citrullinated sites are indicated by underlining: AITT R SGQTLSK (SEQ ID NO:247),ESSSHPGIAEFPS R GK (SEQ ID NO: 248), QFTSSTSYN R GDSTFESK (SEQ ID NO: 249), SGQSED R QPVPGQQMTLK (SEQ ID NO: 250), ASHLGLA R SNLDEDIIAEENIVSR (SEQ ID NO: 251), QLGLPGPPDVPDHAAYHPF RR (SEQ ID NO: 252), GPC R AFIQLWAFDAVK (SEQ ID NO: 253), VGFYESDVMG R GHAR (SEQ ID NO: 254), ATAS R GASQAGAPQGR (SEQ ID NO: 255), L R TEGDGVYTLNDK (SEQ ID NO: 256) and DLLF R DDTVCLAK (SEQ ID NO: 257).

[0099] 55. The method according to any one of embodiments 1-54, wherein the biological sample comprises whole blood, plasma, serum or blood supernatant.

[0100] 56. The method according to embodiment 55, wherein the citrullinated site is: R1391 of proteoglycan 4 (corresponding to R1391 of Q92954), R573 of fibrinogen α chain (corresponding to R573 of P02671), or R591 of fibrinogen α chain (corresponding to R591 of P02671), at R591 or R593 of P02671.

[0101] 57. The method according to embodiment 56, wherein the peptide fragment comprises the following amino acid sequence: AITT R SGQTLSK (SEQ ID NO: 247), ESSSHPGIAEFPS R GK (SEQ ID NO: 248) or QFTSSTSYN R GDSTFESK (SEQ ID NO: 249), where the underlined R is the citrullinated site.

[0102] 58. The method according to any one of embodiments 1-54, wherein the biological sample comprises synovial fluid.

[0103] 59. The method according to embodiment 58, wherein the citrullinated site is: R1391 of proteoglycan 4 (corresponding to R1391 of Q92954), R573 of fibrinogen α chain (corresponding to R573 of P02671), R591 of fibrinogen α chain (corresponding to R591 of P02671) at R591 or R593 of P02671, R573 of complement C3 (corresponding to R573 of P01024), R688 of inter-α-trypsin inhibitor heavy chain H4 (corresponding to R688 of Q14624), or R297 of α-1-microglobulin / bisKunitz inhibitor precursor (corresponding to R297 of P02760).

[0104] 60. The method according to embodiment 59, wherein the sequence of said peptide fragment comprises one of the following sequences, wherein said citrullinated sites are indicated by underscores: AITT R SGQTLSK (SEQ ID NO: 247), ESSSHPGIAEFPS R GK (SEQ ID NO: 248), QFTSSTSYN R GDSTFESK (SEQ ID NO: 249), SGQSED R QPVPGQQMTLK (SEQ ID NO: 250), QLGLPGPPDVPDHAAYHPF R R (SEQ ID NO: 252) and GPC R AFIQLWAFDAVK (SEQ ID NO: 253).

[0105] 61. The method according to any one of embodiments 1-60, wherein the method includes evaluating the citrullination of two or more proteins or peptide fragments thereof.

[0106] 62. The method according to any one of embodiments 1-60, wherein the method further comprises evaluating the citrullination of at least one additional citrullination site listed in Table A or any one of Tables 3-18.

[0107] 63. A method, the method comprising:

[0108] (a) Immunoenriching a biological sample to form an immunoenriched sample by immunoenriching a target protein, wherein the immunoenrichment comprises contacting the sample with an antibody that specifically binds to both citrullinated and uncitrullinated forms of the target protein, or specifically binds to both citrullinated and uncitrullinated forms of a peptide fragment of the target protein, wherein the target protein or the peptide fragment contains a citrullinated site, and

[0109] (b) Evaluate the citrullination at the citrullination sites in the immunoenriched samples.

[0110] 64. A method comprising:

[0111] (a) Immunoenriching a biological sample to form an immunoenriched sample by immunoenriching the target protein, wherein the immunoenrichment comprises contacting the sample with an antibody that binds to both citrullinated and non-citrullinated forms of the target protein, or specifically binds to both citrullinated and non-citrullinated forms of a peptide fragment of the target protein, wherein the target protein or the peptide fragment contains a citrullinated site.

[0112] (b) Enzymatic digestion of the immunoenriched sample to form peptide fragments of the target protein, wherein portions (a) and (b) can be performed in any order, and

[0113] (c) Evaluate the citrullination of the peptide fragment at the citrullination site.

[0114] 65. A method, the method comprising:

[0115] (a) Immunoenrichment of a biological sample by contacting it with a fixed antibody, the fixed antibody binding to both citrullinated and non-citrullinated forms of a target protein, the target protein containing a citrullinated site.

[0116] (b) Elute the proteins bound to the fixed antibodies.

[0117] (d) Enzymatic digestion of the eluted protein to form its peptide fragments, and

[0118] (e) Evaluate the citrullination of the peptide fragment at the citrullination site.

[0119] 66. A method, the method comprising

[0120] (a) Enzymatic digestion of proteins in biological samples to form their peptide fragments.

[0121] (b) Immunoenrichment of the enzymatically digested biological sample by contacting it with a fixed antibody, wherein the fixed antibody specifically binds to both citrullinated and non-citrullinated forms of a peptide fragment containing a citrullinated site.

[0122] (c) Elution of peptides bound to the immobilized antibody, and

[0123] (d) Evaluate the citrullination of the peptide at the citrullination site.

[0124] 67. The method according to any one of embodiments 63-66, wherein the sample comprises whole blood, plasma, serum or blood supernatant.

[0125] 68. A method for evaluating the activity of a PAD4 inhibitor, the method comprising:

[0126] (a) Obtaining biological samples from the subject after treatment with at least one dose of a PAD4 inhibitor, and

[0127] (b) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the biological sample.

[0128] 69. An in vitro method for evaluating the activity of a PAD4 inhibitor, the method comprising:

[0129] (a) Treating a biological sample from a subject with a PAD4 inhibitor to form a treated biological sample, and following said treatment,

[0130] (b) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the biological sample.

[0131] 70. An in vitro method for evaluating the activity of a PAD4 inhibitor, the method comprising:

[0132] (i) The biological samples obtained from the subjects will be divided into multiple biological samples.

[0133] (ii) Contact each of the plurality of biological samples with a different dose of the PAD4 inhibitor, and

[0134] (iii) For each of the plurality of biological samples, assess the citrullination of PAD4-dependent citrullination sites on proteins or peptide fragments present in the biological sample.

[0135] 71. The method according to embodiment 70, the method further comprising calculating the IC50 of the PAD4 inhibitor based on the outcome of the assessment.

[0136] 72. The method according to any one of embodiments 68 or 70-71, the method further comprising selecting or adjusting the dose of the PAD4 inhibitor to be administered to the subject based on the outcome of the assessment.

[0137] 73. An in vitro method for evaluating citrullinated endogenous PAD4, the method comprising:

[0138] (a) Incubate whole blood samples from the subjects at 35ºC–40ºC for the incubation period.

[0139] (b) After the incubation period, plasma or supernatant is separated from the whole blood sample, and

[0140] (c) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the plasma or supernatant.

[0141] 74. An in vitro method for assessing changes in citrullination in a subject, the method comprising:

[0142] (a) Incubate whole blood samples from the subject at 35ºC–40ºC for an incubation period, wherein the samples were obtained from the subject after administration of the PAD4 inhibitor.

[0143] (b) After the incubation period, plasma or supernatant is separated from the whole blood sample, and

[0144] (c) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the plasma or supernatant.

[0145] 75. The method according to embodiment 74, wherein the method further comprises

[0146] (d) Incubate a second whole blood sample from the subject at 35ºC–40ºC for an incubation period, wherein the second whole blood sample was obtained from the subject prior to administration of the PAD4 inhibitor.

[0147] (e) After the incubation period, plasma or supernatant is separated from the second whole blood sample, and

[0148] (f) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in plasma or supernatant from the second whole blood sample.

[0149] 76. The method according to embodiment 74, the method further comprising comparing the outcome of the assessment in step (f) with the outcome of the assessment in step (c).

[0150] 77. A method for evaluating the effect of a PAD4 inhibitor, the method comprising:

[0151] (a) Expose whole blood samples from subjects to PAD4 inhibitors in vitro.

[0152] (b) Incubate the whole blood sample at 35ºC-40ºC for the incubation period.

[0153] (c) After the incubation period, plasma or supernatant is separated from the whole blood sample, and

[0154] (d) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the plasma or supernatant.

[0155] 78. The method according to embodiment 75, wherein the method further comprises

[0156] (e) Incubate the control whole blood sample at 35ºC-40ºC for the incubation period.

[0157] (f) After the incubation period, control plasma or supernatant is separated from the control whole blood sample, and

[0158] (g) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the control plasma or supernatant.

[0159] 79. The method according to embodiment 78, the method further comprising comparing the outcome of the assessment in step (g) with the outcome of the assessment in step (d).

[0160] 80. An in vitro method for evaluating PAD4-dependent citrullination, the method comprising:

[0161] The citrullination of citrullinated sites on proteins or peptide fragments present in plasma or supernatant separated from whole blood samples that have been incubated at 35ºC–40ºC for an incubation period is evaluated.

[0162] 81. The method according to embodiment 88, wherein the plasma or supernatant has been frozen, and the method includes thawing the plasma or supernatant prior to the evaluation.

[0163] 82. A method for assessing citrullination at citrullination sites, the method comprising:

[0164] (i) Measuring a first concentration of citrullinated protein or citrullinated peptide in a biological sample by mass spectrometry (MS), wherein the citrullinated protein or citrullinated peptide is citrullinated at a citrullination site, and

[0165] (ii) Measure the second concentration of the corresponding total protein in the biological sample by MS.

[0166] 83. The method according to embodiment 82, wherein the method comprises (i) measuring a first concentration of a citrullinated peptide from a target protein in a biological sample by means of MS, wherein the citrullinated peptide is citrullinated at a citrullination site, and (ii) measuring a second concentration by means of measuring the concentration of a characteristic peptide from the target protein in the biological sample by means of MS.

[0167] 84. The method according to embodiment 82 or 83, the method further comprising calculating the ratio of the first concentration to the second concentration.

[0168] 85. The method according to any one of embodiments 82-84, wherein the method includes measuring the first concentration and the second concentration of each of a plurality of different proteins or peptides.

[0169] 86. The method according to embodiment 85, wherein the method includes measuring the first concentration of each of two different peptides, each of the peptides being a non-overlapping fragment of the same protein and containing different citrullinated sites.

[0170] 87. The method according to embodiment 85 or 86, wherein the method includes calculating the ratio of the first concentration to the second concentration of each of the plurality of different proteins or peptides.

[0171] 88. The method according to any one of embodiments 63-87, wherein the citrullination site is a site listed in Table A or any one of Tables 3-18.

[0172] 89. The method according to any one of embodiments 63-88, wherein the citrullinated site is selected from one or more of the following:

[0173] R1391 of proteoglycan 4 (corresponding to R1391 of Q92954).

[0174] R573 of the fibrinogen α chain (corresponding to R573 of P02671).

[0175] R591 of the fibrinogen α chain (corresponding to R591 of P02671).

[0176] R573 of complement C3 (corresponding to R573 of P01024).

[0177] R748 of complement C3 (corresponding to R748 of P01024).

[0178] R688 of the heavy chain H4, an inhibitor of inter-α-trypsin (corresponding to R688 of Q14624).

[0179] R297 of the α-1-microglobulin / biskunitz inhibitor precursor (corresponding to R297 of P02760).

[0180] R715 of α-2-macroglobulin (corresponding to R715 of P01023).

[0181] R32 of gel sol protein (corresponding to R32 of P06396).

[0182] R59 of haptoglobin (corresponding to R59 of P00738), and

[0183] R651 of serum transferrin (corresponding to R651 of P02787).

[0184] 90. The method according to embodiment 89, wherein the method includes evaluating citrullination at one of the following peptide sequences, wherein the citrullination site is indicated by underscore: AITT R SGQTLSK (SEQ ID NO:247),ESSSHPGIAEFPS R GK (SEQ ID NO: 248), QFTSSTSYN R GDSTFESK (SEQ ID NO: 249), SGQSED R QPVPGQQMTLK (SEQ ID NO: 250), ASHLGLA R SNLDEDIIAEENIVSR (SEQ ID NO: 251), QLGLPGPPDVPDHAAYHPF R R (SEQ ID NO: 252), GPC R AFIQLWAFDAVK (SEQ ID NO: 253), VGFYESDVMG R GHAR (SEQ ID NO: 254), ATAS R GASQAGAPQGR (SEQ ID NO: 255), L R TEGDGVYTLNDK (SEQ ID NO: 256) and DLLF R DDTVCLAK (SEQ ID NO: 257).

[0185] 91. The method according to any one of embodiments 1-90, wherein the biological sample is obtained from a subject who is ACPA positive.

[0186] 92. The method according to any one of embodiments 1-91, wherein the biological sample is obtained from a subject who is positive for endogenous PAD4 antibody.

[0187] 93. The method according to embodiment 92, wherein the subject is positive for an endogenous PAD4 activation antibody.

[0188] 94. The method according to any one of embodiments 1-93, wherein the biological sample is obtained from a subject who has been diagnosed with citrullination-related disease or is at risk of developing citrullination-related disease.

[0189] 95. The method according to any one of embodiments 1-94, wherein the method further comprises selecting a subject from whom the biological sample is derived for the treatment of a citrullination-related disease based on the outcome of the assessment.

[0190] 96. The method according to embodiment 95, wherein the citrullination-related disease is an autoimmune disorder.

[0191] 97. The method according to embodiment 96, 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).

[0192] 98. The method according to any one of embodiments 1-97, wherein the biological sample is obtained from the subject after the subject has been treated with at least one dose of a PAD4 inhibitor.

[0193] 99. The method according to any one of embodiments 1-98, wherein the biological sample is obtained from a subject who is a normal healthy subject.

[0194] 100. The method according to any one of claims 1-99, wherein the method further comprises administering a PAD4 inhibitor to the subject, and optionally evaluating citrullination of citrullinated sites on proteins or peptide fragments thereof in samples collected from the subject after administration of the PAD4 inhibitor.

[0195] Other exemplary embodiments of this disclosure include the following:

[0196] 1. A method for assessing the activity of a PAD4 modulator, the method comprising assessing citrullination at citrullination sites on proteins or peptide fragments thereof present in a biological sample from a subject, wherein the biological sample has been exposed to a PAD4 modulator, wherein the protein is selected from one or more of the following: α-2-HS-glycoprotein, α-2-macroglobulin, albumin, antithrombin-III, apolipoprotein AI, apolipoprotein A-IV, apolipoprotein B-100, apolipoprotein E, apolipoprotein L1, C4b-binding protein α chain, ceruloplasmin, clusteringin, complement C2, complement C3, complement C4-A, complement C4-B, complement factor B, complement factor H, complement factor I, complement component C9, fibrinogen α chain, fibrinogen β chain, fibrinogen γ chain, fibronectin, galactochondrin-3-binding protein Globularin, haptoglobulin, haptoglobulin-related protein, hemoglobin-binding protein, immunoglobulin heavy chain variable 1-3, immunoglobulin heavy chain variable 1-8, immunoglobulin heavy chain variable 1-46, immunoglobulin heavy chain variable 1-69, immunoglobulin heavy chain variable 1-69D, inter-α-trypsin inhibitor heavy chain H4, immunoglobulin λ-like polypeptide 1, immunoglobulin heavy chain constant μ, inter-α-trypsin inhibitor heavy chain H2, complement C4-A isoform 2, plasminogen, plasminogen-like protein A, α-1-microglobulin / bisKunitz inhibitor precursor (protein AMBP), prothrombin, proteoglycan 4, serum transferrin, serum amyloid A-1 protein, serum amyloid A-2 protein, serum amyloid A-4 protein, lysosome protease-1, vitamin D-binding protein, and porphyrin.

[0197] 2. The method according to embodiment 1, wherein the PAD4 modulator is a PAD4 inhibitor.

[0198] 3. The method according to embodiment 1 or 2, wherein the citrullinated site is selected from one or more citrullinated sites shown in Table A or Tables 3-18.

[0199] 4. The method according to embodiment 3, the method comprising evaluating the citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises the peptide sequences shown in Tables 3-18.

[0200] 5. The method according to embodiment 1 or 2, wherein the citrullinated site is selected from one or more of the following:

[0201] R1391 of proteoglycan 4 (corresponding to R1391 of Q92954).

[0202] R573 of the fibrinogen α chain (corresponding to R573 of P02671).

[0203] R591 of the fibrinogen α chain (corresponding to R591 of P02671).

[0204] R573 of complement C3 (corresponding to R573 of P01024).

[0205] R748 for complement C3 (corresponding to R573 for P01024).

[0206] R688 of the heavy chain H4, an inhibitor of inter-α-trypsin (corresponding to R688 of Q14624).

[0207] R297 of the α-1-microglobulin / biskunitz inhibitor precursor (corresponding to R297 of P02760).

[0208] R715 of α-2-macroglobulin (corresponding to R715 of P01023).

[0209] R32 of gel sol protein (corresponding to R32 of P06396).

[0210] R59 of haptoglobin (corresponding to R59 of P00738), and

[0211] R651 of serum transferrin (corresponding to R651 of P02787).

[0212] 6. The method according to any one of embodiments 1 to 4, the method comprising evaluating citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises one of the following sequences, wherein the citrullination site is indicated by an underscore: AITT R SGQTLSK (SEQ ID NO: 247), ESSSHPGIAEFPS R GK (SEQ ID NO: 248), QFTSSTSYN R GDSTFESK (SEQ ID NO: 249), SGQSED R QPVPGQQMTLK (SEQ ID NO: 250), ASHLGLA R SNLDEDIIAEENIVSR (SEQ ID NO: 251), QLGLPGPPDVPDHAAYHPF R R (SEQ ID NO:252), GPC R AFIQLWAFDAVK (SEQ ID NO: 253), VGFYESDVMG R GHAR (SEQ ID NO: 254), ATAS R GASQAGAPQGR (SEQ ID NO: 255), LR TEGDGVYTLNDK (SEQ ID NO: 256) and DLLF R DDTVCLAK (SEQ ID NO: 257).

[0213] 7. The method according to any one of embodiments 1 to 6, wherein the biological sample has been exposed to the PAD4 modulator in the subject due to administration of the PAD4 modulator to the subject.

[0214] 8. The method according to any one of embodiments 1 to 7, wherein the biological sample comprises synovial fluid.

[0215] 9. The method according to embodiment 8, wherein the citrullination site is selected from one or more citrullination sites shown in Table 11 and / or the sequence of the peptide fragment includes the peptide sequences listed in Table 11.

[0216] 10. The method according to embodiment 8, wherein the citrullinated site is selected from one or more citrullinated sites shown in Table 11 or 12, or wherein the sequence of the peptide fragment includes one of the following sequences, wherein the citrullinated site is indicated by underlining: AITT R SGQTLSK (SEQ ID NO: 247), ESSSHPGIAEFPS R GK (SEQ ID NO: 248), QFTSSTSYN R GDSTFESK (SEQ ID NO: 249), SGQSED R QPVPGQQMTLK (SEQ IDNO: 250), QLGLPGPPDVPDHAAYHPF R R (SEQ ID NO: 252) and GPC R AFIQLWAFDAVK (SEQ ID NO:253).

[0217] 11. The method according to any one of embodiments 1 to 7, wherein the biological sample comprises blood or blood-derived samples.

[0218] 12. The method according to any one of embodiments 1 to 7, wherein the biological sample comprises serum.

[0219] 13. The method according to any one of embodiments 1 to 7, wherein the biological sample comprises plasma.

[0220] 14. The method according to any one of embodiments 1 to 11, wherein the biological sample comprises blood supernatant.

[0221] 15. The method according to any one of embodiments 1 to 6 or 8 to 14, wherein the biological sample has been exposed to the PAD4 regulator by ex vivo contact of the biological sample with the PAD4 regulator.

[0222] 16. The method according to any one of embodiments 1 to 15, wherein the method comprises freezing the biological sample and thawing the biological sample prior to evaluating the citrullination of the citrullination site.

[0223] 17. The method according to any one of embodiments 1 to 16, wherein evaluating the citrullination of the citrullination site includes mass spectrometry (MS).

[0224] 18. The method according to any one of embodiments 1 to 16, wherein evaluating the citrullination of the citrullination site includes liquid chromatography and mass spectrometry (LC-MS).

[0225] 19. The method according to any one of embodiments 1-18, wherein the method includes preparing the biological sample for evaluation prior to the evaluation.

[0226] 20. The method according to embodiment 19, wherein the preparation includes enzymatic digestion of proteins in the biological sample.

[0227] 21. The method according to embodiment 19 or 20, wherein the preparation includes depleting proteins from the biological sample that are not the target of the evaluation.

[0228] 22. The method according to any one of embodiments 19 to 21, wherein the preparation includes diluting the biological sample.

[0229] 23. The method according to any one of embodiments 19 to 22, wherein the preparation includes denaturing proteins in the biological sample.

[0230] 24. The method according to any one of embodiments 19 to 23, wherein the preparation comprises enriching the biological sample with a protein or peptide fragment thereof containing the citrullinated site to be evaluated.

[0231] 25. The method according to embodiment 24, wherein the preparation includes contacting the biological sample with an antibody that specifically binds to both citrullinated and non-citrullinated forms of a protein or peptide containing the citrullinated site to be evaluated.

[0232] 26. The method according to any one of embodiments 1 to 25, wherein the method includes contacting the biological sample with exogenous PAD4.

[0233] 27. The method according to embodiment 26, wherein the method includes incubating the exogenous PAD4 together with the biological sample.

[0234] 28. The method according to embodiment 27, wherein the method includes incubating the exogenous PAD4 with the biological sample at a temperature of 36ºC-38ºC.

[0235] 29. The method according to embodiment 27 or 28, wherein the incubation lasts for 1-3 hours.

[0236] 30. The method according to any one of embodiments 27 to 29, wherein the method comprises adding EDTA to the biological sample after incubation with exogenous PAD4.

[0237] 31. The method according to any one of embodiments 1 to 30, wherein the method includes incubating the biological sample for an incubation period prior to evaluating citrullination.

[0238] 32. The method according to embodiment 31, wherein the incubation is carried out at 35ºC-40ºC.

[0239] 33. The method according to embodiment 31 or 32, wherein the incubation period is 48 to 96 hours.

[0240] 34. The method according to any one of embodiments 1 to 33, wherein evaluating the citrullination of the citrullination site includes measuring a first concentration of the citrullinated protein or peptide and a second concentration of the corresponding total protein in the biological sample.

[0241] 35. The method according to embodiment 34, wherein evaluating the citrullination of the citrullination site includes determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.

[0242] 36. The method according to embodiment 35, wherein the method includes comparing the citrullination ratio with a reference citrullination ratio.

[0243] 37. The method according to embodiment 36, wherein the reference citrullination ratio is a citrullination ratio determined for a control biological sample.

[0244] 38. The method according to embodiment 37, wherein the control biological sample is a biological sample that has not been exposed to the PAD4 modulator.

[0245] 39. The method according to any one of embodiments 36-38, wherein the evaluation includes determining the difference between the citrullination ratio of the biological sample and the reference citrullination ratio.

[0246] 40. A method for evaluating the activity of a PAD4 regulator, the method comprising:

[0247] (a) Obtaining biological samples from the subject after treatment with at least one dose of a PAD4 modulator, and

[0248] (b) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the biological sample.

[0249] 41. An in vitro method for evaluating the activity of a PAD4 modulator, the method comprising:

[0250] (a) Treating a biological sample from a subject with a PAD4 modifier to form a treated biological sample, and following said treatment,

[0251] (b) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the biological sample.

[0252] 42. The method according to embodiment 40 or 41, the method further comprising evaluating citrullination at the citrullination site on the protein or peptide fragment in a control biological sample.

[0253] 43. The method according to embodiment 42, the method further comprising comparing the outcome of assessing citrullination in the biological sample with the corresponding outcome of assessing citrullination in the control biological sample.

[0254] 44. The method according to any one of embodiments 40-43, wherein the evaluation comprises (i) measuring a first concentration of citrullinated protein or peptide in the sample, and (ii) measuring a second concentration of the corresponding total protein in the sample, wherein the citrullinated protein or peptide is citrullinated at the citrullination site, and the corresponding total protein encompasses both modified and unmodified forms of the protein, and optionally (iii) determining a citrullination ratio, the citrullination ratio being the ratio of the first concentration to the second concentration.

[0255] 45. The method according to embodiment 44, wherein the method includes comparing the citrullination ratio of the biological sample with the citrullination ratio of a control biological sample.

[0256] 46. ​​The method according to any one of embodiments 42-43 or 45, wherein the control biological sample and the biological sample are from the same subject.

[0257] 47. The method according to embodiment 46, wherein the control biological sample is a biological sample obtained from the subject prior to treatment with the PAD4 modulator.

[0258] 48. The method according to any one of embodiments 42-43 or 45-46, wherein the control biological sample has not been exposed to the PAD4 modulator.

[0259] 49. The method according to any one of embodiments 42-43 or 45-46, wherein the control biological sample has been exposed to a treatment different from that of the biological sample.

[0260] 50. The method according to any one of embodiments 40-49, wherein the PAD4 modulator is a PAD4 inhibitor.

[0261] 51. The method according to any one of embodiments 40 or 42-50, the method further comprising selecting or adjusting the dose of the PAD4 modulator based on the outcome of the assessment.

[0262] 52. An in vitro method for evaluating the activity of a PAD4 modulator, the method comprising:

[0263] (i) The biological samples obtained from the subjects will be divided into multiple biological samples.

[0264] (ii) Contact each of the plurality of biological samples with a different dose of the PAD4 modulator, and

[0265] (iii) For each of the plurality of biological samples, assess the citrullination of PAD4-dependent citrullination sites on proteins or peptide fragments present in the biological sample.

[0266] 53. The method according to embodiment 52, wherein the PAD4 modulator is a PAD4 inhibitor.

[0267] 54. The method according to embodiment 52 or 53, the method further comprising calculating the IC50 of the PAD4 modifier based on the outcome of the assessment.

[0268] 55. The method according to any one of embodiments 52-54, the method further comprising selecting or adjusting the dose of the PAD4 modulator to be administered to the subject based on the outcome of the assessment.

[0269] 56. An in vitro method for evaluating the citrullination of endogenous PAD4, the method comprising:

[0270] (a) Incubate whole blood samples from the subjects at 35ºC–40ºC for the incubation period.

[0271] (b) After the incubation period, plasma or supernatant is separated from the whole blood sample, and

[0272] (c) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the plasma or supernatant.

[0273] 57. The method according to embodiment 56, wherein the incubation period is 48 to 96 hours.

[0274] 58. The method according to any one of embodiments 56 or 57, wherein the incubation is carried out at a temperature of 36ºC to 39ºC, at a temperature of 36ºC to 38ºC, or at a temperature of 37ºC.

[0275] 59. The method according to any one of embodiments 56-58, wherein the method comprises enzymatically digesting the polypeptide in the plasma or supernatant prior to the evaluation.

[0276] 60. The method according to any one of embodiments 56-59, the method further comprising (i) incubating the plasma or supernatant together with a protein-depleting resin, and (ii) recovering the depleted plasma or supernatant flowing through the resin to obtain a depleted flow, optionally wherein the recovery comprises centrifugation.

[0277] 61. The method according to embodiment 60, wherein the method comprises enzymatically digesting the polypeptide in the depleted flow prior to the evaluation.

[0278] 62. The method according to embodiment 60 or 61, the method further comprising cleaning the enzymatically digested plasma or supernatant prior to the evaluation.

[0279] 63. The method according to any one of embodiments 56-62, wherein the whole blood sample and the plasma or supernatant separated therefrom have not been frozen and thawed.

[0280] 64. The method according to any one of embodiments 56-62, wherein the whole blood sample and / or the plasma or supernatant separated therefrom has been frozen and thawed.

[0281] 65. The method according to embodiment 64, wherein the method includes freezing the plasma or supernatant after the separation, and then thawing the plasma or supernatant prior to the evaluation.

[0282] 66. The method according to any one of embodiments 56-65, wherein the whole blood sample has been exposed to the PAD4 modifier.

[0283] 67. The method according to embodiment 66, wherein the whole blood sample has been exposed to the PAD4 modulator in vitro.

[0284] 68. The method according to embodiment 66, wherein the whole blood sample has been exposed to the PAD4 modulator in the subject due to administration of the PAD4 modulator to the subject.

[0285] 69. An in vitro method for assessing changes in citrullination in a subject, the method comprising:

[0286] (a) Incubate whole blood samples from the subject at 35ºC–40ºC for an incubation period, wherein the samples are obtained from the subject after administration of the PAD4 modulator.

[0287] (b) After the incubation period, plasma or supernatant is separated from the whole blood sample, and

[0288] (c) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the plasma or supernatant.

[0289] 70. The method according to embodiment 69, wherein the method further comprises

[0290] (d) Incubate a second whole blood sample from the subject at 35ºC–40ºC for an incubation period, wherein the second whole blood sample was obtained from the subject prior to administration of the PAD4 modulator to the subject.

[0291] (e) After the incubation period, plasma or supernatant is separated from the second whole blood sample, and

[0292] (f) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in plasma or supernatant from the second whole blood sample.

[0293] 71. The method according to embodiment 70, the method further comprising comparing the outcome of the assessment in step (f) with the outcome of the assessment in step (c).

[0294] 72. A method for evaluating the effect of PAD4 regulators, the method comprising:

[0295] (a) Expose whole blood samples from subjects to a PAD4 modulator in vitro.

[0296] (b) Incubate the whole blood sample at 35ºC-40ºC for the incubation period.

[0297] (c) After the incubation period, plasma or supernatant is separated from the whole blood sample, and

[0298] (d) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the plasma or supernatant.

[0299] 73. The method according to embodiment 72, wherein the method further comprises

[0300] (e) Incubate the control whole blood sample at 35ºC-40ºC for the incubation period.

[0301] (f) After the incubation period, control plasma or supernatant is separated from the control whole blood sample, and

[0302] (g) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the control plasma or supernatant.

[0303] 74. The method according to embodiment 73, the method further comprising comparing the outcome of the assessment in step (g) with the outcome of the assessment in step (d).

[0304] 75. An in vitro method for evaluating PAD4-dependent citrullination, the method comprising:

[0305] The citrullination of citrullinated sites on proteins or peptide fragments present in plasma or supernatant separated from whole blood samples that have been incubated at 35ºC–40ºC for an incubation period is evaluated.

[0306] 76. The method according to embodiment 75, wherein the plasma or supernatant has been frozen, and the method includes thawing the plasma or supernatant prior to the evaluation.

[0307] 77. A method, the method comprising:

[0308] (a) Immunoenriching a sample with a target protein to form an immunoenriched sample, wherein the immunoenrichment comprises contacting the sample with an antibody that specifically binds to both the citrullinated and non-citrullinated forms of the target protein, the target protein comprising a citrullinated site, and

[0309] (b) Evaluate the citrullination at the citrullination site of the immunoenriched sample, wherein the sample is optionally a serum or plasma sample.

[0310] 78. A method comprising:

[0311] (a) Immunoenriching a sample with a target protein or peptide to form an immunoenriched sample, wherein the immunoenrichment comprises contacting the sample with an antibody that binds to both the citrullinated and non-citrullinated forms of the target protein or peptide, the target protein or peptide containing a citrullinated site.

[0312] (b) Enzymatically digesting the immunoenriched sample before or after the immunoenrichment to form digested peptides from the target protein, and

[0313] (c) Evaluate the citrullination of the digested peptide at the citrullination site, optionally wherein the sample is a serum or plasma sample.

[0314] 79. A method, the method comprising:

[0315] (a) Immunoenrichment of the sample by contacting it with a fixed antibody, the fixed antibody binding to both citrullinated and non-citrullinated forms of the target protein, the target protein containing citrullinated sites.

[0316] (b) Elute the proteins bound to the fixed antibodies.

[0317] (d) Enzymatic digestion of eluted proteins to form digestible peptides, and

[0318] (e) Evaluate the citrullination of the digested peptide at the citrullination site, optionally wherein the sample is a serum or plasma sample.

[0319] 80. The method according to embodiment 77, 78 or 79, wherein the immunoenrichment comprises incubating the sample with the immobilized antibody for at least 30 minutes.

[0320] 81. The method according to embodiment 80, wherein the incubation is carried out at a temperature of 22ºC-28ºC.

[0321] 82. The method according to embodiments 80-81, wherein the incubation lasts for a period of 30 to 90 minutes.

[0322] 83. The method according to any one of embodiments 77-82, wherein the immunoenrichment includes oscillation during the incubation.

[0323] 84. The method according to any one of embodiments 77-83, wherein the immunoenrichment comprises removing the fixed antibody from the sample and washing the fixed antibody prior to the elution.

[0324] 85. The method according to any one of embodiments 79-84, wherein the elution comprises washing the fixed antibody with an elution composition under acidic conditions.

[0325] 86. The method according to embodiment 85, wherein the elution composition comprises a detergent.

[0326] 87. The method according to embodiment 86, wherein the detergent is an amphoteric detergent.

[0327] 88. The method according to any one of embodiments 85-87, wherein the method includes neutralizing the eluted protein in a buffer before evaluation and optionally before enzymatic digestion.

[0328] 89. The method according to any one of embodiments 77-88, wherein the antibody is immobilized by attaching to a solid surface.

[0329] 90. The method according to any one of embodiments 77-89, wherein the evaluation of citrullination includes measuring a first concentration of citrullinated peptides in the digested peptide and a second concentration of characteristic peptides in the digested peptide, and optionally determining a citrullination ratio, wherein the citrullination ratio is the ratio of the first concentration to the second concentration.

[0330] 91. The method according to any one of embodiments 77-90, wherein the method includes denaturing the proteins in the immunoenriched sample prior to the evaluation.

[0331] 92. The method according to any one of embodiments 79-90, wherein the method includes denaturing the eluted protein prior to the evaluation.

[0332] 93. The method according to embodiment 92, wherein the method includes denaturing the eluted protein prior to enzymatic digestion of the eluted protein.

[0333] 94. The method according to any one of embodiments 77-93, the method comprising diluting the sample prior to the immunoenrichment, and performing the immunoenrichment on the diluted sample.

[0334] 95. The method according to embodiment 94, wherein the method includes diluting the sample by 2 to 1000 times.

[0335] 96. The method according to embodiment 94 or 95, wherein the volume of the diluted sample is at least 5 μl.

[0336] 97. The method according to any one of embodiments 40 to 96, wherein the evaluation includes mass spectrometry (MS).

[0337] 98. The method according to embodiment 97, wherein the evaluation includes liquid chromatography and mass spectrometry (LC-MS).

[0338] 99. The method according to embodiment 98, wherein the evaluation includes selective reaction monitoring and chromatographic separation.

[0339] 100. The method according to any one of embodiments 77-99, wherein the method includes evaluating the citrullination of two or more target proteins, and the antibody includes an antibody that specifically binds to the citrullinated and non-citrullinated forms of each of the two or more target proteins.

[0340] 101. A method, the method comprising

[0341] (i) Expose biological samples from the subject to exogenous PAD4, and

[0342] (ii) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in biological samples.

[0343] 102. The method according to embodiment 101, wherein the biological sample comprises plasma or serum.

[0344] 103. The method according to embodiment 101 or 102, wherein the method includes preparing the biological sample for evaluation prior to the evaluation.

[0345] 104. The method according to embodiment 103, wherein the preparation includes enzymatic digestion of proteins in the biological sample to produce digested peptides and optionally cleaning up the digested peptides.

[0346] 105. The method according to embodiment 103 or 104, wherein the preparation includes depleting proteins from the biological sample that are not the target of the evaluation.

[0347] 106. The method according to any one of embodiments 105, wherein depletion includes depletion of human serum albumin.

[0348] 107. The method according to any one of embodiments 101-106, wherein the preparation includes using an antibody to immunoenrich the biological sample with the protein or a peptide fragment thereof, the antibody binding to a citrullinated or non-citrullinated form of the protein or peptide.

[0349] 108. The method according to any one of embodiments 40 to 107, wherein the protein or its peptide fragment or the target protein is selected from one or more of the following: α-2-HS-glycoprotein, α-2-macroglobulin, albumin, antithrombin-III, apolipoprotein AI, apolipoprotein A-IV, apolipoprotein B-100, apolipoprotein E, apolipoprotein L1, C4b-binding protein α chain, ceruloplasmin, clusteringin, complement C2, complement C3, complement C4-A, complement C4-B, complement factor B, complement factor H, complement factor I, complement component C9, fibrinogen α chain, fibrinogen β chain, fibrinogen γ chain, fibronectin, galactochondrin-3-binding protein, coagulant, haptoglobin, haptoglobin-related protein, hemoglobin Immunoglobulin binding protein, immunoglobulin heavy chain variable 1-3, immunoglobulin heavy chain variable 1-8, immunoglobulin heavy chain variable 1-46, immunoglobulin heavy chain variable 1-69, immunoglobulin heavy chain variable 1-69D, inter-α-trypsin inhibitor heavy chain H4, immunoglobulin λ-like polypeptide 1, immunoglobulin heavy chain constant μ, inter-α-trypsin inhibitor heavy chain H2, complement C4-A isoform 2, plasminogen, plasminogen-like protein A, α-1-microglobulin / biskunitz inhibitor precursor (protein AMBP), prothrombin, proteoglycan 4, serum transferrin, serum amyloid A-1 protein, serum amyloid A-2 protein, serum amyloid A-4 protein, matrix lysozyme-1, vitamin D binding protein, and porphyrin.

[0350] 109. The method according to any one of embodiments 40-107, wherein the citrullinated site is selected from one or more citrullinated sites shown in Table A or Tables 3-18.

[0351] 111. The method according to any one of embodiments 40-107, wherein the citrullinated site is selected from one or more of the following:

[0352] R1391 of proteoglycan 4 (corresponding to R1391 of Q92954).

[0353] R573 of the fibrinogen α chain (corresponding to R573 of P02671).

[0354] R591 of the fibrinogen α chain (corresponding to R591 of P02671).

[0355] R573 of complement C3 (corresponding to R573 of P01024).

[0356] R748 for complement C3 (corresponding to R573 for P01024).

[0357] R688 of the heavy chain H4, an inhibitor of inter-α-trypsin (corresponding to R688 of Q14624).

[0358] R297 of protein AMBP (corresponding to R297 of P02760).

[0359] R715 of α-2-macroglobulin (corresponding to R715 of P01023).

[0360] R32 of gel sol protein (corresponding to R32 of P06396).

[0361] R59 of haptoglobin (corresponding to R59 of P00738), and

[0362] R651 of serum transferrin (corresponding to R651 of P02787).

[0363] 112. The method according to any one of embodiments 40-107,

[0364] The method includes evaluating citrullination of a peptide fragment, wherein the sequence of the peptide fragment includes one of the following sequences, wherein the citrullination site is indicated by an underscore: AITT R SGQTLSK (SEQ ID NO: 247), ESSSHPGIAEFPS R GK (SEQ ID NO: 248), QFTSSTSYN R GDSTFESK (SEQ ID NO: 249), SGQSED R QPVPGQQMTLK (SEQ ID NO: 250), ASHLGLA R SNLDEDIIAEENIVSR (SEQ ID NO: 251), QLGLPGPPDVPDHAAYHPF R R (SEQ ID NO: 252), GPC R AFIQLWAFDAVK (SEQ ID NO: 253), VGFYESDVMG R GHAR (SEQ ID NO: 254), ATAS R GASQAGAPQGR (SEQ ID NO: 255), L R TEGDGVYTLNDK (SEQ ID NO: 256) and DLLF R DDTVCLAK (SEQ ID NO: 257).

[0365] 113. The method according to any one of embodiments 40-107, wherein the protein or its peptide fragment or the target protein comprises fibrinogen α chain or its peptide fragment and / or colloid protein or its peptide fragment.

[0366] 114. The method according to embodiment 113, wherein the protein or a peptide fragment thereof or the target protein is a fibrinogen α chain or a peptide fragment thereof.

[0367] 115. The method according to embodiment 114, wherein the citrullination site is at R591 or R593 of P02671.

[0368] 116. The method according to embodiment 114 or 115, wherein the method includes (a) evaluating a QFTSSTSYN R Citrullination of the peptide containing the amino acid sequence of GDSTFESK (SEQ ID NO: 249), wherein the underlined R is the citrullination site, or (b) evaluation of peptides containing ESSSHHPGIAEFPS. R Citrullinated peptides of the amino acid sequence GK (SEQ ID NO: 248), wherein the underlined R is the citrullinated site.

[0369] 117. The method according to any one of embodiments 40-107, wherein the protein or a peptide fragment thereof or the target protein comprises a gel sol protein or a peptide fragment thereof.

[0370] 118. The method according to embodiment 117, wherein the citrullinated site is at R32 of P06396.

[0371] 119. The method according to embodiment 117 or 118, wherein the method includes evaluating ATAS. R Citrullinated peptide of the amino acid sequence GASQAGAPQGR (SEQ ID NO: 255), wherein the underlined R is the citrullinated site.

[0372] 120. A method for assessing citrullination at citrullination sites, the method comprising:

[0373] (i) Measuring a first concentration of citrullinated protein or citrullinated peptide in a biological sample by mass spectrometry (MS), wherein the citrullinated protein or citrullinated peptide is citrullinated at a citrullination site, and

[0374] (ii) Measure the second concentration of the corresponding total protein in the biological sample by MS.

[0375] 121. The method according to embodiment 120, wherein the method comprises (i) measuring a first concentration of a citrullinated peptide from a target protein in a biological sample by means of MS, wherein the citrullinated peptide is citrullinated at a citrullination site, and (ii) measuring a second concentration by means of measuring the concentration of a characteristic peptide from the target protein in the biological sample by means of MS.

[0376] 122. The method according to embodiment 120 or 121, the method further comprising calculating the ratio of the first concentration to the second concentration.

[0377] 123. The method according to any one of embodiments 120 to 122, wherein the method includes measuring the first concentration and the second concentration of each of a plurality of different proteins or peptides.

[0378] 124. The method according to embodiment 123, wherein the method includes measuring the first concentration of each of two different peptides, each of the peptides being a non-overlapping fragment of the same protein and containing different citrullinated sites.

[0379] 125. The method according to embodiment 123 or 124, wherein the method includes calculating the ratio of the first concentration to the second concentration of each of the plurality of different proteins or peptides.

[0380] 126. The method according to any one of embodiments 120 to 125, wherein the citrullination site is a site listed in Table A or one or more of Tables 3-18.

[0381] 127. The method according to any one of embodiments 120 to 126, wherein (i) the citrullinated protein is selected from α-2-HS-glycoprotein, α-2-macroglobulin, albumin, antithrombin-III, apolipoprotein AI, apolipoprotein A-IV, apolipoprotein B-100, apolipoprotein E, apolipoprotein L1, C4b-binding protein α chain, ceruloplasmin, clusteringin, complement C2, complement C3, complement C4-A, complement C4-B, complement factor B, complement factor H, complement factor I, complement component C9, fibrinogen α chain, fibrinogen β chain, fibrinogen γ chain, fibronectin, galactochondrin-3-binding protein, coagulant, haptoglobin, haptoglobin-related protein, hemoglobin-binding protein, immunoglobulin heavy chain variable 1-3, immunoglobulin Protein heavy chains variable 1-8, immunoglobulin heavy chains variable 1-46, immunoglobulin heavy chains variable 1-69, immunoglobulin heavy chains variable 1-69D, inter-α-trypsin inhibitor heavy chain H4, immunoglobulin λ-like polypeptide 1, immunoglobulin heavy chain constant μ, inter-α-trypsin inhibitor heavy chain H2, complement C4-A variant 2, plasminogen, plasminogen-like protein A, α-1-microglobulin / bisKunitz inhibitor precursor, prothrombin, proteoglycan 4, serum transferrin, serum amyloid A-1 protein, serum amyloid A-2 protein, serum amyloid A-4 protein, matrix lysozyme-1, vitamin D-binding protein, and porphyrin; or (ii) the citrullinated peptide is a peptide fragment of the protein listed in (i).

[0382] 128. The method according to any one of embodiments 120 to 127, wherein the citrullinated peptide comprises a sequence selected from the peptide sequences listed in Tables 3-18.

[0383] 129. The method according to any one of embodiments 120 to 128, wherein (i) the citrullinated protein is selected from proteoglycan 4, fibrinogen α chain, complement C3, inter-α-trypsin inhibitor heavy chain H4, protein AMBP, α-2 macroglobulin, colloidin, haptoglobin and serum transferrin, or (ii) the citrullinated peptide is a peptide fragment of a protein listed in (i).

[0384] 130. The method according to any one of embodiments 120 to 129, wherein the citrullinated site is selected from...

[0385] R1391 of proteoglycan 4 (corresponding to R1391 of Q92954).

[0386] R573 of the fibrinogen α chain (corresponding to R573 of P02671).

[0387] R591 of the fibrinogen α chain (corresponding to R591 of P02671).

[0388] R573 of complement C3 (corresponding to R573 of P01024).

[0389] R748 for complement C3 (corresponding to R573 for P01024).

[0390] R688 of the heavy chain H4, an inhibitor of inter-α-trypsin (corresponding to R688 of Q14624).

[0391] R297 of protein AMBP (corresponding to R297 of P02760).

[0392] R715 of α-2-macroglobulin (corresponding to R715 of P01023).

[0393] R32 of gel sol protein (corresponding to R32 of P06396).

[0394] R59 of haptoglobin (corresponding to R59 of P00738), and

[0395] R651 of serum transferrin (corresponding to R651 of P02787).

[0396] 131. The method according to any one of embodiments 120 to 130, wherein the method comprises measuring the amount of a citrullinated peptide comprising a peptide sequence selected from AITT. R SGQTLSK (SEQ ID NO: 247), ESSSHPGIAEFPS R GK (SEQ ID NO: 248), QFTSSTSYN R GDSTFESK (SEQ ID NO: 249), SGQSED R QPVPGQQMTLK (SEQ ID NO: 250), ASHLGLA R SNLDEDIIAEENIVSR (SEQ ID NO: 251), QLGLPGPPDVPDHAAYHPF R R (SEQ ID NO: 252), GPC R AFIQLWAFDAVK (SEQ ID NO: 253), VGFYESDVMG R GHAR (SEQ ID NO: 254), ATAS R GASQAGAPQGR (SEQ ID NO: 255), L RTEGDGVYTLNDK (SEQ ID NO: 256) and DLLF R DDTVCLAK (SEQ ID NO: 257),

[0397] In each case, the underlined R is the citrullination site.

[0398] 132. The method according to any one of embodiments 120-131, wherein the citrullinated protein or the citrullinated peptide comprises a fibrinogen α chain and / or a gelling protein or a peptide fragment thereof.

[0399] 133. The method according to embodiment 132, wherein the citrullinated protein or the citrullinated peptide is a fibrinogen α chain or a peptide fragment thereof.

[0400] 134. The method according to embodiment 133, wherein the citrullination site is at R591 or R593 of P02671.

[0401] 135. The method according to embodiment 133 or 134, wherein the citrullinated peptide comprises QFTSSTSYN R The amino acid sequence of GDSTFESK (SEQ ID NO: 249), wherein the underlined R is the citrullinated site, or wherein the citrullinated peptide contains ESSSHHPGIAEFPS. R The amino acid sequence of GK (SEQ ID NO: 248), wherein the underlined R is the citrullinated site.

[0402] 136. The method according to embodiment 132, wherein the citrullinated protein or the citrullinated peptide comprises a gel sol protein or a peptide fragment thereof.

[0403] 137. The method according to embodiment 136, wherein the citrullinated site is at R32 of P06396.

[0404] 138. The method according to embodiment 136 or 137, wherein the citrullinated peptide comprises ATAS R The amino acid sequence of GASQAGAPQGR (SEQ ID NO: 255), wherein the underlined R is the citrullinated site.

[0405] 139. The method according to any one of embodiments 120-138, wherein the biological sample is obtained from a subject.

[0406] 140. The method according to embodiment 139, wherein the biological sample is whole blood, plasma, serum, blood supernatant, or synovial fluid.

[0407] 141. The method according to embodiment 139 or 140, wherein the method further comprises comparing the first concentration and the second concentration with a first concentration and the second concentration obtained from a control biological sample.

[0408] 142. The method according to embodiments 139, 140 or 141, wherein the method further comprises determining one or more of the following based on the first concentration and the second concentration or the ratio of the first concentration and the second concentration: probability of clinical outcome, risk of developing citrullination-related disease and diagnosis of citrullination-related disease.

[0409] 143. The method according to embodiment 142, wherein the method further comprises selecting a subject from whom the biological sample is derived for the treatment of a citrullination-related disease.

[0410] 144. The method according to embodiment 143, wherein the PAD4 modulator is a PAD4 inhibitor.

[0411] 145. The method according to any one of embodiments 1-144, wherein the biological sample is obtained from a subject who is ACPA positive.

[0412] 146. The method according to any one of embodiments 1-145, wherein the biological sample is obtained from a subject who is positive for endogenous PAD4 antibody.

[0413] 147. The method according to embodiment 146, wherein the subject is positive for an endogenous PAD4 activation antibody.

[0414] 148. The method according to any one of embodiments 1-147, wherein the biological sample is obtained from a subject who has been diagnosed with citrullination-related disease or is at risk of developing citrullination-related disease.

[0415] 149. The method according to embodiment 148, wherein the citrullination-related disease is an autoimmune disorder.

[0416] 150. The method according to embodiment 148, 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).

[0417] 151. The method according to any one of embodiments 1-144, wherein the biological sample is obtained from a subject who is a normal healthy subject.

[0418] In other embodiments, any of the method embodiments listed above that include a PAD4 modulator can also be performed using other therapeutic agents. Non-limiting examples of such other therapeutic agents include agents that can be used to treat citrullination-related diseases, and agents that target molecules involved in biological pathways that may also involve PAD4 (such as, for example, NETosis or METosis). Therefore, in such methods described above, this other therapeutic agent can replace the PAD4 modulator. In any embodiment of the above embodiments, where a PAD4 modulator is included in the method, the PAD4 modulator may, in some cases, be a PAD4 inhibitor, examples of which are provided throughout this disclosure.

[0419] In any of the embodiments described above, in some cases, the assessment of citrullination can be performed, for example, by MS (including by LC-MS). In any of the embodiments described above, unless a specific citrullination site or protein or peptide is listed, the citrullination sites used for assessment can be as listed, for example, in the embodiments described above. In any method herein, unless otherwise stated above, the biological sample can be any such sample listed herein, including but not limited to biological fluid samples such as blood, plasma, serum, blood supernatant, synovial fluid, lymph, pleural effusion, interstitial fluid, sweat, tears, sputum, or urine, and tissue samples. Also as stated above, in any method herein, the citrullination ratio (described below) and / or the concentration of citrullinated proteins or peptides and the corresponding total protein concentration can be determined as part of the method. Additionally, in any method described above, the biological sample can be prepared in various ways prior to assessment, such as dilution, freezing and thawing, protein denaturation, enzymatic digestion of proteins, separation of supernatant from the sample for analysis, etc. The above implementation scheme can also be applied to control samples as well as to target biological samples, for example, to allow for comparison of citrullination assessment between the target sample and the control.

[0420] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive on the claims. References cited herein are incorporated in their entirety by reference, and the amino acid sequences of proteins available herein, accessible at www.uniprot.org via their UniProt accession numbers, are also incorporated herein by reference. Attached Figure Description

[0421] Figure 1The antibody hz13-5 D31E showed dose-dependent inhibition of human PAD4 at four concentrations ranging from 13.5 nM (1 µg / mL) to 108 nM (8 µg / mL).

[0422] Figures 2A-2F Reduced extracellular citrullinated histone 3 in human CD14+ monocytes stimulated with lipopolysaccharide (LPS) treated with a specified concentration of the indicated antibody or with an isotype control antibody. Figure 2A The levels of citrullinated histone 3 in monocytes incubated with hz13-5 are shown. Figure 2B The levels of citrullinated histone 3 in monocytes incubated with hz13-5 D31E are shown. Figure 2C The levels of citrullinated histone 3 in monocytes incubated with hz20-2 are shown. Figure 2D The levels of citrullinated histone 3 in monocytes incubated with hz20-7 are shown. Figure 2E The levels of citrullinated histone 3 in monocytes incubated with hz13-3 are shown. Figure 2F The levels of citrullinated histone 3 in monocytes incubated with hz13-12 are shown.

[0423] Figures 3A-3E This demonstrates the efficacy of anti-human PAD4 mAb and its humanized derivatives in the LPS ALI PD model. Figure 3A , Figure 3B and Figure 3C The effects of clone 20, clone 20-based humanized hz20-2 antibody, and clone 20-based humanized hz20-7 antibody on extracellular citrullinated H3 at the indicated doses are shown relative to total extracellular H3; the percentage reduction indicates the reduction relative to the isotype control condition. Figure 3D and Figure 3E The effects of the clone 20-based hz20-2 antibody and the clone 20-based hz20-7 antibody on extracellular citrullinated ITIH4 at the indicated doses are shown relative to total extracellular ITIH4; the percentage reduction indicates the reduction relative to the isotype control condition. "Mpk" = mg / kg. "IC" = isotype control antibody. "Untreated" = not nebulized with LPS.

[0424] Figures 4A-4E Demonstrates the efficacy of anti-human PAD4 mAb clone 13 and humanized derivatives in the LPS ALI PD model. Figure 4A , Figure 4B and Figure 4CThe effects of clone 13, clone 13-based humanized hz13-12 antibody, and clone 13-based humanized hz13-5 antibody on extracellular citrullinated H3 at the indicated doses are shown relative to total extracellular H3; the percentage reduction indicates the reduction relative to the isotype control condition. Figure 4D and Figure 4E The effects of the clone 13-based hz13-12 antibody and the clone 13-based hz13-5 antibody on extracellular citrullinated ITIH4 at the indicated doses are shown relative to total extracellular ITIH4; the percentage reduction indicates the reduction relative to the isotype control condition. "Mpk" = mg / kg. "IC" = isotype control antibody. "Untreated" = not nebulized with LPS.

[0425] Figures 5A-5E This shows the experimental design and human PAD4 endpoint of the LPS AJI PD study using Hu-PAD4 knock-in mice. Figure 5A Show the research timeline. Figure 5B This section shows an overview of the experimental procedure, including treatment at the knee joint and extraction of citrullinated proteins from the explant tissue. It also shows extracellular Cit-PRG4 in the patellar explant supernatant. Figure 5C ), extracellular Cit-ITIH4 ( Figure 5D ) and PAD4 ( Figure 5E ).

[0426] Figures 6A-6C Demonstrates the efficacy of anti-human PAD4 antibody in LPS AJI PD studies. Shows the efficacy of clone 13-based hz13-12 antibody relative to total extracellular ITIH4. Figure 6A ), HZ13-5 antibody based on clone 13 ( Figure 6B ) and HZ20-2 antibody based on clone 20 ( Figure 6C Effect of the drug on extracellular citrullinated ITIH4 at the indicated dose; percentage reduction indicates reduction relative to isotype control conditions. "Mpk" = mg / kg. "IC" = isotype control antibody. "Untreated" = no pterostilbene injection. "PBS" = untreated, phosphate-buffered saline was injected instead of LPS.

[0427] Figures 7A-7C Demonstrates the efficacy of anti-human PAD4 antibody in the LPS AJI PD study. Shows extracellular Cit-PRG4 in patellar graft supernatant. Shows the relative concentration of hz13-12 antibody based on clone 13 compared to total extracellular PRG4. Figure 7A ), HZ13-5 antibody based on clone 13 ( Figure 7B ) and HZ20-2 antibody based on clone 20 ( Figure 7CEffect of the drug on extracellular citrullinated PRG4 at the indicated dose; percentage reduction indicates reduction relative to isotype control conditions. "Mpk" = mg / kg. "IC" = isotype control antibody. "PBS" = phosphate-buffered saline. "Untreated" = no LPS injection.

[0428] Figures 8A-8B This demonstrates the efficacy of anti-human PAD4 antibodies hz13-5 and hz13-5 D31E in the LPS AJI study. The antibodies inhibit ITIH4 (… Figure 8A ) and PRG4 ( Figure 8B Citrullination of HZ13-5 D31E antibody was observed; the results showed dose-dependent inhibition of the antibody. "Mpk" = mg / kg. "IC" = isotype control antibody. "Untreated" = no LPS injection.

[0429] Figures 9A-9D The in vitro induction and inhibition of citrullination in incubated blood are shown. Whole blood samples were treated with Ab hz13-5 D31E, PBS (control), or isotype control and incubated at 37ºC for 48 h to induce citrullination. PBS-0h shows the citrullination level before induction. Samples were analyzed using whole proteomics. Citrullination of the peptides listed in Table 10 was evaluated. The mean citrullination % for each peptide is shown for 6 donors, calculated as the average ratio of citrullinated peptide intensity to total protein intensity * 100%, where the total protein intensity was measured by measuring the intensity of the characteristic peptide. Black bars: first blood draw; gray bars: repeat blood draw after 1 week. Figure 9A Results showing the gel sol protein (GSN) peptide. Figure disclosed SEQ ID NO: 405. Figure 9B Results showing complement C3 (C3) peptide. Figure disclosed SEQ ID NO:406. Figure 9C The results show the results of the serine inhibitory protein C1 peptide. Figure disclosed is SEQ ID NO: 407. Figure 9D Results showing fibrinogen A (FGA) peptide. Figure disclosed SEQ ID NO: 408.

[0430] Figures 10A-10C The results showed that citrullination of endogenous serum proteins by exogenous PAD4 was inhibited by hz13-5 D31E in a dose-dependent manner. Samples were analyzed using targeted proteomics methods on the nanoLC-TimsTOF platform. Citrullination of the peptides listed in Table 15 was evaluated. The citrullination percentage and its targeting are shown. Figures 9A-9D The calculation is based on the above. Figure 10A Display FGA peptide ESSSHHPGIAEFPS RInhibition of citrullination of GK (SEQ ID NO: 248). Figure 10B Display FGA peptide QFTSSTSYN R Inhibition of citrullination of GDSTFESK (SEQ ID NO: 249). Figure 10C This indicates inhibition of citrullination of the PRG4 peptide.

[0431] Figures 11A-11B The in vitro induction and inhibition of citrullination were confirmed using targeted proteomics in incubated blood (from the first blood draw). Whole blood samples were treated with Ab hz13-5 D31E, PBS (control), or an isotype control and incubated at 37ºC for 48 h. PBS-0h showed the citrullination level before induction. Samples were analyzed using targeted proteomics. Citrullination of GSN and FGA peptides was assessed; the sequences of these peptides are provided in Table 16. Figures 9A-9D (N = 6 donors) The average citrullination percentage is calculated as described above. The error bars are the standard error of the average. Figure 11A The results show the GSN peptide. Figure 11B The results show the FGA peptide.

[0432] Figure 12 This study demonstrates targeted proteomics analysis of ex vivo citrullinated FGA in TruCulture samples following immunocapture enrichment. The FGA peptide QFTSSTSYN was measured on an LC-TripleQuad 7500 system. R GDSTFESK (SEQ ID NO:249). Representative chromatograms from two different donor samples are shown, with citrullination percentages measured at 0.5% (left inset) and 2.6% (right inset). (See also: For example, regarding...) Figures 9A-9D The above is used to calculate citrullination. Figure 12 It is also publicly disclosed as "QFTSSTSYN" as SEQ ID NO: 409. R GDSTFES.

[0433] Figures 13A-13B The results showed that the hz13-5 D31E antibody maintained its efficacy in the presence of endogenous PAD4 antibodies from RA patients. Figure 13A This image shows purified IgG PAD4 autoantibodies measured by OD450 in ELISA from serum samples of 21 RA patients and 10 healthy control subjects (NHV). Figure 13BThe image shows the inhibition of H3 citrullination by hz13-5 D31E in the presence of purified IgG from RA (solid square) or NHV (hollow square) serum. The dashed line indicates H3 citrullination of PAD4 in the presence of purified IgG from RA donors (bottom line) and NHV donors (top line) in the absence of hz13-5 D31E.

[0434] Figure 14 This chart shows the plasma concentrations (in pM) of citrullinated proteoglycan 4 (Cit-PRG4) from patients with rheumatoid arthritis (RA) and healthy volunteers (NHV). Error bars represent mean ± SD. For this dataset, 30 RA and 30 NHV samples were analyzed. Detailed Implementation

[0435] I. Definition

[0436] Unless otherwise defined, scientific and technical terms used in connection with this invention should have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms should include plural terms, and plural terms should include singular terms.

[0437] In this application, unless otherwise stated, the use of “or” means “and / or”. In the context of multiple dependent claims, the use of “or” is only used alternatively to refer back to more than one of the foregoing independent or dependent claims. Similarly, terms such as “element” or “component” cover both elements and components comprising one unit and elements and components comprising more than one subunit, unless otherwise expressly stated.

[0438] Exemplary techniques used in conjunction with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, liposome transfection), enzymatic reactions and purification techniques are described in, for example, the following literature: Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), and others.

[0439] As used herein, the term "about" refers to a numerical value, including, for example, integers, fractions, and percentages, whether explicitly stated or not. The term "about" generally refers to a range of numerical values ​​(e.g., + / - 5% to 10% of the listed range) that would be considered equivalent to the listed values ​​(e.g., having the same function or result). When the term "about" precedes a list of numerical values ​​or ranges, the term modifies all values ​​or ranges provided in the list. In some cases, the term "about" may include a numerical value rounded to the nearest significant figure.

[0440] The terms “subject” and “patient” are used interchangeably in this document to refer to humans unless otherwise explicitly indicated (i.e., mouse subjects, etc.).

[0441] In this document, "biological sample" refers to a sample obtained from a biological source, such as from a subject. A "biological sample" can include fluids and / or tissues, and therefore can be a "biological fluid sample" or a "tissue sample," and can be processed, such as by centrifugation or mixing with other agents, to facilitate analysis. For example, some biological fluid samples may contain a supernatant after separation of particulate and fluid substances. In some cases, a biological sample contains, for example, blood or blood components, such as whole blood, serum, plasma, or blood supernatant. In some cases, it may contain synovial fluid. In other cases, a biological sample may be a tissue sample, such as a tumor sample or a lesion sample.

[0442] The term "peptide" refers to a polymer of amino acid residues and is not limited to a minimum length. A "protein" may contain one or more peptides. Such polymers of amino acid residues may contain native or non-native amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. The definition covers full-length proteins and fragments thereof. The term also includes post-expression modifications of peptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Furthermore, for the purposes of this invention, "peptide" or "protein" refers to peptides or proteins that include modifications to the native sequence, such as deletions, additions, and substitutions (often of conserved nature), provided the protein maintains the desired activity. These modifications may be intentional (e.g., by site-directed mutagenesis) or accidental (e.g., by mutations in the host producing the protein or errors due to PCR amplification). A protein may contain two or more peptides.

[0443] The terms "peptide fragment" or "peptide" (e.g., "protein or peptide fragment thereof") refer to a portion of the polypeptide or protein. In some cases, analysis of the chemical modifications of amino acid residues in a polypeptide can be performed, for example, by analyzing a peptide fragment of the polypeptide that includes the amino acid residues in question.

[0444] Some amino acids within a protein or its peptide fragments can be “citrullinated.” As used herein, this term refers to a protein or its peptide fragment containing at least one amino acid that has been converted from one of 20 naturally occurring amino acids to a citrullinated amino acid. For example, certain arginine (Arg, R) residues can be chemically modified to convert their side chains into the side chains of the amino acid citrulline (Cit). Therefore, such arginine residues and the corresponding peptides or proteins in which they are found are said to be citrullinated.

[0445] Amino acid residues converted to citrulline may also be referred to herein as “citrullination sites.” For example, a residue at a specific amino acid position within a protein can be citrullinated. In this case, that specific amino acid position may be referred to as a “citrullination site.” In some cases, citrullination sites herein can be identified based on an exemplary reference amino acid sequence of the protein. In cases 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 the citrullination site, and the site can be identified in other naturally occurring variants of the protein by sequence alignment with the reference sequence provided herein. When more than one residue in a given protein is found to be citrullinated, the protein may also have one or more such citrullination sites.

[0446] "PAD" or "protein arginine deiminoase" refers to an enzyme that catalyzes the conversion of arginine in proteins or peptides to citrulline under certain conditions. Examples of PADs include, for example, PAD4 and PAD2.

[0447] As used herein, “PAD4” or “protein arginine deiminase 4” or “peptidyl arginine deiminase 4” refers to human PAD4 (huPAD4; UniProt ID: Q9UM07) unless otherwise explicitly stated (i.e., mouse PAD4, cynomolgus monkey PAD4, etc.). Exemplary human PAD4 amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0448] As used herein, a “PAD4 modulator” refers to a compound (e.g., a small molecule or macromolecule or biological product) 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. Modulation of PAD4 activity can be achieved through any mechanism and can be observed in vitro and / or in vivo. In some cases, a PAD4 modulator can modulate the activity of other proteins besides PAD4 (e.g., other PAD enzymes, such as PAD2); while in other cases, activity modulation may be specific to PAD4, and the PAD4 modulator does not modulate the activity of other proteins (e.g., other PAD enzymes, such as PAD2).

[0449] As used herein, the term "agonist" refers to a compound that increases the activity or function of at least one molecule bound to it, or otherwise activates or assists in the activation of said molecule. As used herein, the terms "antagonist" or "inhibitor" refer to a compound that decreases the activity or function of at least one molecule bound to it, or otherwise blocks or inhibits the activity or function of at least one molecule.

[0450] The terms “inhibition” or “inhibit” more generally refer to the reduction or cessation of any event (such as protein ligand binding), or the reduction or cessation of any phenotypic trait, or the reduction or cessation of the occurrence, extent, or likelihood of that trait. “Reduction” or “inhibit” means a reduction, decrease, or prevention of activity, function, and / or quantity compared to a reference. Inhibition or reduction is not necessarily complete. For example, in some embodiments, “reduction” or “inhibit” means the ability to cause an overall reduction of 20% or more. In another embodiment, “reduction” or “inhibit” means the ability to cause an overall reduction of 50% or more. In yet another embodiment, “reduction” or “inhibit” means the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more.

[0451] In some cases, PAD4 modulators can be small molecule compounds, or they can be large molecules such as antibodies, or they can be another type of biological product. The terms “anti-PAD4 antibody,” “PAD4-antibody,” “antibody that specifically binds to PAD4,” or “antibody that binds to PAD4,” and similar phrases refer to antibodies that specifically bind to PAD4 as defined herein. The term “antibody” as used herein refers to a molecule comprising at least complementary determining regions (CDRs) 1, CDR2, and CDR3 of a heavy chain and at least CDR1, CDR2, and CDR3 of a light chain, wherein said molecule is capable of binding to an antigen. The term is used in the broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, biantibodies, etc.), full-length antibodies, single-chain antibodies, antibody conjugates, and antibody fragments, provided they exhibit the desired PAD4-specific binding activity.

[0452] "Isolated" antibodies are antibodies that have been separated from components of their native environment. In some respects, antibodies are purified to a purity greater than 95% or 99%, as determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods used to assess antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0453] An "antigen" is the target of an antibody, that is, a molecule that specifically binds to an antibody. The term "epitope" refers to a site on a protein- or non-protein antigen that binds to an antibody. Epitopes on proteins can form from continuous amino acid extensions (linear epitopes) or contain discontinuous amino acids (conformational epitopes), for example, due to antigen folding (i.e., through the tertiary folding of protein antigens) that makes them spatially proximate. Linear epitopes are typically still bound to antibodies after protein antigens are exposed to denaturing agents, while conformational epitopes are typically destroyed after treatment with denaturing agents.

[0454] The term "heavy chain" refers to a polypeptide containing at least a heavy chain variable region, with or without a leader sequence. In some embodiments, the heavy chain contains at least a portion of the heavy chain constant region. The term "full-length heavy chain" refers to a polypeptide containing both a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0455] The term "light chain" refers to a polypeptide containing at least a light chain variable region and having or not having a leader sequence. In some embodiments, the light chain contains at least a portion of a light chain constant region. The term "full-length light chain" refers to a polypeptide containing both a light chain variable region and a light chain constant region and having or not having a leader sequence.

[0456] As used herein, a “complementarity-determining region” (“CDR”) refers to each of the highly variable regions of an antibody that determines antigen-binding specificity. Typically, an antibody contains six CDRs: three in the VH region (CDR-H1 or heavy chain CDR1, CDR-H2, CDR-H3) and three in the VL region (CDR-L1, CDR-L2, CDR-L3). Unless otherwise specified, CDRs are determined based on the sequence listing herein.

[0457] "Frame" or "FR" refers to residues in the variable region that are not part of the complementarity-determining region (CDR). The FRs in the variable region are typically composed of four FRs: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences usually appear in the VH (or VL) in the following sequence: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0458] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that participates in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A variable domain may contain a heavy chain (HC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and / or FR4; and a 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, as long as it retains antigen-binding activity. A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to specific antigens can be isolated by screening libraries containing complementary VL or VH domains using either the VH or VL domain of the antibody binding the antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0459] The "constant regions" of an antibody's light and heavy chains refer to additional sequence portions outside the FR and CDR and variable regions. Some antibody fragments may lack all or some of the constant regions. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH) (also called a variable heavy chain domain or heavy chain variable region), followed by three constant heavy chain domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL) (also called a variable light chain domain or light chain variable region), followed by a constant light chain (CL) domain.

[0460] The term "Fc region" used herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes both native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to Gly446 and Lys447 (EU number) at the C-terminus of the heavy chain. Antibodies produced by host cells may undergo post-translational cleavage of one or more (particularly one or two) amino acids at the C-terminus of the heavy chain. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain may include the full-length heavy chain, or it may include cleaved variants of the full-length heavy chain. This could be the case where the last two C-terminal amino acids of the heavy chain are glycine and lysine, respectively. Therefore, the C-terminal lysine or C-terminal glycine and lysine of the Fc region may or may not be present. Therefore, a “full-length heavy chain constant region” or “full-length antibody” (e.g., a human IgG1 antibody) includes IgG1 with both C-terminal glycine and lysine, IgG1 without C-terminal lysine, or IgG1 without both C-terminal glycine and lysine. Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in the following reference: Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0461] "Effective functions" refer to those biological activities attributable to the Fc region of an antibody, which vary across antibody isotypes. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0462] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The antibody light chain can be assigned to one of two types called kappa (κ) and lambda (λ) based on the amino acid sequence of its constant domain.

[0463] "Antibody fragment" refers to a molecule other than a complete antibody, which contains the portion of the complete antibody that binds to the antigen bound by the complete antibody (i.e., PAD4). Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of some antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0464] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this document to refer to antibodies that have a structure substantially similar to that of natural antibodies, or, in the case of IgG antibodies, heavy chains containing an Fc region as defined above.

[0465] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a specific source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0466] A “humanized” antibody is a chimeric antibody comprising amino acid residues from a nonhuman CDR and amino acid residues from a human FR. In some respects, a humanized antibody will comprise substantially all of at least one (and typically two) variable domains, wherein all or substantially all CDRs correspond to the CDRs of the nonhuman antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Optionally, a humanized antibody may comprise at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a nonhuman antibody) refers to an antibody that has undergone humanization.

[0467] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical and / or bind to the same epitopes, except for possible variant antibodies, such as those containing naturally occurring mutations or those generated during the production of the monoclonal antibody formulation, which are typically present in small quantities. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method.

[0468] "Multispecific" antibodies are antibodies that specifically bind to more than one target antigen, while "bispecific" antibodies are antibodies that specifically bind to two antigens. "Antibody conjugates" are antibodies conjugated to one or more heterologous molecules (including but not limited to therapeutic agents or tags).

[0469] The "percentage of amino acid sequence identity (%)" and "homology" for peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence after sequence alignment and the introduction of vacancies (if necessary) to achieve maximum sequence identity percentage. Alignments used to determine the percentage of amino acid sequence identity can be performed in a variety of ways well known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0470] The term "signal sequence" or "leader sequence" refers to the amino acid residue sequence located at the N-terminus of a polypeptide that facilitates the secretion of the polypeptide from mammalian cells. The leader sequence can be cleaved after the polypeptide is exported from mammalian cells, thereby forming the mature protein. Leader sequences can be natural or synthetic, and they can be heterologous or homologous to the protein to which they are attached. Non-limiting exemplary leader sequences also include leader sequences derived from heterologous proteins. In some embodiments, the antibody lacks a leader sequence. In some embodiments, the antibody comprises at least one leader sequence, which can be selected from natural antibody leader sequences and heterologous leader sequences.

[0471] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of: a base, specifically a purine or pyrimidine base (i.e., cytosine I, guanine (G), adenine (A), thymine (T), or uracil (U)); a sugar (i.e., deoxyribose or ribose); and a phosphate group. Typically, nucleic acid molecules are described by their base sequence, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are typically represented from 5' to 3'. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. Additionally, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases or chemically modified residues linked to a derived sugar or phosphate backbone. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the present invention in vitro and / or in vivo (e.g., in a host or patient). Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified.

[0472] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include those normally found in cells containing nucleic acid molecules, but which are located outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0473] "Isolated nucleic acid encoding anti-PAD4 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of anti-PAD4 antibody, including one or more such nucleic acid molecules in a single vector or a single vector, and one or more such nucleic acid molecules present at one or more locations in a host cell.

[0474] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell that has already been introduced therein. Some vectors are capable of guiding the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0475] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which foreign nucleic acids have been introduced, including the progeny of such cells.

[0476] In this disclosure, the terms “binds” or “binding” or “specific binding” and similar terms, when referring to, for example, proteins and their ligands or antibodies and their antigen targets or some other binding pair, mean that the binding affinity between the members of the binding pair is strong enough that no interaction occurs due to random molecular association (i.e., “non-specific binding”). Such binding typically requires a dissociation constant (K0). D The K0 is 1 μM or lower, and can typically involve 100 nM or lower. D .

[0477] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, 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 expressed as the dissociation constant (K0). D The affinity of an antibody for an antigen can be measured, for example, by methods commonly known in the art, such as surface plasmon resonance (SPR).

[0478] As used herein, “treatment” encompasses any administration or application of a therapeutic agent for a human disease, and includes suppressing the progression of a disease or a disease or one or more symptoms of a disease, suppressing or slowing the progression of a disease or a disease or one or more symptoms of a disease, preventing its development, partially or completely alleviating a disease or one or more symptoms of a disease, or preventing the recurrence of one or more symptoms of a disease.

[0479] As used herein, “autoimmune disease” or “autoimmune disorder” encompasses diseases characterized by a subject’s immune system attacking its own normal cells and tissues, and also encompasses immune-mediated diseases that may or may not be characterized by the presence of autoantibodies. Numerous non-limiting examples of autoimmune diseases are provided throughout this disclosure. Some non-limiting 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, an autoimmune disease is characterized by the presence of autoantibodies. The term “effective amount” or “therapeutic effective amount” refers to an amount of drug that is effective in treating a subject’s disease or disorder (e.g., partially or completely relieving one or more symptoms). In some embodiments, an effective amount refers to an amount that, at the necessary dose and time period, effectively achieves the desired therapeutic or preventative outcome.

[0480] Additional definitions of terms or phrases used in this document are provided in the following sections.

[0481] II. Methods for assessing citrullination

[0482] This disclosure specifically covers several methods for evaluating the citrullination modification activity of PAD4 modulators or other therapeutic agents, as well as methods for evaluating citrullination in biological samples from subjects. One or more citrullination sites may be located on a protein or peptide. In some cases, a peptide is a peptide fragment of a protein, for example, a digested peptide produced by the enzymatic digestion of a protein. Therefore, the expression "evaluating citrullination of a citrullination site" encompasses evaluating one citrullination site on a protein or peptide, as well as evaluating more than one citrullination site on a protein or peptide, and evaluating multiple citrullination sites. And therefore, although the disclosure herein may enumerate citrullination sites on proteins or peptides, "a (a)" in this expression means "one or more" citrullination sites, and citrullination of more than one protein or peptide may also be evaluated. Evaluating multiple citrullination sites may encompass evaluating citrullination sites on multiple proteins or multiple peptides. For example, multiple citrullination sites may be multiple citrullination sites on a protein or a peptide fragment of such a protein, and / or multiple citrullination sites may be multiple citrullination sites on multiple different proteins or peptides. While the methods for evaluating citrullination disclosed herein can be used to evaluate citrullination at any citrullination site, in some embodiments, the citrullination site (e.g., multiple citrullination sites) is the citrullination site disclosed herein. Any and all such combinations are considered herein.

[0483] The methods described herein particularly include a method for assessing citrullination at citrullination sites using mass spectrometry (MS). In some cases, the method includes assessment by liquid chromatography followed by MS (LC-MS). For example, liquid chromatography (e.g., HPLC, RPLC, nLC, µLC, etc.) can be used to separate peptides or proteins for analysis by MS. In some cases, the method includes measuring in a biological sample: (i) a first concentration of citrullinated protein or citrullinated peptide (which may be derived 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 in the sample (which includes both modified and unmodified forms of the protein). In some embodiments, the concentration of the corresponding total protein is measured by measuring the concentration of a characteristic peptide derived from the protein. The characteristic peptide is a peptide present in both modified and unmodified forms of the protein, and the characteristic peptide is itself an unmodified peptide. Therefore, when measuring the concentration of characteristic peptides present in a well sample by mass spectrometry, for example, the concentration of characteristic peptides represents the total protein concentration in the well. In some embodiments, the methods for measuring concentrations (i) and (ii) are performed by MS (such as LC-MS). In some embodiments, the method further includes calculating the citrullination ratio.

[0484] As used herein, “citrullination ratio” refers to the ratio of a first concentration (i) to a second concentration (ii). The citrullination ratio can be expressed in various ways, for example, as a percentage, or as any other fraction proportional to the aforementioned ratio of the first concentration to the second concentration.

[0485] In some cases, the method includes assessing citrullination at more than one citrullination site. Therefore, the method may include measuring a first concentration and a second concentration of each of a plurality of (meaning two or more) different proteins or peptides, each containing a citrullination site. In some such cases, the method includes measuring a first concentration and a second concentration of each of the plurality of proteins or peptides. In some such cases, at least two of the plurality of different peptides are non-overlapping fragments of the same protein and contain different citrullination sites; in those cases, the second concentration is the same for peptides that are fragments of the same protein. In some cases, the method includes calculating the ratio of the first concentration to the second concentration of each of the plurality of different proteins or peptides. Exemplary citrullination sites, proteins, and their peptide fragments are discussed below and provided in Tables A and 3-18 herein. In some cases, the biological sample is obtained from a subject. In some cases, the biological sample is whole blood, plasma, serum, blood supernatant, or synovial fluid. In other cases, the biological sample is a tissue sample. In some cases, the method further includes comparing the first concentration and the second concentration, or the ratio of the first concentration to the second concentration, with a first concentration and a second concentration, or the ratio of the first concentration to the second concentration, obtained from a control.

[0486] In assessing citrullination in a biological sample from a subject, the method further includes determining one or more of the following based on a first concentration and a second concentration or a ratio of the first concentration and the second concentration: the probability of a clinical outcome, the risk of developing a citrullination-related disease, a diagnosis of a citrullination-related disease, and the selection of the subject from whom the biological sample is derived for the treatment of a citrullination-related disease. For example, in some cases, a subject has a citrullination-related disease or may be at risk of developing one, and the assessment of citrullination in the biological sample according to the method described herein can be used to confirm the diagnosis of such disease or to determine whether the subject is at risk of developing such disease. In some cases, the method may allow monitoring of citrullination in a subject's biological sample to determine the probability of a clinical outcome, for example, based on a comparison with a reference subject whose clinical outcome is known, or to determine whether a PAD4 modulator or another therapeutic agent is suitable for treating the subject, or to determine whether a PAD4 modulator or other therapeutic agent currently being administered to the subject is acting on the subject to reduce citrullination. Therefore, for example, in one embodiment, the method further includes determining, based on a first concentration and a second concentration or a ratio of the first concentration and the second concentration, whether a subject should receive treatment, for example, in some cases, for the treatment of citrullination-related diseases. In some cases, the method further includes determining, based on a first concentration and a second concentration or a ratio of the first concentration and the second concentration, whether a subject should receive treatment with a therapeutic agent. Non-limiting examples of such agents include those that can be used to treat citrullination-related diseases, and those that target biological pathways that may also involve PAD4 (such as, for example, NETosis or METosis), such as, for example, anti-histone antibodies (e.g., CIT-013) and PAD4 modulators (such as PAD4 inhibitors). In other cases, the method includes determining whether a subject who has already received treatment with a therapeutic agent or other therapy should have their treatment adjusted, such as increasing or decreasing the dose of the administered therapeutic agent, or initiating treatment with an agent such as a PAD4 modulator or an agent that targets molecules involved in biological pathways involving PAD4. In some cases, the method further includes determining whether a subject should receive treatment with a PAD4 modulator, or whether a subject already receiving treatment with a PAD4 modulator should have their treatment adjusted, such as increasing or decreasing the dose of the PAD4 modulator or discontinuing its use. In some cases, the PAD4 modulator is a PAD4 inhibitor. Exemplary PAD4 modulators are provided in this disclosure and include, for example, therapeutic agents that can affect the expression or activity of PAD4, including citrullinated activity of PAD4. Such molecules include antibodies (such as anti-PAD4 antibodies) and small molecules (such as small molecule PAD4 inhibitors) and other such molecules described in this disclosure.

[0487] This disclosure also covers a method for evaluating the activity of a therapeutic agent, including but not limited to agents that can be used to treat citrullination-related diseases, agents that target biological pathways involving PAD4, agents that target NETosis and / or METosis, anti-histone antibodies, and PAD4 modulators (such as PAD4 inhibitors). In some cases, such therapeutic agents may comprise macromolecules (such as antibodies or fusion proteins) or may comprise small molecules. In some embodiments, the method for evaluating the activity of a therapeutic agent (such as a PAD4 modulator) includes evaluating citrullination at citrullination sites on proteins or peptide fragments thereof present in a biological sample from a subject. In such methods, the biological sample has been exposed to the PAD4 modulator or other therapeutic agent, either directly by adding the PAD4 modulator or other therapeutic agent to the sample prior to evaluation, or indirectly by the subject having received at least one dose of the PAD4 modulator or other therapeutic agent prior to receiving the sample. In some cases, the therapeutic agent being evaluated is a PAD4 modulator. In some cases, the PAD4 modulator is a PAD4 inhibitor.

[0488] In any of the methods described herein, in some embodiments, at least two distinct citrullination sites are evaluated. In some cases, at least three distinct sites are evaluated. In some cases, at least four distinct sites are evaluated. In some cases, citrullination of at least two different proteins is evaluated. In some cases, citrullination of at least three different proteins is evaluated. In some cases, citrullination of at least four different proteins is evaluated. In some cases, 2-4 citrullination sites and / or citrullination of 2-4 different proteins are evaluated.

[0489] In any of the methods described herein, in some cases, the protein containing the citrullinated site used for evaluation is selected from one or more of the following: α-2-HS-glycoprotein, α-2-macroglobulin, albumin, antithrombin-III, apolipoprotein AI, apolipoprotein A-IV, apolipoprotein B-100, apolipoprotein E, apolipoprotein L1, C4b-binding protein α chain, ceruloplasmin, clusteringin, complement C2, complement C3, complement C4-A, complement C4-B, complement factor B, complement factor H, complement factor I, complement component C9, fibrinogen α chain, fibrinogen β chain, fibrinogen γ chain, fibronectin, galactagogue-3-binding protein, coagulin, haptoglobin, haptoglobin-related protein, hemoglobin Synthetic proteins, immunoglobulin heavy chain variable 1-3, immunoglobulin heavy chain variable 1-8, immunoglobulin heavy chain variable 1-46, immunoglobulin heavy chain variable 1-69, immunoglobulin heavy chain variable 1-69D, inter-α-trypsin inhibitor heavy chain H4, immunoglobulin λ-like polypeptide 1, immunoglobulin heavy chain constant μ, inter-α-trypsin inhibitor heavy chain H2, complement C4-A isoform 2, plasminogen, plasminogen-like protein A, α-1-microglobulin / bisKunitz inhibitor precursor, prothrombin, proteoglycan 4, serum transferrin, serum amyloid A-1 protein, serum amyloid A-2 protein, serum amyloid A-4 protein, matrix lysozyme-1, vitamin D-binding protein, and porphyrin. Therefore, in some embodiments, citrullinated sites from the above-described proteins or their peptide fragments are evaluated. For example, in some embodiments, the methods described herein include a method for assessing the activity of a PAD4 modulator or other therapeutic agent, the method comprising assessing citrullination of a protein or peptide fragment thereof present in a biological sample from a subject, wherein the biological sample has been exposed to the PAD4 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 A below.

[0490] In some cases, the citrullination sites to be evaluated are selected from one or more citrullination sites shown in Table A below. In the table below, the amino acid residues corresponding to the citrullination sites are recorded in the left column, and the reference accession number that can be used to locate the citrullination sites in publicly available UniProt databases is listed in the right column. The UniProt accession number for each listed protein is provided in the table. Public databases provide the amino acid and gene sequences corresponding to the accession numbers (e.g., P01023 for α-2-macroglobulin). It should be understood that each protein listed in the table below may have more than one naturally occurring amino acid sequence. Different allotypes of proteins may arise, for example, due to the natural presence of different alleles reflecting substitutions, insertions, and / or deletions in the amino acid sequence, and due to variable promoters, alternative splicing, and / or variable translation initiation sites. Therefore, the accession numbers and associated sequences listed for a given protein in Table A and other tables that also provide citrullination sites and reference protein accession numbers or sequences herein are for reference only. They can be used to identify citrullination sites in proteins with the same sequence as the reference sequence, and to identify citrullination sites in naturally occurring variant proteins, for example, by comparing variant sequences with reference sequences. In some embodiments, only one citrullination site in Table A is evaluated, while in others, at least two, at least three, at least five, at least ten, at least twenty, or at least fifty sites are evaluated, such as 2-5, 2-10, 2-20, 2-50, 10-50, 5-50, 10-50, or 5-20 sites. In some cases, citrullination sites of only one protein are evaluated, while in others, citrullination sites of two or more proteins (e.g., at least 2, 3, 5, 6, 7, or 8 proteins) are evaluated. In some cases, citrullination sites of 2-5, 2-10, 5-10, 2-20, 5-10, or 10-20 different proteins are evaluated.

[0491] Table A provided below lists, for example, exemplary citrullination sites, i.e., residues of specific human proteins that may be citrullinated in biological samples, and lists in the right column sites such as those located within protein sequences published in the UniProt database (accessible at www.uniprot.org).

[0492] Table A - Exemplary Citrullination Sites

[0493]

[0494] In some cases, the citrullination site is an underlined site on one or more peptides shown in Tables 3-18. In some cases, the method includes evaluating the citrullination of a peptide fragment of a protein, wherein the sequence of said peptide fragment includes the peptide sequences shown in any of Tables 3-18. In some cases, the method includes evaluating the citrullination of a peptide fragment of a protein, wherein the sequence of said peptide fragment includes the peptide sequences shown in Table 3. In some cases, at least two peptide sequences as shown in Table 3, or two, three, four, or all sequences as shown in Table 3 are evaluated. In some cases, the method includes evaluating the citrullination of a peptide fragment, wherein the sequence of said peptide fragment includes the peptide sequences shown in Table 4. In some cases, at least two peptide fragment sequences as shown in Table 4, or two, three, four, or all sequences as shown in Table 4 are evaluated. In some cases, the method includes evaluating the citrullination of a peptide fragment, wherein the sequence of said peptide fragment includes the peptide sequences shown in Table 5. In some cases, at least two peptide fragment sequences as shown in Table 5, or two, three, four, or all sequences as shown in Table 5 are evaluated. In some cases, the method includes evaluating citrullinated peptide fragments, wherein the sequence of the peptide fragments comprises the peptide sequences shown in Table 6. In some cases, at least two peptide fragment sequences as shown in Table 6, or 2, 3, 4, or all sequences as shown in Table 6 are evaluated. In some cases, the method includes evaluating citrullinated peptide fragments, wherein the sequence of the peptide fragments comprises the peptide sequences shown in Table 7. In some cases, at least two peptide fragment sequences as shown in Table 7, or 2, 3, 4, or all sequences as shown in Table 7 are evaluated. In some cases, the method includes evaluating citrullinated peptide fragments, wherein the sequence of the peptide fragments comprises the peptide sequences shown in Table 8. In some cases, at least two peptide fragment sequences as shown in Table 8, or 2, 3, 4, or all sequences as shown in Table 8 are evaluated. In some cases, the method includes evaluating citrullinated peptide fragments, wherein the sequence of the peptide fragments comprises the peptide sequences shown in Table 9. In some cases, at least two peptide fragment sequences as shown in Table 9, or 2, 3, 4, or all sequences as shown in Table 9 are evaluated. In some cases, the method includes evaluating citrullinated peptide fragments, wherein the sequence of the peptide fragments comprises the peptide sequences shown in Table 11. In some cases, at least two peptide fragment sequences as shown in Table 11, or two, three, four, or all sequences as shown in Table 11 are evaluated. In some cases, the method includes evaluating citrullinated peptide fragments, wherein the sequence of the peptide fragments comprises the peptide sequences shown in Table 11. In some cases, at least two peptide fragment sequences as shown in Table 11, or two, three, four, or all sequences as shown in Table 11 are evaluated. In some cases, the method includes evaluating citrullinated peptide fragments, wherein the sequence of the peptide fragments comprises the peptide sequences shown in Table 12.In some cases, at least two peptide fragment sequences as shown in Table 12, or two, three, four, or all sequences as shown in Table 12, are evaluated. In some cases, the method includes evaluating citrullinated peptide fragments, wherein the sequence of the peptide fragments includes the peptide sequences shown in Table 13. In some cases, at least two peptide fragment sequences as shown in Table 13, or two, three, four, or all sequences as shown in Table 13, are evaluated. In some cases, the method includes evaluating citrullinated peptide fragments, wherein the sequence of the peptide fragments includes the peptide sequences shown in Table 14. In some cases, at least two peptide fragment sequences as shown in Table 14, or two, three, four, or all sequences as shown in Table 14, are evaluated. In some cases, the method includes evaluating citrullinated peptide fragments, wherein the sequence of the peptide fragments includes the peptide sequences shown in Table 15. In some cases, at least two peptide fragment sequences as shown in Table 15, or two, three, four, or all sequences as shown in Table 15, are evaluated. In some cases, the method includes evaluating citrullinated peptide fragments, wherein the sequence of the peptide fragments comprises the peptide sequences shown in Table 16. In some cases, at least two peptide fragment sequences as shown in Table 16, or 2, 3, 4, or all sequences as shown in Table 16 are evaluated. In some cases, the method includes evaluating citrullinated peptide fragments of a protein, wherein the sequence of the peptide fragments comprises the peptide sequences shown in Table 17. In some cases, at least two peptide sequences as shown in Table 17, or 2, 3, 4, or all sequences as shown in Table 17 are evaluated. In some cases, the method includes evaluating citrullinated peptide fragments, wherein the sequence of the peptide fragments comprises the peptide sequences shown in Table 18. In some cases, at least two peptide fragment sequences as shown in Table 18, or 2, 3, 4, or all sequences as shown in Table 18 are evaluated.

[0495] In some cases, the citrullinated protein or peptide being evaluated may contain chemical modifications other than citrullination, particularly such as urea methylation of cysteine, oxidation of methionine, and N-terminal modifications. These chemical modifications can be detected, for example, by mass spectrometry.

[0496] In some cases, the citrullination site is selected from one or more of the following: R1391 of proteoglycan 4 (corresponding to arginine residue R1391 in the protein sequence found in the UniProt database with accession number Q92954), R573 of the fibrinogen α chain (corresponding to R573 of P02671), R591 of the fibrinogen α chain (corresponding to R591 of P02671), R573 of complement C3 (corresponding to R573 of P01024), and R688 of the inter-α-trypsin inhibitor heavy chain H4. (R688 corresponding to Q14624), R297 of protein AMBP (R297 corresponding to P02760), R715 of α-2-macroglobulin (R715 corresponding to P01023), R32 of colloidin (R32 corresponding to P06396), R59 of haptoglobin (R59 corresponding to P00738), and R651 of serum transferrin (R651 corresponding to P02787), where all codes beginning with Q or P followed by a number are the UniProt accession numbers of the proteins. In some cases, these citrullinated sites can be assessed from one or more peptide fragments of the protein, wherein the sequence of said peptide fragment includes (where citrullinated sites are bolded and underlined): AITT R SGQTLSK (SEQ ID NO: 247), ESSSHPGIAEFPS R GK (SEQ ID NO: 248), QFTSSTSYN R GDSTFESK (SEQ IDNO: 249), SGQSED R QPVPGQQMTLK (SEQ ID NO: 250), ASHLGLA R SNLDEDIIAEENIVSR (SEQ IDNO: 251), QLGLPGPPDVPDHAAYHPF R R (SEQ ID NO: 252), GPC R AFIQLWAFDAVK (SEQ ID NO:253), VGFYESDVMG R GHAR (SEQ ID NO: 254), ATAS R GASQAGAPQGR (SEQ ID NO: 255), L R TEGDGVYTLNDK (SEQ ID NO: 256) or DLLF R DDTVCLAK (SEQ ID NO: 257). In some cases, citrullination sites are evaluated on one or more peptide fragments of a protein, wherein the sequence of said peptide fragment includes (wherein the citrullination sites are bolded and underlined): AITTR SGQTLSK (SEQ ID NO: 247), ESSSHPGIAEFPS R GK (SEQ IDNO: 248), QFTSSTSYN R GDSTFESK (SEQ ID NO: 249), SGQSED R QPVPGQQMTLK (SEQ ID NO:250), QLGLPGPPDVPDHAAYHPF R R (SEQ ID NO: 252) or GPC R AFIQLWAFDAVK (SEQ ID NO: 253). In some cases, the citrullination site is one or more of the following: R715 of α-2-macroglobulin (corresponding to R715 of P01023), R297 of protein AMBP (corresponding to R297 of P02760), R250 of cycloclastin (corresponding to R250 of P10909-2), R23 of cycloclastin (corresponding to R23 of P10909-3), R165 of cycloclastin (corresponding to R165 of P10909-4), R209 of cycloclastin (corresponding to R209 of P10909-5), and R178 of cycloclastin. (R178 corresponding to P10909-6), R198 of clusterin (R198 corresponding to P10909), R32 of colloidin (R32 corresponding to P06396), R59 of haptoglobin (R59 corresponding to P00738-2 or P00738), R60 of haptoglobin-associated protein (R60 corresponding to P00739), R97 of haptoglobin-associated protein (R97 corresponding to P00739-2), or R651 of serum transferrin (R651 corresponding to P02787). In any of the above cases, in some embodiments, only one member within the above citrullinated site group is evaluated, while in other cases, at least two, at least three, at least five, (and within the larger group above) at least ten or at least twenty sites are evaluated, such as 2-5, or 2-10, or 2 to all sites. In some cases, citrullination sites on only one protein are evaluated, while in others, citrullination sites on two or more proteins (e.g., 2-5, 2-10, 5-10, 2-20, 5-10, or 10-20 different proteins) are evaluated. In some such cases, the biological sample is whole blood, serum, plasma, blood supernatant, or synovial fluid.

[0497] In some cases where biological samples include synovial fluid, the citrullination sites to be evaluated are selected from one or more citrullination sites shown in Table 11. In some cases, at least two peptide fragment sequences as shown in Table 11, or two, three, four, or all sequences as shown in Table 11, are evaluated. In some such cases, the citrullination sites are selected from one or more citrullination sites shown in Table 11, or citrullination sites located on peptide fragments containing one of the following sequences, wherein the citrullination sites are indicated by underscore: AITT R SGQTLSK (SEQ ID NO: 247), ESSSHPGIAEFPS R GK (SEQ ID NO: 248), QFTSSTSYN R GDSTFESK (SEQ ID NO: 249), SGQSED R QPVPGQQMTLK (SEQ ID NO: 250), QLGLPGPPDVPDHAAYHPF R R (SEQ ID NO: 252) or GPC R AFIQLWAFDAVK (SEQ ID NO: 253). In some cases, at least two peptide fragment sequences as shown in Table 11, or 2, 3, 4 or all sequences as shown in Table 11, are evaluated.

[0498] In some cases, the target protein includes fibrinogen α-chains and / or gelosin. In such cases, the citrullination site used for evaluation may include the fibrinogen citrullination site located at R591 of P02671, which in some cases can be identified via the sequence QFTSSTSYN. R The peptide of GDSTFESK (SEQ ID NO: 249) is used for evaluation, where R is a citrullination site. In some cases, the citrullination site used for evaluation may include the colloid citrullination site located at R32 of P06396, which in some cases can be evaluated via sequence ATAS. R The peptide GASQAGAPQGR (SEQ ID NO: 255) is used for evaluation, where the underlined R is the citrullinated site. In some cases where the citrullinated site originates from fibrinogen and / or coagulants, the sample is whole blood, serum, plasma, blood supernatant, or synovial fluid. In some such cases, the sample is whole blood, serum, plasma, or blood supernatant.

[0499] In the methods described herein, "evaluation" encompasses a variety of ways of examining citrullination, such as, but not limited to, qualitatively determining whether citrullination has occurred at a specific citrullination site on a protein or peptide, and quantitatively determining citrullination of a protein or peptide. In some embodiments, evaluation includes quantitatively determining citrullination at a specific citrullination site (e.g., the citrullination sites disclosed herein). In some embodiments, quantitatively determining citrullination at a specific citrullination site includes determining a "citrullination ratio" as defined above.

[0500] In some cases, mass spectrometry (MS) is used to assess citrullination at citrullination sites. MS can detect the difference in molecular weight between citrullinated proteins or peptides and their uncitrullinated counterparts. As described in the examples herein, the intensity measurements obtained using MS can be used to measure the concentration of the citrullinated peptide. MS can also be used to measure the total concentration of the corresponding protein from which the citrullinated peptide is derived. For example, this total protein concentration can be measured by measuring the concentration of a characteristic peptide. As used herein, this “characteristic peptide” is an unmodified peptide present in both modified and unmodified forms of the protein; therefore, the concentration of the characteristic peptide represents the concentration of the corresponding total protein, including both modified and unmodified forms. For example, in the case of FGA, an exemplary characteristic peptide is GSESGIFTNTK (SEQ ID NO: 258; amino acids 548-558). This peptide is unmodified post-translationally in the human matrix tested and is located at the citrullinated peptide ESSSHHPGIAEFPS. R GK (SEQ ID NO: 248; amino acids 559-575) and QFTSSTSYN R The GDSTFESK (SEQ ID NO: 249; amino acids 582-600) is located near the protein, thus accurately representing the total FGA protein concentration. The selection of characteristic peptides is known in the art and described, for example, in the following literature: Qiu, XI et al., Signature peptide selection workflow for biomarker quantification using LC-MS based targeted proteomics. Bioanalysis (April 11, 2023) 10.4155 / bio-2022-0241C. In some cases, assessing citrullination at citrullination sites involves performing liquid chromatography to aid in the separation of protein or peptide species, followed by mass spectrometry (LC-MS). For example, liquid chromatography (e.g., HPLC, RPLC, nLC, µLC, etc.) can be used to separate peptides or proteins for analysis by MS.

[0501] In the methods described herein, samples can be evaluated directly after acquisition, or alternatively, samples can be prepared in some way before evaluation of citrullination, for example, to improve the detection of the target protein or peptide in the sample. For example, the sample can be diluted with a buffer, or samples containing cells can be treated to lyse the cells or separate the cells from the sample fluid, such as by centrifugation or filtration. For example, the target protein or its peptide fragment can be enriched before evaluation of citrullination by separating it from other components in the starting sample. One method for enriching the target protein or peptide, for example, is to separate it from the sample. Thus, the protein or peptide can be exposed to an affinity reagent (such as an antibody), thereby being targeted for purification or removal from the sample. In some cases, such an affinity reagent can be immobilized on a solid surface (such as beads, chips, plates, or the wells of plates) to facilitate this enrichment. In some cases, the protein in the sample can also be enzymatically digested to form peptide fragments before evaluation, can be denatured to remove tertiary structures, and / or the sample can be diluted or treated to remove or separate certain components. In some cases, other components (such as nucleic acids) can be degraded or removed from the sample, for example by enzymatic degradation or by alcohol precipitation.

[0502] In some cases, samples may also be treated prior to evaluation to promote the release of endogenous PAD4 from the sample, or may be contacted with exogenous PAD4 to enhance the signal from the sample, as well as other optional treatments. In some cases, such as to enhance overall PAD4 activity in a biological sample, the sample may be incubated with exogenous PAD4 prior to evaluating citrullination. For example, in some cases, concentrations of PAD4 from 5 nM to 15 nM may be added, such as 7 nM to 14 nM, 7 nM to 10 nM, 10 nM to 15 nM; in others, concentrations of 10 nM or about 10 nM of exogenous PAD4 may be added. In some cases, exogenous calcium ions (Ca2+) may also be added to the sample. In some cases, when citrullination from another PAD protein is also being evaluated, said PAD protein may also be added exogenously, such as PAD2, which may be added, for example, at 2-4 nM (e.g., 3 nM or about 3 nM). In other cases, the sample is not exposed to exogenous PAD4, or to any exogenous PAD protein. Therefore, in some cases, all PAD4 activity in the biological sample derives from endogenous PAD4 present in the sample. In some cases, the biological sample can be incubated for a period of time, which may induce the release of endogenous PAD4 within the sample. This incubation can last 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 lasts for 48–96 hours. In some cases, this incubation is performed at 34ºC–40ºC (e.g., at 36ºC–39ºC, 36ºC–38ºC, or 37ºC). In some cases, incubation is performed in TruCulture® empty tubes (Rules Based Medicine, Austin, Texas) or similar containers containing culture medium for preserving or analyzing biological samples. In some cases where the method involves evaluating exogenous PAD4 modifiers, the PAD4 modifier may be added before incubation of the sample. In other cases, the PAD4 modifier is added after incubation. When exogenous PAD4 is added to the sample, the sample may also be incubated with exogenous PAD4 before evaluating citrullination, for example, for 1–3 hours, 1–2 hours, or 1, 2, or 3 hours. In some cases, this incubation is performed at 34ºC–40ºC (e.g., at 36ºC–39ºC, 36ºC–38ºC, or 37ºC). In some cases, exogenous PAD4 and exogenous PAD4 modifier may be added together. In other cases, the PAD4 modifier may be added after incubation with exogenous PAD4.In some cases, incubation is performed in a CO2 incubator. In other cases, the activity of exogenous PAD4 added to the sample is suppressed by adding EDTA to the sample. Thus, for example, in some cases, the method includes adding EDTA to the biological sample after incubation with exogenous PAD4 to suppress the activity of PAD4.

[0503] In some cases, samples can be treated prior to analysis to expose one or more proteins or peptide fragments thereof containing citrullinated sites for subsequent evaluation. For example, proteins in the sample can be denatured or digested with enzymes. For instance, proteins can be denatured to remove tertiary structures, for example, by incubating at temperatures above physiological temperatures (e.g., 60ºC–90ºC, such as 60ºC–80ºC, 70ºC–85ºC, or 80ºC) for a period of 5–30 minutes (e.g., 10–30, 10–20, 15–25, or 20 minutes). In other cases, chemical denaturing agents, such as urea, can be applied. Enzymatic digestion can be performed using proteolytic enzymes (such as trypsin, LysC, rLysC, LysN, GluC, AspN, rAspN) or combinations of proteolytic enzymes (such as more than one of trypsin, LysC, rLysC, LysN, GluC, AspN, and rAspN) to form peptide fragments of the target protein for subsequent evaluation.

[0504] Therefore, in summary, this paper presents multiple methods for preparing samples for citrullination evaluation.

[0505] This document uses the assessment of citrullination of biological samples to evaluate implementation schemes for PAD4 modulators or other therapeutic agents. Several other methods are described herein for exposing biological samples to PAD4 modulators or other therapeutic agents. In some cases, the sample is incubated directly with the PAD4 modulator or other therapeutic agent (i.e., exogenous PAD4 modulator), such as by ex vivo contact of a biological fluid with the PAD4 modulator or other therapeutic agent. In other cases, the biological sample has already been exposed to the PAD4 modulator or other therapeutic agent in the subject due to administration to the subject. For example, the PAD4 modulator or its metabolites may pass through blood, serum, plasma, synovial fluid, or other body fluids or tissues after administration to the subject, or may come into contact with cells in the body that subsequently travel to such body fluids or certain body tissues.

[0506] Biological samples as used herein may include biological fluids (i.e., biological fluid samples), such as, but not limited to, blood, plasma, serum, blood supernatant, synovial fluid, lymph, pleural effusion, interstitial fluid, sweat, tears, sputum, or urine. In other cases, biological samples may include tissues, such as those derived from joints, fibroids, or tumors. In some methods described herein, the biological sample includes synovial fluid. In some methods, the biological sample includes blood or blood-derived samples; in some cases, the biological sample includes serum, plasma, or blood supernatant (e.g., supernatant formed after centrifuging or filtering blood). In some cases, the biological sample is fresh, meaning it has not been frozen and thawed prior to use. In other cases, it has been frozen and thawed prior to use in the method. Thus, in some cases, the method includes freezing the biological sample and thawing it prior to evaluating citrullination at citrullination sites. For example, the sample may be frozen at -70ºC or lower or -80ºC or lower. In some embodiments, the biological sample is prepared for evaluation prior to evaluation, as performed according to the methods provided above. For example, in some cases, samples are treated by enzymatic digestion of proteins (e.g., with one or more proteases). In some cases, proteins containing citrullination sites are enriched, in some cases this is done by removal from the sample (e.g., with antibodies or affinity reagents), and / or in some cases other proteins or contaminants are removed from the sample. For example, in some cases, antibodies that specifically bind to the protein to be evaluated can be used to enrich that protein for subsequent evaluation of citrullination. In some cases, antibodies or affinity reagents can be immobilized, such as by placing them on a matrix (e.g., beads or wells of a chip or plate, other solid surfaces). In some cases, biological samples can be diluted before evaluating citrullination. In some cases, proteins in biological samples can be denatured to remove tertiary structures before evaluating citrullination. In some cases, any combination of the above treatments can be applied to the sample.

[0507] In some cases, assessing citrullination at citrullination sites involves measuring the citrullination ratio, as described above. In some cases, the method includes comparing a citrullination assessment (such as a citrullination ratio or the concentration of citrullinated protein or its peptide fragments) with a reference or control citrullination assessment. For example, in some cases, the comparison is made with a reference citrullination ratio, which may be obtained, for example, from a reference sample, or may be a value or range of values ​​representing those obtained from a reference sample. Thus, in some cases, the method includes assessing the difference between determining the citrullination ratio of a biological sample and a reference citrullination ratio.

[0508] The terms "reference" and "control" are used interchangeably when referring to biological samples, comparing the measurement of the sample of interest with the biological sample. In some cases, such a control is a sample that has not been exposed to PAD4 but is otherwise comparable to the target sample. In some cases, the control is a "baseline" sample, which is a sample taken from the subject before a target event (e.g., before treatment with a therapeutic agent (e.g., a PAD4 modulator)) and can be compared, for example, with a sample taken from the same subject after said event (e.g., after treatment with said therapeutic agent). In some cases, the control is a sample taken from the subject before treatment with a PAD4 modulator. In some cases, the control is a sample taken from the subject before changing the PAD4 modulator treatment. Therefore, in some cases, it is possible to determine how treatment with a PAD4 modulator affects the subject by comparing with 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 exposure to a different PAD4 modulator or a different therapeutic agent. In some cases, the control biological sample has been exposed to a different dose of a PAD4 modulator or other therapeutic agent than the biological sample. In some cases, the control is a sample from a healthy subject, which may be compared to a sample from a subject with a disability or a subject treated with PAD4. In other cases, the control biological sample is a sample that has been exposed to a different therapeutic agent or PAD4 modulator than the target sample, or exposed to a different dose of the same therapeutic agent or PAD4 modulator. Therefore, in some embodiments, the citrullination assessment is compared with the citrullination assessment of one or more control samples (e.g., baseline samples or samples from the same subject at other time points, or samples from other subjects), or with values ​​for one or more control samples (e.g., from a library of healthy subjects). For example, in some cases, the citrullination assessment of the biological sample herein may be compared with a set of control samples that represent typical values ​​found in a particular type of subject, or values ​​found in a range of subjects.

[0509] In some embodiments, this disclosure includes a method for assessing citrullination in a subject's biological sample, the method comprising assessing citrullination at citrullination sites on proteins or peptide fragments thereof present in the biological sample. In some cases, the subject has not yet received treatment with any PAD4 modulator. In other cases, the subject has received treatment with a PAD4 modulator. In some cases, the subject has not yet received treatment with any therapeutic agent. In other cases, the subject has received treatment with a therapeutic agent. This disclosure also covers a method for assessing the activity of a PAD4 modulator or other therapeutic agent, the method comprising obtaining a biological sample from the subject after treatment with at least one dose of a PAD4 modulator or other therapeutic agent, and assessing citrullination at citrullination sites on proteins or peptide fragments thereof present in the biological sample. In some cases, these methods further comprise comparing the citrullination assessment with a citrullination assessment of a control biological sample, as described above. In some cases, where the subject has already been treated with a PAD4 modulator or other therapeutic agent, the control biological sample is a baseline sample obtained from the subject prior to treatment with the PAD4 modulator or other therapeutic agent. In some cases, the method includes obtaining a baseline biological sample from the subject prior to treatment with a PAD4 modulator or other therapeutic agent, assessing citrullination at citrullination sites on proteins or peptide fragments present in the baseline biological sample, and optionally comparing the assessed citrullination (e.g., citrullination ratio) of the biological sample to that of the baseline biological sample. In some cases, citrullination assessment is performed to determine whether the subject should receive treatment with a PAD4 modulator, or as a baseline for subsequent monitoring of citrullination after treatment with a PAD4 modulator or other therapeutic agent.

[0510] In some cases, the method includes (a) contacting a biological sample from a subject with exogenous PAD4, and (b) assessing citrullination at citrullination sites on proteins or peptide fragments 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 assessment. In some cases, the concentration of exogenous PAD4 may be as provided above (e.g., 5 nM to 15 nM PAD4, such as 7 nM to 14 nM, 7 nM to 10 nM, 10 nM to 15 nM). In some cases, another PAD protein (e.g., PAD2) is also added. In some cases, calcium ions (Ca2+) are also added to the sample. For example, in some cases, the sample is treated to enrich the target protein, and / or denatures and / or enzymatically digests the target protein for subsequent assessment, and / or depletes the protein (e.g., serum albumin). In some cases, the target protein is enriched using immunoenrichment techniques as described below, for example, the techniques may include using affinity reagents to separate or remove the target protein.

[0511] In some embodiments, the method includes an in vitro method for assessing the activity of a PAD4 modulator or other therapeutic agent, the in vitro method comprising (a) treating a biological sample from a subject with a PAD4 modulator or other therapeutic agent to form a treated biological sample, and, following treatment, (b) assessing citrullination at citrullination sites on proteins or peptide fragments present in the biological sample. In some cases, such a method further comprises assessing citrullination at citrullination sites on proteins or peptide fragments in a control biological sample, as described above. The method herein also includes, for example, an in vitro method for assessing the activity of a PAD4 modulator or other therapeutic agent, the in vitro method comprising (i) aliquoting a biological sample obtained from a subject into multiple biological samples, (ii) contacting each of the multiple biological samples with a different dose of a PAD4 modulator or other therapeutic agent, and (iii) for each of the multiple biological samples, assessing citrullination at PAD4-dependent citrullination sites on proteins or peptide fragments present in the biological sample. In some cases, this method includes calculating the IC50 of the PAD4 modulator or other therapeutic agent based on the assessed outcome. The methods described herein may also include, for example, an in vitro method for assessing citrullinated endogenous PAD4, said in vitro method comprising incubating a biological sample (or multiple biological samples) from a subject for an incubation period (e.g., to trigger the release of endogenous PAD4), and assessing citrullinated sites on proteins or peptide fragments present in the sample. The methods described herein also include, for example, an in vitro method for assessing citrullinated endogenous PAD4, said in vitro method comprising (a) incubating a whole blood sample from a subject at 34ºC–40ºC for an incubation period (e.g., to trigger the release of endogenous PAD4), (b) after the incubation period, separating plasma or supernatant from the whole blood sample, and (c) assessing citrullinated sites on proteins or peptide fragments present in the plasma or supernatant. In some cases, incubation is performed in TruCulture® empty tubes. This incubation can last 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 incubation is carried out at a temperature of 35ºC to 40ºC, 36ºC to 39ºC, 36ºC to 38ºC, or 37ºC. In some cases, the sample is prepared for evaluation of citrullination, such as enrichment of the protein or peptide to be evaluated. In some cases, the method includes, for example, separating plasma or supernatant from a whole blood sample, such as by centrifugation or filtration of the whole blood sample.These methods may also include (i) incubating plasma or supernatant with a protein-depleting resin, and (ii) recovering the depleted plasma or supernatant flowing through the resin to obtain a depleted flowstream, optionally wherein the recovery includes centrifugation. They may also include enzymatically digesting peptides in the depleted flowstream prior to evaluation, and optionally cleaning the enzymatically digested peptides, such as with an iST-BCT kit (PreOmics, Prannoy / Martin Red, Germany) or a similar kit. In some embodiments, such kits may be used to purify samples prior to MS analysis and / or to minimize artificial protein modifications (such as deamidation and oxidative modifications) induced by reagents added to the sample. 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 includes freezing the plasma or supernatant after separating it from the whole blood, and then thawing the plasma or supernatant prior to evaluation.

[0512] The published text of this document also covers an in vitro method for assessing changes in citrullination in a subject, the in vitro method comprising (a) incubating a whole blood sample from the subject at 34ºC–40ºC for an incubation period (e.g., at 36ºC–39ºC, at 36ºC–38ºC, or at 37ºC 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 after administration of a PAD4 modulator, (b) separating plasma or supernatant from the whole blood sample after the incubation period, and (c) assessing citrullination at citrullination sites on proteins or peptide fragments present in the plasma or supernatant. In some cases, the method further includes (d) incubating a second whole blood sample from the subject at 34ºC–40ºC for an incubation period (e.g., at 36ºC–39ºC, at 36ºC–38ºC, or at 37ºC 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 prior to administration of the PAD4 modifier to the subject; (e) after the incubation period, separating plasma or supernatant from the second whole blood sample; and (f) assessing citrullination of citrullinated sites on proteins or peptide fragments present in the plasma or supernatant from the second whole blood sample. In some cases, the outcome assessed in step (f) is compared with the outcome assessed in step (c). The methods described herein also include, for example, a method for assessing the effect of a PAD4 modulator, the method comprising (a) exposing a whole blood sample from a subject to the PAD4 modulator in vitro, (b) incubating the whole blood sample at 34ºC–40ºC for an incubation period (e.g., at 36ºC–39ºC, at 36ºC–38ºC, or at 37ºC 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 citrullinated sites on proteins or peptide fragments present in the plasma or supernatant. In some embodiments, the method further includes the step of performing a parallel group of at least one control whole blood sample.In some embodiments, the method further includes (e) incubating the control whole blood sample at 34ºC–40ºC for an incubation period (e.g., at 36ºC–39ºC, at 36ºC–38ºC, or at 37ºC 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 citrullinated sites on proteins or peptide fragments present in the control plasma or supernatant. In some cases, the outcome assessed in step (g) is compared with the outcome assessed in step (d). The disclosure herein also covers, for example, an in vitro method for assessing PAD4-dependent citrullination, the method comprising assessing citrullination at citrullination sites on proteins or peptide fragments thereof present in plasma or supernatant separated from whole blood samples, the whole blood samples having been incubated at 34ºC–40ºC for an incubation period (e.g., at 36ºC–39ºC, at 36ºC–38ºC, or at 37ºC 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 may be plasma or supernatant from a whole blood sample. In some cases, the plasma or supernatant sample has not been frozen before evaluation, while in other cases the sample has been frozen and thawed, and in some cases the method further includes freezing the plasma or supernatant after separation from whole blood, but thawing the plasma or supernatant before evaluation.

[0513] In some cases, samples may be contacted with reagents (such as affinity reagents) to enrich or purify proteins or peptide fragments thereof having citrullinated sites to be analyzed. For example, in some cases, the method includes “immunoenriching” the sample with a protein or peptide fragment of interest having a citrullinated site for evaluation. This “immunoenrichment” may include contacting the sample with an antibody reagent that specifically binds to proteins containing citrullinated sites and / or their peptide fragments (e.g., containing epitopes of the target protein). In some cases, the antibody reagent binds to proteins or peptides in both non-citrullinated and citrullinated forms. Thus, proteins or peptide fragments thereof can be enriched prior to citrullination evaluation. In some cases, antibodies or other affinity reagents may be immobilized, such as by attaching to a solid surface (e.g., beads, chips, plates, or wells of a plate). This immobilization, for example, can help separate the target protein for citrullination evaluation from other proteins in the sample. In some cases, the reagent used for immunoenrichment is an antibody attached to a solid surface (e.g., beads, such as magnetic beads, or chips, plates, or wells of a plate). In some such cases, the sample may be a sample containing or as a sample containing: whole blood, serum, plasma, synovial fluid, pleural effusion, interstitial fluid, sputum, urine, or another biological sample.

[0514] Therefore, the methods disclosed herein also include, for example, a method comprising: (a) immunoenriching a sample with a target protein or a peptide fragment thereof to form an immunoenriched sample, wherein immunoenrichment comprises contacting a serum or plasma sample with an antibody that specifically binds to both a citrullinated and uncitrullinated form of the target protein or a protein fragment thereof, the target protein or peptide comprising a citrullinated site; and (b) evaluating citrullination at the citrullinated site in the immunoenriched sample. In some cases, the methods disclosed herein comprise: (a) immunoenriching a sample with a target protein to form an immunoenriched sample, wherein immunoenrichment comprises contacting a serum or plasma sample with an antibody that binds to both a citrullinated and uncitrullinated form of the target protein or a peptide fragment thereof, the target protein or a peptide fragment thereof comprising a citrullinated site; (b) enzymatically digesting the immunoenriched sample to form a digested peptide from the target protein, wherein steps (a) and (b) may have any order; and (c) evaluating citrullination at the citrullinated site in the digested peptide. In other cases, the method includes (a) immunoenriching the sample by contacting it with a fixed antibody that binds to both citrullinated and uncitrullinated forms of the target protein, the target protein containing citrullinated sites; (b) eluting the protein bound to the fixed antibody; (d) enzymatically digesting the eluted protein to form digested peptides; and (e) evaluating the citrullination of the digested peptides at the citrullinated sites. In any of the above methods, the sample may be diluted prior to the immunoenrichment step, such as by a dilution of 2-1000 times. In any of the above methods, the sample may be whole blood, plasma, serum, or synovial fluid. In some embodiments, it is a plasma or serum sample. In some cases, immunoenrichment is performed before enzymatic digestion of the target protein. In other cases, the target protein is first enzymatically digested, and then one or more peptide fragments are immunoenriched. In some cases, the sample may be diluted to a volume of at least 5 μl, for example, to volumes of 5 μl–100 μl, 5 μl–50 μl, 5 μl–25 μl, or 5 μl–15 μl, or for example, to a volume of 10 μl. In any of the methods described above, immunoenrichment may include incubating the sample with the immobilized antibody for at least 30 minutes, such as a time period of 30–90 minutes. In some cases, the incubation may last for 30–60 minutes, 40–80 minutes, 50–70 minutes, 55–65 minutes, or 60 minutes. Incubation may be performed at a temperature of 22°C–28°C or at room temperature. In some cases, immunoenrichment may include shaking during incubation (e.g., at 800–1200 rpm or 1000 rpm).In some cases, immunoenrichment includes removing fixed antibodies from the sample and washing the fixed antibodies prior to elution, for example, with a wash buffer (e.g., a wash buffer containing PBS (phosphate-buffered saline)). In some cases, elution includes washing the fixed antibodies with an elution composition. A variety of elution compositions can be used, including, for example, elution compositions that elute the target protein based on pH changes (e.g., acidic elution compositions) and / or elution compositions containing detergents (e.g., zwitterionic detergents). In some cases, the elution buffer may contain an acidic solution, optionally further containing a buffer or ions (e.g., zwitterionic). In some cases, the buffer is changed before assessing citrullination, and optionally before intermediate steps (e.g., enzymatic digestion, if such steps are performed). For example, if an acidic elution composition is used, the buffer may be changed, for example, to a neutral pH range for subsequent steps. In some cases, the protein, which has been immunoenriched according to the methods described herein, is denatured and / or enzymatically digested before assessing citrullination. For example, proteins can be denatured to remove tertiary structures, for instance, by incubating at temperatures above physiological levels (e.g., 60ºC–90ºC, such as 60ºC–80ºC, 70ºC–85ºC, or 80ºC) for 5–30 minutes (e.g., 10–30, 10–20, 15–25, or 20 minutes). In other cases, chemical denaturing agents, such as urea, can be applied. Enzymatic digestion can be performed using proteases (e.g., trypsin, LysC, rLysC, LysN, GluC, AspN, rAspN) or combinations of proteases (e.g., more than one of trypsin, LysC, rLysC, LysN, GluC, AspN, and rAspN).

[0515] In these methods described above, the target protein may include any of the proteins listed herein (such as those listed in Table A above), and the citrullination site to be evaluated may include any one or more of those listed, for example, in Table A or Tables 3-18. In some cases, the target protein includes fibrinogen α chains and / or colloid proteins. In such cases, the citrullination site for evaluation may include the fibrinogen citrullination site located at R591 of P02671, which in some cases may be via the sequence QFTSSTSYN. R The peptide of GDSTFESK (SEQ ID NO: 249) can be evaluated, where R is a citrullination site. In such cases, the citrullination site used for evaluation may include the gel citrullination site at R32 of P06396, which in some cases can be evaluated via sequence ATAS. RThe peptide GASQAGAPQGR (SEQ ID NO: 255) is used for evaluation, where the underlined R is a citrullinated site. Exemplary antibodies that can be used for the immunoenrichment of fibrinogen may be, for example, recombinant antifibrinogen antibodies, such as Abcam antibody 244646. Exemplary antibodies that can be used for the immunoenrichment of sol-gel proteins may be, for example, recombinant antisol-gel protein antibodies, such as antibody 247406. In some such embodiments, immunoenrichment of the sample is performed prior to enzymatic digestion.

[0516] In any of the methods mentioned above, including immunoenrichment and enzymatic digestion, the assessment of citrullination can be performed by mass spectrometry (MS), for example by liquid chromatography followed by mass spectrometry (such as tandem mass spectrometry (LC-MS or LC-MS / MS)).

[0517] The methods described above can be used in a wide variety of situations and with a wide range of PAD4 modulators (such as PAD4 inhibitors and PAD4 agonists), as well as other therapeutics, such as those used to treat citrullination-related diseases, those targeting biological pathways involving PAD4 (such as NETosis and / or METosis), and those that, in other cases, can affect citrullination activity through PAD4 or PAD4 expression. For example, the methods can be used to test novel PAD4 modulators or other agents to determine their effect on citrullination at certain sites. Alternatively, the methods can be used to examine the activity of known PAD4 modulators or other agents. Or, the methods can be used to monitor the effects of PAD4 modulators or other therapeutics after administration to a patient, such as their effect on citrullination levels. Therefore, in some cases, the effects of PAD4 modulators or other therapeutics that have not yet been administered to a subject can be tested using biological samples, and the PAD4 modulators or other therapeutics can be exposed to the biological sample by incubating the sample with the modulator. In other cases, the biological sample may be obtained from a subject who has previously been administered a PAD4 modulator or other therapeutic agent, and thus the sample has been exposed to the PAD4 modulator by administering the PAD4 modulator or other therapeutic agent to the subject from whom the sample was obtained. In some cases, the PAD4 modulator or other therapeutic agent is an anti-histone antibody, such as CIT-013. In some such cases, the PAD4 modulator or other therapeutic agent is a PAD4 modulator. In some cases, it is a PAD4 inhibitor, such as an anti-PAD4 antibody or a small molecule PAD4 inhibitor. In some cases, the PAD4 modulator does not significantly affect the activity of any protein species other than PAD4. In some such cases, the PAD4 modulator does not significantly affect the activity of, for example, PAD2. In other cases, the PAD4 modulator modulates the activity of PAD4 and at least one other protein, such as PAD2. Certain exemplary PAD4 modulators compatible with the methods described herein are described below.

[0518] PAD4 modulators can be administered to subjects who, for example, have citrullination-related diseases (such as autoimmune diseases, infectious diseases, or cancer) or are at risk of developing such diseases. These diseases and related subjects are discussed in more detail in the following sections. In some cases, the subject has been diagnosed with an autoimmune disorder or is at risk of developing one. In some cases, citrullination-related diseases are 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 of developing it. However, in other cases, the subject has not been diagnosed with an autoimmune disease (such as RA) or is considered not at risk of developing it. Therefore, in some cases, the subject is a normal healthy subject. In some cases, the subject is positive for anti-citrullinated protein antibodies (ACPA positive). In some cases, subjects may test positive for anti-PAD4 activating antibodies. A positive ACPA test or a positive anti-PAD4 activating antibody test may also indicate a risk of developing an autoimmune disorder (such as RA).

[0519] This method can also be used, for example, to assess citrullination levels in subjects who are not currently receiving therapeutic treatment or are not taking PAD4 modulators. Therefore, in some cases, the subject is a normal, healthy individual. In some cases, the subject is positive for antibodies against citrullinated proteins (ACPA positive). In some cases, the subject is considered to be at risk of developing an autoimmune disorder or a citrullination-related disease.

[0520] The methods described herein can also be used to evaluate, for example, citrullination of sites via mouse PAD4 citrullination in laboratory animals (e.g., mice), for example, by evaluating mouse citrullination sites and the citrullination of related peptides, for example, to test mouse PAD4 modulators and inhibitors. See, for example, International Patent Publication No. WO2024 / 020579, which describes an exemplary mouse PAD4 antibody.

[0521] III. Exemplary PAD4 Regulator

[0522] Exemplary small molecule PAD4 modulators

[0523] Examples of PAD4 modulators include a variety of small molecule compounds that can increase or decrease PAD4 activity (such as citrullination of amino acid residues). Examples of small molecule PAD4 modulators include compounds derived from benzimidazole and heteroaryl compounds, as well as peptides (such as macrocyclic peptides). Exemplary small molecule PAD4 modulators include, for example, the compounds disclosed in International Patent Publication No. WO2014 / 015905, which describes benzimidazole-derived PAD4 modulators, such as PAD4 inhibitors, for example, 2-(azaindol-2-yl)benzimidazole. Other small molecule PAD4 modulators (such as PAD4 inhibitors) are also described in, for example, the following documents: International Patent Publication Nos. WO2016 / 185279, WO2017 / 007405, WO2017 / 100601, WO2017 / 100594, WO2017 / 147102, WO2018 / 022897, WO2018 / 049296, WO2019 / 058393、WO2020 / 033490、WO2020 / 033514、WO2020 / 033520、WO2020 / 033488、WO2021 / 158 840, WO2021 / 163254, WO2022 / 173722, WO2022 / 140428, WO2022 / 221642 and WO2023 / 083365. Other small molecule PAD4 modulators include compounds disclosed in the following applications: US Patent Application or Publication Nos. US11198681, US20220402950, ​​US11524959, US20230203039, US20220348562, US11878965, US20220227787, and US11976083. Other small molecule PAD4 modulators include, for example, GSK121, GSK199, AFM-30a, and GSK 484, described in Lewis et al. (2015) NatChem 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; JBI-1044, 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 streptavidin, 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) JAm Chem Soc 128(4): 1092-1093, and its modifications, including BB-F-amidine (Muth et al. (2017) J MedChem 60(7): 3198-3211), and oF-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) Int J Mol Sci 20(9): 2174; NSC95397, Ruthenium Red and Sanguisorbine, described in Lewallen et al. (2014) ACS Chem Biol 9(4): 913-921; Chlortetracycline, 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 inhibitors described in the following literature: WO2017 / 027967; WO2014 / 019092; Zhu et al. (2023) Eur J Med 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. (2017) Bioorg Med Chem 25(9):2643-2656; 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.

[0524] In other cases, antibodies can be used as PAD4 modulators. Examples of such antibodies include those described in the following literature: 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. Additionally, Sims et al., doi 10.1136 / annrheumdis-2024-eular.3510 (2024) and Chen et al., doi 10.1136 / annrheumdis-2023-eular.2894 (2023) describe a PAD2 / PAD4 bispecific antibody, and WO2016 / 155745 describes a cross-reactive antibody capable of binding to both PAD2 and PAD4. Other examples of anti-PAD4 antibodies are described in this section and the following sections, as well as in WO2024 / 020579, and the amino acid sequences of the antibodies described below are also provided herein.

[0525] The publications mentioned above are each incorporated into this article in their entirety by reference.

[0526] Clones 13 and related anti-PAD4 antibodies

[0527] A set of exemplary anti-PAD4 antibodies is based on a mouse anti-human antibody called “clone 13,” which was prepared in its initial mouse anti-human form and then humanized to produce a series of antibodies called hz13-1 to hz13-12, wherein hz13-5 and hz13-12 were further modified at positions D31 to D31E (antibodies hz13-5 D31E and hz13-12 D31E). Cryo-EM studies of the PAD4-binding clone 13 Fab and complementary site mappings used to identify the portions of the clone 13 variable region and its humanized variants that directly contact PAD4 provide further structural information about the portions of the clone 13 variable region that determine PAD4 binding and those portions of their associated humanized antibodies. Antibody hz13-5 was also further modified to identify antibodies exhibiting pH-dependent binding to PAD4. Clone 13 antibodies and their humanized derivatives are described in International Patent Publication WO2024 / 020579 and in Provisional Priority Application No. 63 / 528,323 filed on July 21, 2023, the contents of which are incorporated herein by reference.

[0528] For example, in some embodiments, the PAD4 modulator or PAD4 inhibitor comprises an anti-PAD4 antibody that specifically binds to protein arginine deiminase 4 (PAD4) and includes a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 4, an HCDR2 containing the amino acid sequence of SEQ ID NO: 5, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 6; and a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 7, an LCDR2 containing the amino acid sequence of SEQ ID NO: 8, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 9.

[0529] In some embodiments, the antibody specifically binds to protein arginine deiminase 4 (PAD4) and includes a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 62, an HCDR2 containing the amino acid sequence of SEQ ID NO: 5, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 6; and a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 7, an LCDR2 containing the amino acid sequence of SEQ ID NO: 8, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 9.

[0530] In some embodiments, the antibody comprises a heavy chain variable region (VH) containing a heavy chain complementarity-determining region 1 (HCDR1) with the amino acid sequence of SEQ ID NO: 4 or 62 and an HCDR3 with the amino acid sequence of SEQ ID NO: 6; and a light chain variable region (VL) containing a light chain complementarity-determining region 1 (LCDR1) with the amino acid sequence of SEQ ID NO: 7. Specifically, complementary site mapping and structural analysis of antibodies containing the above-described heavy and light chain CDR groups reveal that HCDR1, HCDR3, and LCDR1 are contacted with PAD4. (See also...) Figures 8A-8BTherefore, in some embodiments, the antibodies described herein comprise the group of the three CDRs described above. In some embodiments, the antibody comprises VH, which comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 4 and HCDR3 containing the amino acid sequence of SEQ ID NO: 6; and VL, which comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 7. In other embodiments, the antibody comprises VH, which comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 62 and HCDR3 containing the amino acid sequence of SEQ ID NO: 6; and VL, which comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 7.

[0531] In some embodiments, the antibody comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 221 or 225 and the amino acid sequence of SEQ ID NO: 222; and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 223. Each of these complementary regions is identified as a contact PAD4, as described in priority application 63 / 528,323 and international patent publication WO2024 / 020579. In some embodiments, the antibody further comprises a light chain constant region containing the amino acid sequence of SEQ ID NO: 224.

[0532] In some embodiments, the antibody contains VH, which contains glycine at position 94 of the Kabat (Gly94). Thus, for example, the initially identified mouse anti-human clone 13 antibody contains glycine at position 94 of the Kabat, but several human frame regions do not. Therefore, in some humanized antibodies, a reversion mutation is necessary to obtain glycine at position 94 of the Kabat. Gly94 is an amino acid residue in the loop adjacent to VH CDR3 (i.e., the VH CDR3 loop). In some embodiments, Gly94 relates to the flexibility and / or geometry of the VH CDR3 loop. In some embodiments, Gly94 can interact with VH CDR3. In some embodiments, Gly94 is associated with the activity of anti-PAD4 antibodies. In some embodiments, the mutation of Gly94 to a different amino acid (such as threonine) results in reduced flexibility of the VH CDR3 ring, changes in the geometry of the VH CDR3 ring, reduced interaction between the amino acid at position 94 and the VH CDR3 ring, reduced binding of the anti-PAD4 antibody to PAD4, decreased activity of the anti-PAD4 antibody, increased binding of the anti-PAD4 antibody to extracellular matrix (ECM) proteins, changes in the secondary structure of the anti-PAD4 antibody, decreased stability of the anti-PAD4 antibody, or any combination thereof. Therefore, in some embodiments, the VH of the antibody is contained in glycine at position 94 of the Kabat (position 98 of SEQ ID NO: 10). (See Priority Application No. 63 / 528,323) Figure 1 E and International Patent Publication No. WO2024 / 020579).

[0533] In some embodiments, the antibody comprises VH, said VH being at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO: 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, or 68. In some embodiments, the antibody comprises VL, said VL being at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO: 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, or 70. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO: 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, or 68; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO: 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, or 70. In some embodiments, the antibody comprises VH, said VH comprising an amino acid sequence of any one of SEQ ID Nos: 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, or 68 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, said VL comprising an amino acid sequence of any one of SEQ ID Nos: 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, or 70 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises an amino acid sequence modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL ...In some such cases, antibodies VH and VL further comprise (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 4 or 62, HCDR3 containing the amino acid sequence of SEQ ID NO: 6, and LCDR1 containing the amino acid sequence of SEQ ID NO: 7; (b) HCDR1 containing the amino acid sequence of SEQ ID NO: 4 or 62, HCDR2 containing the amino acid sequence of SEQ ID NO: 5, HCDR3 containing the amino acid sequence of SEQ ID NO: 6, LCDR1 containing the amino acid sequence of SEQ ID NO: 7, LCDR2 containing the amino acid sequence of SEQ ID NO: 8, and LCDR3 containing the amino acid sequence of SEQ ID NO: 9; or (c) the amino acid sequence of SEQ ID NO: 221 or 225 in VH and the amino acid sequence of SEQ ID NO: 222 in VL, further optionally having a light chain constant region containing the amino acid sequence of SEQ ID NO: 224. Therefore, in such cases, the changes in VH and VL are located outside these specific CDR or complementary sequences compared to the sequence identifiers listed above. In some embodiments, the antibody comprises VH, which contains the amino acid sequence of any one of SEQ ID No: 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, or 68. In some embodiments, the antibody comprises VL, which contains the amino acid sequence of any one of SEQ ID No: 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, or 70.

[0534] In other embodiments, the antibody comprises both VH and VL, wherein VH comprises the amino acid sequence of any one of SEQ ID No: 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, or 68; and VL comprises the amino acid sequence of any one of SEQ ID No: 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, or 70. Therefore, for example, the following exemplary antibodies are within the scope of this disclosure:

[0535] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 10 and VL containing the amino acid sequence of SEQ ID NO: 12;

[0536] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 16;

[0537] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 18 and VL containing the amino acid sequence of SEQ ID NO: 20;

[0538] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 22 and VL containing the amino acid sequence of SEQ ID NO: 24;

[0539] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 26 and VL containing the amino acid sequence of SEQ ID NO: 28;

[0540] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 30 and VL containing the amino acid sequence of SEQ ID NO: 32;

[0541] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 34 and VL containing the amino acid sequence of SEQ ID NO: 36;

[0542] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 38 and VL containing the amino acid sequence of SEQ ID NO: 40;

[0543] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 42 and VL containing the amino acid sequence of SEQ ID NO: 44;

[0544] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 46 and VL containing the amino acid sequence of SEQ ID NO: 48;

[0545] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 50 and VL containing the amino acid sequence of SEQ ID NO: 52;

[0546] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 54 and VL containing the amino acid sequence of SEQ ID NO: 56;

[0547] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 58 and VL containing the amino acid sequence of SEQ ID NO: 60;

[0548] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 68 and VL containing the amino acid sequence of SEQ ID NO: 70.

[0549] In some cases, any of the antibodies described above bind to epitopes on PAD4 containing SEQ ID NO: 217 and SEQ ID NO: 218.

[0550] In some embodiments described herein, the anti-PAD4 antibody is an IgA, IgG, or IgM antibody. In some cases, the antibody is an IgG antibody, such as a human IgG1, IgG2, IgG3, or IgG4 antibody, or a mouse IgG1 or IgG2 antibody. In some cases, the antibody contains a wild-type, human IgG1, IgG2, or IgG4 heavy chain constant region. In some embodiments, the antibody contains a full-length heavy chain and / or a full-length light chain. In other cases, the antibody lacks a C-terminal lysine residue at the end of the heavy chain constant region. In still other cases, the antibody lacks a C-terminal glycine-lysine residue at the end of the heavy chain constant region. In some cases, the antibody is an antibody fragment, such as Fv, single-chain Fv (scFv), Fab, Fab', or (Fab')2.

[0551] In some embodiments, the anti-PAD4 antibody is a bispecific or multispecific antibody, or it is covalently or non-covalently conjugated to at least one other molecule. In some embodiments, the antibody is covalently or non-covalently conjugated to at least one other molecule, wherein said at least one other molecule includes a detection marker and / or a drug.

[0552] In some embodiments, the antibody comprises a human IgG1 heavy chain constant region containing at least 90%, at least 95%, at least 97%, or at least 99% of the amino acid sequence identical to any one of SEQ ID No: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192. In some embodiments, the antibody comprises a light chain constant region containing at least 90%, at least 95%, at least 97%, or at least 99% of the amino acid sequence identical to that of SEQ ID NO: 194. In some embodiments, the antibody comprises both a human IgG1 heavy chain constant region and a light chain constant region, wherein the human IgG1 heavy chain constant region comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of any one of SEQ ID No: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; and the light chain constant region comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 194. In some cases, the antibody comprises a heavy chain constant region comprising an amino acid sequence modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids compared to the amino acid sequence of any one of SEQ ID No: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192. In some cases, the antibody comprises a light chain constant region, which contains an amino acid sequence modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids compared to the amino acid sequence of SEQ ID NO: 194. In some cases, the antibody comprises both a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region contains an amino acid sequence modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids compared to the amino acid sequence of any one of SEQ ID NO: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; and the light chain constant region contains an amino acid sequence modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids compared to the amino acid sequence of SEQ ID NO: 194. In some embodiments, the antibody comprises a human IgG1 heavy chain constant region comprising the amino acid sequence of any one of SEQ ID No: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192. In some embodiments, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 194.In some embodiments, the antibody comprises both a heavy chain constant region and a light chain constant region of human IgG1, wherein the heavy chain constant region of human IgG1 comprises the amino acid sequence of any one of SEQ ID No: 174, 176, 178, 180, 182, 184, 186, 188, 190 or 192; and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 194.

[0553] Clone 20 and related antibodies

[0554] The methods described herein can also be performed using anti-PAD4 antibodies derived from second mouse anti-human antibody clone 20 and its humanized variants hz20-1 to hz20-14, as described in International Patent Publication WO2024 / 020579 and Provisional Priority Application No. 63 / 528,323 filed July 21, 2023, the contents of which are incorporated herein by reference. Therefore, in some embodiments, the anti-PAD4 antibody comprises a separated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 72, an HCDR2 containing the amino acid sequence of SEQ ID NO: 73, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 74; and a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 75, an LCDR2 containing the amino acid sequence of SEQ ID NO: 76, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 77.

[0555] In some embodiments, the antibody comprises VH, said VH being at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO: 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134. In some embodiments, the antibody comprises VL, said VL being at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO: 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136. In some embodiments, the antibody comprises VH, which comprises an amino acid sequence of any one of SEQ ID Nos: 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions; and wherein the antibody comprises VL, which comprises an amino acid sequence of any one of SEQ ID Nos: 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some such cases, antibody VH further comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 72, HCDR2 containing the amino acid sequence of SEQ ID NO: 73, or HCDR3 containing the amino acid sequence of SEQ ID NO: 74. In some cases, antibody VL further comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 75, LCDR2 containing the amino acid sequence of SEQ ID NO: 76, or LCDR3 containing the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody comprises VH, which contains the amino acid sequence of any one of SEQ ID No: 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134. In some embodiments, the antibody comprises a VL containing an amino acid sequence of any one of SEQ ID No: 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136.

[0556] In yet another embodiment, the antibody comprises VH, which comprises the amino acid sequence of any one of SEQ ID No: 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134; and comprises VL, which comprises the amino acid sequence of any one of SEQ ID No: 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136. Therefore, for example, the following exemplary antibodies are within the scope of this disclosure:

[0557] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 78 and VL containing the amino acid sequence of SEQ ID NO: 80;

[0558] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 82 and VL containing the amino acid sequence of SEQ ID NO: 84;

[0559] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 86 and VL containing the amino acid sequence of SEQ ID NO: 88;

[0560] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 90 and VL containing the amino acid sequence of SEQ ID NO: 92;

[0561] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 94 and VL containing the amino acid sequence of SEQ ID NO: 96;

[0562] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 98 and VL containing the amino acid sequence of SEQ ID NO: 100;

[0563] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 102 and VL containing the amino acid sequence of SEQ ID NO: 104;

[0564] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 106 and VL containing the amino acid sequence of SEQ ID NO: 108;

[0565] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 110 and VL containing the amino acid sequence of SEQ ID NO: 112;

[0566] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 114 and VL containing the amino acid sequence of SEQ ID NO: 116;

[0567] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 118 and VL containing the amino acid sequence of SEQ ID NO: 120;

[0568] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 122 and VL containing the amino acid sequence of SEQ ID NO: 124;

[0569] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 126 and VL containing the amino acid sequence of SEQ ID NO: 128;

[0570] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 130 and VL containing the amino acid sequence of SEQ ID NO: 132;

[0571] - An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO: 134 and VL containing the amino acid sequence of SEQ ID NO: 136.

[0572] In some cases, the antibody in any of the above antibodies binds to epitopes on PAD4 containing SEQ ID NO: 219 and SEQ ID NO: 220.

[0573] In some embodiments described herein, the antibody is an IgA, IgG, or IgM antibody. In some cases, the antibody is an IgG antibody, such as a human IgG1, IgG2, IgG3, or IgG4 antibody, or a mouse IgG1 or IgG2 antibody. In some cases, the antibody contains a wild-type, human IgG1, IgG2, or IgG4 heavy chain constant region. In some aspects, the antibody belongs to the human IgG1 isotype. In some aspects, the antibody belongs to the human IgG1 isotype and has P329G, L234A, and L235A (LALAPG; EU number) mutations to reduce Fc region effector function. In other aspects, the antibody belongs to the human IgG2 isotype. In some aspects, the antibody belongs to the IgG4 isotype and has an S228P mutation (EU number) in the hinge region to improve the stability of the IgG4 antibody. In some aspects, the antibody (e.g., a non-humanized antibody) may have a non-human IgG constant region and may be, for example, a mouse IgG2a antibody, such as a mouse IgG2a LALAPG antibody. In some embodiments, the antibody contains a full-length heavy chain and / or a full-length light chain. In other cases, the antibody lacks a C-terminal lysine residue at the end of the constant region of the heavy chain. In still other cases, the antibody lacks a C-terminal glycine-lysine residue at the end of the constant region of the heavy chain. In some cases, the antibody is an antibody fragment, such as Fv, single-chain Fv (scFv), Fab, Fab', or (Fab')2.

[0574] In some embodiments, the antibody is a bispecific or multispecific antibody, or it is covalently or non-covalently conjugated to at least one other molecule. In some embodiments, the antibody is covalently or non-covalently conjugated to at least one other molecule, wherein said at least one other molecule includes a detection marker and / or a drug.

[0575] In some embodiments, the antibody comprises a human IgG1 heavy chain constant region containing at least 90%, at least 95%, at least 97%, or at least 99% of the amino acid sequence identical to any one of SEQ ID No: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192. In some embodiments, the antibody comprises a light chain constant region containing at least 90%, at least 95%, at least 97%, or at least 99% of the amino acid sequence identical to that of SEQ ID NO: 194. In some embodiments, the antibody comprises both a human IgG1 heavy chain constant region and a light chain constant region, wherein the human IgG1 heavy chain constant region comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of any one of SEQ ID No: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; and the light chain constant region comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 194. In some cases, the antibody comprises a heavy chain constant region comprising an amino acid sequence modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids compared to the amino acid sequence of any one of SEQ ID No: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192. In some cases, the antibody comprises a light chain constant region, which contains an amino acid sequence modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids compared to the amino acid sequence of SEQ ID NO: 194. In some cases, the antibody comprises both a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region contains an amino acid sequence modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids compared to the amino acid sequence of any one of SEQ ID NO: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; and the light chain constant region contains an amino acid sequence modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids compared to the amino acid sequence of SEQ ID NO: 194. In some embodiments, the antibody comprises a human IgG1 heavy chain constant region comprising the amino acid sequence of any one of SEQ ID No: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192. In some embodiments, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 194.In some embodiments, the antibody comprises both a heavy chain constant region and a light chain constant region of human IgG1, wherein the heavy chain constant region of human IgG1 comprises the amino acid sequence of any one of SEQ ID No: 174, 176, 178, 180, 182, 184, 186, 188, 190 or 192; and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 194.

[0576] pH-dependent clone 13-related antibodies

[0577] Other examples of anti-PAD4 antibodies to which the methods described herein are applied include variants of the parental antibody clone 13 or hz13-5, for example, to affect the pH dependence of PAD4 binding. These antibodies are also described in International Patent Publication WO2024 / 020579 and in Provisional Priority Application No. 63 / 528,323, filed July 21, 2023, the contents of which are incorporated herein by reference.

[0578] In some such embodiments, the antibody comprises VH, which comprises HCDR1 containing amino acid sequences at positions 26-35 of SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, or SEQ ID NO: 168; HCDR2 containing amino acid sequences at positions 50-66; and HCDR3 containing amino acid sequences at positions 99-108. In some embodiments described above, the antibody further comprises a VL containing an LCDR1 having amino acid sequences of residues 24-38 of SEQ ID NO: 170, an LCDR2 having amino acid sequences of residues 54-60 of SEQ ID NO: 170, and an LCDR3 having amino acid sequences of residues 93-101 of SEQ ID NO: 170. In other embodiments described above, the antibody further comprises a VL containing an LCDR1 having amino acid sequences of residues 24-38 of SEQ ID NO: 172, an LCDR2 having amino acid sequences of residues 54-60 of SEQ ID NO: 172, and an LCDR3 having amino acid sequences of residues 93-101 of SEQ ID NO: 172.

[0579] In some implementations, the antibody comprises:

[0580] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 138, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 138, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 138, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 170;

[0581] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 140, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 140, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 140, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 170;

[0582] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 142, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 142, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 142, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 170;

[0583] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 144, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 144, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 144, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 170;

[0584] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 146, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 146, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 146, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 170;

[0585] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 148, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 148, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 148, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 170;

[0586] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 150, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 150, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 150, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 170;

[0587] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 152, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 152, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 152, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 170;

[0588] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 154, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 154, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 154, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 170;

[0589] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 156, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 156, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 156, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 170;

[0590] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 158, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 158, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 158, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 170;

[0591] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 160, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 160, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 160, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 170;

[0592] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 162, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 162, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 162, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 170;

[0593] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 164, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 164, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 164, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 170;

[0594] - VH, wherein VH comprises HCDR1 containing amino acid sequences at positions 26-35 of SEQ ID NO: 166, HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 166, and HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 166, and VL, wherein VL comprises LCDR1 containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170; or

[0595] - VH, wherein VH comprises HCDR1 containing amino acid sequences at positions 26-35 of SEQ ID NO: 168, HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 168, and HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 168, and VL, wherein VL comprises LCDR1 containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170.

[0596] In some implementations, the antibody comprises:

[0597] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 138, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 138, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 138, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 172;

[0598] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 140, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 140, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 140, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 172;

[0599] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 142, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 142, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 142, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 172;

[0600] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 144, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 144, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 144, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 172;

[0601] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 146, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 146, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 146, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 172;

[0602] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 148, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 148, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 148, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 172;

[0603] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 150, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 150, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 150, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 172;

[0604] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 152, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 152, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 152, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 172;

[0605] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 154, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 154, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 154, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 172;

[0606] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 156, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 156, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 156, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 172;

[0607] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 158, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 158, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 158, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 172;

[0608] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 160, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 160, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 160, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 172;

[0609] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 162, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 162, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 162, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 172;

[0610] - VH, wherein VH comprises HCDR1 containing amino acid sequence at positions 26-35 of SEQ ID NO: 164, HCDR2 containing amino acid sequence at positions 50-66 of SEQ ID NO: 164, and HCDR3 containing amino acid sequence at positions 99-108 of SEQ ID NO: 164, and VL, wherein VL comprises LCDR1 containing amino acid sequence at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequence at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequence at residues 93-101 of SEQ ID NO: 172;

[0611] - VH, wherein VH comprises HCDR1 containing amino acid sequences at positions 26-35 of SEQ ID NO: 166, HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 166, and HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 166, and VL, wherein VL comprises LCDR1 containing amino acid sequences at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 172; or

[0612] - VH, wherein VH comprises HCDR1 containing amino acid sequences at positions 26-35 of SEQ ID NO: 168, HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 168, and HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 168, and VL, wherein VL comprises LCDR1 containing amino acid sequences at residues 24-38 of SEQ ID NO: 172, LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 172, and LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 172.

[0613] In some embodiments described above, the antibody comprises VH, said VH comprising an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to the amino acid sequence of SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, or SEQ ID NO: 168. In some embodiments described above, the antibody comprises VL, said VL comprising an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to the amino acid sequence of SEQ ID NO: 170. In other embodiments, the antibody comprises a VL containing an amino acid sequence that is at least 90%, at least 95%, or at least 97% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody further comprises the corresponding HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as provided above.

[0614] In some embodiments described above, the antibody comprises a VH containing the amino acid sequence of SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, or SEQ ID NO: 168. In some embodiments described above, the antibody comprises a VL containing the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID NO: 172. In some embodiments described above, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, or SEQ ID NO: 168, and VL, which comprises the amino acid sequence of SEQ ID NO: 170. In some embodiments described above, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, or SEQ ID NO: 168, and VL, which comprises the amino acid sequence of SEQ ID NO: 172.

[0615] In some embodiments, this disclosure relates to an isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) containing amino acid sequences at positions 26-35 of SEQ ID NO: 138, an HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 138, and an HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 138; and a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LCDR1) containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, an LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and an LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 138, and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID No: 138 modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids. In some embodiments, the antibody comprises VL, which comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 138 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172.In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 138; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID No: 138 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 138 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions.

[0616] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 138. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 138; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 138 and a VL containing the amino acid sequence of SEQ ID No: 170.

[0617] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 138. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 138; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 138 and a VL containing the amino acid sequence of SEQ ID No: 172.

[0618] In some embodiments, this disclosure relates to an isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) containing amino acid sequences at positions 26-35 of SEQ ID NO: 140, an HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 140, and an HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 140; and a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LCDR1) containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, an LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and an LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 140; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID No: 140 modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids. In some embodiments, the antibody comprises VL, which comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein VH comprises the amino acid sequence of SEQ ID No: 140 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and VL comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172.In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 140; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID No: 140 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 140 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions.

[0619] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 140. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 140; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 140; and a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 140 and a VL containing the amino acid sequence of SEQ ID No: 170.

[0620] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 140. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 140; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 140 and a VL containing the amino acid sequence of SEQ ID No: 172.

[0621] In some embodiments, this disclosure relates to an isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) containing amino acid sequences at positions 26-35 of SEQ ID NO: 142, an HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 142, and an HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 142; and a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LCDR1) containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, an LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and an LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 142. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 142; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID No: 142 modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids. In some embodiments, the antibody comprises VL, which comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein VH comprises the amino acid sequence of SEQ ID No: 142 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and VL comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172.In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 142; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID No: 142 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 142 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions.

[0622] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 142. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 142; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 142 and a VL containing the amino acid sequence of SEQ ID No: 170.

[0623] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 142. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 142; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 142 and a VL containing the amino acid sequence of SEQ ID No: 172.

[0624] In some embodiments, this disclosure relates to an isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) containing amino acid sequences at positions 26-35 of SEQ ID NO: 144, an HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 144, and an HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 144; and a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LCDR1) containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, an LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and an LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 144. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 144; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID No: 144 modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids. In some embodiments, the antibody comprises VL, which comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein VH comprises the amino acid sequence of SEQ ID No: 144 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and VL comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172.In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 144; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID No: 144 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 144 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions.

[0625] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 144. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 144; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 144; and a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 144 and a VL containing the amino acid sequence of SEQ ID No: 170.

[0626] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 144. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 144; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 144 and a VL containing the amino acid sequence of SEQ ID No: 172.

[0627] In some embodiments, this disclosure relates to an isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) containing amino acid sequences at positions 26-35 of SEQ ID NO: 146, an HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 146, and an HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 146; and a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LCDR1) containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, an LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and an LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 146. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 146, and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID No: 146 modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids. In some embodiments, the antibody comprises VL, which comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 146 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172.In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 146; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID No: 146 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 146 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions.

[0628] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 146. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 146; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 146 and a VL containing the amino acid sequence of SEQ ID No: 170.

[0629] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 146. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 146; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 146 and a VL containing the amino acid sequence of SEQ ID No: 172.

[0630] In some embodiments, this disclosure relates to an isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) containing amino acid sequences at positions 26-35 of SEQ ID NO: 148, an HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 148, and an HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 148; and a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LCDR1) containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, an LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and an LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 148. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 148; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID No: 148 modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids. In some embodiments, the antibody comprises VL, which comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein VH comprises the amino acid sequence of SEQ ID No: 148 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and VL comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172.In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 148; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID No: 148 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 148 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions.

[0631] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 148. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 148; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 148 and a VL containing the amino acid sequence of SEQ ID No: 170.

[0632] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 148. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 148; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 148 and a VL containing the amino acid sequence of SEQ ID No: 172.

[0633] In some embodiments, this disclosure relates to an isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (HCDR1) containing amino acid sequences at positions 26-35 of SEQ ID NO: 150, an HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 150, and an HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 150; and a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (LCDR1) containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, an LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and an LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 150. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 150; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID No: 150 modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids. In some embodiments, the antibody comprises VL, which comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein VH comprises the amino acid sequence of SEQ ID No: 150 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and VL comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172.In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 150; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID No: 150 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 150 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions.

[0634] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 150. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 150; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 150; and a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 150 and a VL containing the amino acid sequence of SEQ ID No: 170.

[0635] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 150. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 150; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 150 and a VL containing the amino acid sequence of SEQ ID No: 172.

[0636] In some embodiments, the method can be used to evaluate an antibody that specifically binds to protein arginine deiminase 4 (PAD4) and comprises a heavy chain variable region (VH) containing a heavy chain complementarity-determining region 1 (HCDR1) containing amino acid sequences at positions 26-35 of SEQ ID NO: 152, an HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 152, and an HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 152; and a light chain variable region (VL) containing a light chain complementarity-determining region 1 (LCDR1) containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, an LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and an LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 152. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 152; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID No: 152 modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids. In some embodiments, the antibody comprises VL, which comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein VH comprises the amino acid sequence of SEQ ID No: 152 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and VL comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172.In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:152; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:172. In some embodiments, the antibody comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID No:152 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID No:172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 152 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions.

[0637] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 152. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 152; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 152 and a VL containing the amino acid sequence of SEQ ID No: 170.

[0638] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 152. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 152; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 152 and a VL containing the amino acid sequence of SEQ ID No: 172.

[0639] In some embodiments, the antibody comprises a heavy chain variable region (VH) containing a heavy chain complementarity-determining region 1 (HCDR1) of amino acid sequences at positions 26-35 of SEQ ID NO: 154, an HCDR2 of amino acid sequences at positions 50-66 of SEQ ID NO: 154, and an HCDR3 of amino acid sequences at positions 99-108 of SEQ ID NO: 154; and a light chain variable region (VL) containing a light chain complementarity-determining region 1 (LCDR1) of amino acid sequences at residues 24-38 of SEQ ID NO: 170, an LCDR2 of amino acid sequences at residues 54-60 of SEQ ID NO: 170, and an LCDR3 of amino acid sequences at residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises a VH that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 154. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 154; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID No: 154 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, which comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 154 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172.In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 154; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID No: 154 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 154 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions.

[0640] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 154. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 154; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 154; and a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 154 and a VL containing the amino acid sequence of SEQ ID No: 170.

[0641] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 154. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 154; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 154 and a VL containing the amino acid sequence of SEQ ID No: 172.

[0642] In some embodiments, the method can be used to evaluate antibodies that specifically bind to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a heavy chain variable region (VH) containing a heavy chain complementarity-determining region 1 (HCDR1) containing amino acid sequences at positions 26-35 of SEQ ID NO: 156, an HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 156, and an HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 156; and a light chain variable region (VL) containing a light chain complementarity-determining region 1 (LCDR1) containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, an LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and an LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 156, and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID No: 156 modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids. In some embodiments, the antibody comprises VL, which comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein VH comprises the amino acid sequence of SEQ ID No: 156 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and VL comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172.In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 156; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID No: 156 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 156 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions.

[0643] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 156. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 156; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 156 and a VL containing the amino acid sequence of SEQ ID No: 170.

[0644] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 156. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 156; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 156 and a VL containing the amino acid sequence of SEQ ID No: 172.

[0645] In some embodiments, the antibody specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a heavy chain variable region (VH), the heavy chain variable region comprising a heavy chain complementarity-determining region 1 (HCDR1) containing amino acid sequences at positions 26-35 of SEQ ID NO: 158, an HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 158, and an HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 158; and a light chain variable region (VL), the light chain variable region comprising a light chain complementarity-determining region 1 (LCDR1) containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, an LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and an LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 158. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 158, and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID No: 158 modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids. In some embodiments, the antibody comprises VL, which comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein VH comprises the amino acid sequence of SEQ ID No: 158 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and VL comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172.In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 158; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID No: 158 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 158 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions.

[0646] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 158. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 158; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 158 and a VL containing the amino acid sequence of SEQ ID No: 170.

[0647] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 158. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 158; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 158 and a VL containing the amino acid sequence of SEQ ID No: 172.

[0648] In some embodiments, the antibody specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a heavy chain variable region (VH), the heavy chain variable region comprising a heavy chain complementarity-determining region 1 (HCDR1) containing amino acid sequences at positions 26-35 of SEQ ID NO: 160, an HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 160, and an HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 160; and a light chain variable region (VL), the light chain variable region comprising a light chain complementarity-determining region 1 (LCDR1) containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, an LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and an LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 160; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID No: 160 modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids. In some embodiments, the antibody comprises VL, which comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein VH comprises the amino acid sequence of SEQ ID No: 160 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and VL comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172.In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 160; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID No: 160 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 160 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions.

[0649] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 160. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 160; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 160 and a VL containing the amino acid sequence of SEQ ID No: 170.

[0650] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 160. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 160; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 160 and a VL containing the amino acid sequence of SEQ ID No: 172.

[0651] In some embodiments, the antibody specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a heavy chain variable region (VH), the heavy chain variable region comprising a heavy chain complementarity-determining region 1 (HCDR1) containing amino acid sequences at positions 26-35 of SEQ ID NO: 162, an HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 162, and an HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 162; and a light chain variable region (VL), the light chain variable region comprising a light chain complementarity-determining region 1 (LCDR1) containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, an LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and an LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 162. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 162; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID No: 162 modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids. In some embodiments, the antibody comprises VL, which comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein VH comprises the amino acid sequence of SEQ ID No: 162 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and VL comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172.In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 162; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID No: 162 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID No: 162 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions.

[0652] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 162. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 162; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 162 and a VL containing the amino acid sequence of SEQ ID No: 170.

[0653] In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 162. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 162; and a VL that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VL containing the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH containing the amino acid sequence of SEQ ID No: 162 and a VL containing the amino acid sequence of SEQ ID No: 172.

[0654] In some embodiments, the antibody specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a heavy chain variable region (VH), the heavy chain variable region comprising a heavy chain complementarity-determining region 1 (HCDR1) containing amino acid sequences at positions 26-35 of SEQ ID NO: 164, an HCDR2 containing amino acid sequences at positions 50-66 of SEQ ID NO: 164, and an HCDR3 containing amino acid sequences at positions 99-108 of SEQ ID NO: 164; and a light chain variable region (VL), the light chain variable region comprising a light chain complementarity-determining region 1 (LCDR1) containing amino acid sequences at residues 24-38 of SEQ ID NO: 170, an LCDR2 containing amino acid sequences at residues 54-60 of SEQ ID NO: 170, and an LCDR3 containing amino acid sequences at residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 164. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 164; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody comprises VH, which comprises the amino acid sequence of SEQ ID No: 164 modified by substitution of 1-10 amino acids, 1-5 amino acids, or 1-3 amino acids. In some embodiments, the antibody comprises VL, which comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein VH comprises the amino acid sequence of SEQ ID No: 164 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions, and VL comprises the amino acid sequence of SEQ ID No: 170 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, which is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172.In some embodiments, the antibody comprises both VH and VL, wherein the VH is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 164; and the VL is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID No: 164 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID No: 172 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises both VH and VL, wherein the VH comprises th...

Claims

1. A method for determining citrullination of a protein or a peptide fragment thereof, the method comprising evaluating citrullination at citrullination sites on a protein or a peptide fragment thereof in a biological sample from a subject, wherein the biological sample has been exposed to a PAD4 inhibitor, wherein the citrullination sites are selected from one or more of the following: R1391 of proteoglycan 4 (corresponding to R1391 of Q92954). R573 of the fibrinogen α chain (corresponding to R573 of P02671). R591 of the fibrinogen α chain (corresponding to R591 of P02671). R573 of complement C3 (corresponding to R573 of P01024). R748 of complement C3 (corresponding to R748 of P01024). R688 of the heavy chain H4, an inhibitor of inter-α-trypsin (corresponding to R688 of Q14624). R297 of the α-1-microglobulin / biskunitz inhibitor precursor (corresponding to R297 of P02760). R715 of α-2-macroglobulin (corresponding to R715 of P01023). R32 of gel sol protein (corresponding to R32 of P06396). R59 of haptoglobin (corresponding to R59 of P00738), and R651 of serum transferrin (corresponding to R651 of P02787).

2. A method for evaluating the activity of a PAD4 inhibitor, the method comprising evaluating citrullination at citrullination sites on a protein or peptide fragment thereof present in a biological sample from a subject, wherein the biological sample has been exposed to the PAD4 inhibitor, wherein the citrullination sites are selected from one or more of the following: R1391 of proteoglycan 4 (corresponding to R1391 of Q92954). R573 of the fibrinogen α chain (corresponding to R573 of P02671). R591 of the fibrinogen α chain (corresponding to R591 of P02671). R573 of complement C3 (corresponding to R573 of P01024). R748 of complement C3 (corresponding to R748 of P01024). R688 of the heavy chain H4, an inhibitor of inter-α-trypsin (corresponding to R688 of Q14624). R297 of the α-1-microglobulin / biskunitz inhibitor precursor (corresponding to R297 of P02760). R715 of α-2-macroglobulin (corresponding to R715 of P01023). R32 of gel sol protein (corresponding to R32 of P06396). R59 of haptoglobin (corresponding to R59 of P00738), and R651 of serum transferrin (corresponding to R651 of P02787).

3. The method of claim 2, wherein the PAD4 inhibitor reduces citrullination at the citrullination site in a dose-dependent manner.

4. The method according to claim 1, 2 or 3, wherein the method includes evaluating citrullination of a peptide fragment of the protein, wherein the peptide fragment contains the citrullination site.

5. The method according to any one of claims 1-4, wherein the biological sample has been exposed to the PAD inhibitor in the subject due to administration of the PAD inhibitor to the subject.

6. The method according to any one of claims 1-4, wherein the biological sample has been exposed to the PAD inhibitor by ex vivo contact of the biological sample with the PAD inhibitor.

7. The method according to any one of claims 1-6, wherein the evaluation of citrullination at the citrullination site comprises mass spectrometry (MS).

8. The method according to any one of claims 1-6, wherein the evaluation of citrullination at the citrullination site comprises liquid chromatography and mass spectrometry (LC-MS).

9. The method according to any one of claims 1-6, wherein the evaluation of citrullination includes selective reaction monitoring and chromatographic separation.

10. The method according to any one of claims 1-9, wherein evaluating the citrullination of the citrullination site comprises measuring a first concentration of the citrullinated protein or a peptide fragment thereof and a second concentration of the corresponding total protein in the biological sample.

11. The method of claim 10, wherein evaluating the 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.

12. The method of claim 11, wherein the method comprises comparing the citrullination ratio with a reference citrullination ratio.

13. The method of claim 12, wherein the reference citrullination ratio is a citrullination ratio determined for a control biological sample.

14. The method of claim 13, wherein the control biological sample is a biological sample that: (a) has not been exposed to a PAD4 modulator or a PAD4 inhibitor, (b) is from the same subject, and / or (c) is from the same subject prior to treatment with the PAD4 inhibitor.

15. The method according to any one of claims 12-14, wherein the evaluation includes determining the difference between the citrullination ratio of the biological sample and the reference citrullination ratio.

16. The method according to any one of claims 1-15, wherein the method comprises contacting the biological sample with exogenous PAD4.

17. A method, the method comprising: (i) Expose biological samples from the subject to exogenous PAD4, and (ii) Assess citrullination at citrullination sites on proteins or their peptide fragments present in biological samples; The citrullination site is selected from one or more of the following: R1391 of proteoglycan 4 (corresponding to R1391 of Q92954). R573 of the fibrinogen α chain (corresponding to R573 of P02671). R591 of the fibrinogen α chain (corresponding to R591 of P02671). R573 of complement C3 (corresponding to R573 of P01024). R748 of complement C3 (corresponding to R748 of P01024). R688 of the heavy chain H4, an inhibitor of inter-α-trypsin (corresponding to R688 of Q14624). R297 of the α-1-microglobulin / biskunitz inhibitor precursor (corresponding to R297 of P02760). R715 of α-2-macroglobulin (corresponding to R715 of P01023). R32 of gel sol protein (corresponding to R32 of P06396). R59 of haptoglobin (corresponding to R59 of P00738), and R651 of serum transferrin (corresponding to R651 of P02787).

18. The method of claim 16 or 17, wherein the method comprises incubating the exogenous PAD4 together with the biological sample.

19. The method of claim 18, wherein the method comprises incubating the exogenous PAD4 with the biological sample at a temperature of 36ºC-38ºC.

20. The method according to claim 18 or 19, wherein the incubation lasts for 1-3 hours.

21. The method according to any one of claims 18-20, wherein the method comprises adding EDTA to the biological sample after incubation with exogenous PAD4.

22. The method according to any one of claims 1-21, wherein the method comprises incubating the biological sample for an incubation period prior to evaluating citrullination.

23. The method of claim 22, wherein the incubation is performed at 35ºC-40ºC.

24. The method according to claim 22 or 23, wherein the incubation period is 48 to 96 hours.

25. The method according to any one of claims 1-24, the method comprising preparing the biological sample for evaluation prior to the evaluation.

26. The method of claim 25, wherein the preparation comprises enzymatically digesting the protein in the biological sample to form its peptide fragments.

27. The method of claim 25 or 26, wherein the preparation comprises depleting the biological sample of proteins or peptide fragments thereof that are not the target of the evaluation.

28. The method according to any one of claims 25-27, wherein the preparation includes diluting the biological sample.

29. The method according to any one of claims 25-28, wherein the preparation comprises denaturing a protein or a peptide fragment thereof in the biological sample.

30. The method according to any one of claims 25-29, wherein the preparation comprises enriching the biological sample with a protein or peptide fragment thereof containing the citrullination site to be evaluated.

31. The method of claim 30, wherein the preparation comprises contacting the biological sample with an antibody that specifically binds to both citrullinated and non-citrullinated forms of the protein or a peptide fragment thereof containing the citrullinated site to be evaluated.

32. The method according to any one of claims 1-31, wherein the method comprises freezing the biological sample and thawing the biological sample prior to evaluating the citrullination of the citrullination site.

33. The method according to any one of claims 1-32, the method comprising: (a) Immunoenriching the sample with the protein or a peptide fragment thereof to form an immunoenriched sample, wherein the immunoenrichment comprises contacting the sample with an antibody that binds to both the citrullinated and non-citrullinated forms of the protein containing the citrullinated site, and / or to both the citrullinated and non-citrullinated forms of the peptide fragment of the protein containing the citrullinated site. (b) Enzymatically digesting the proteins in the sample before or after the immunoenrichment to form the peptide fragments, and (c) Evaluate the citrullination of the peptide fragment at the citrullination site.

34. The method of claim 33, wherein the immunoenrichment comprises contacting the sample with a fixed antibody, the fixed antibody binding to both the citrullinated and uncitrullinated forms of the protein containing the citrullinated site, and / or binding to both the citrullinated and uncitrullinated forms of a peptide fragment of the protein containing the citrullinated site, and eluting the protein and / or peptide bound to the fixed antibody.

35. The method of claim 33 or 34, wherein the enzymatic digestion is performed prior to the immunoenrichment.

36. The method of claim 33 or 34, wherein the enzymatic digestion is performed after the immunoenrichment.

37. The method according to any one of claims 33-36, wherein the immunoenrichment comprises incubating the sample with the immobilized antibody for at least 30 minutes.

38. The method of claim 37, wherein the incubation is performed at a temperature of 22ºC-28ºC.

39. The method of claim 37 or 38, wherein the incubation lasts for a period of 30 to 90 minutes.

40. The method according to any one of claims 33-38, wherein the immunoenrichment comprises oscillation during the incubation.

41. The method according to any one of claims 34-40, wherein the immunoenrichment comprises removing the fixed antibody from the sample and washing the fixed antibody prior to the elution.

42. The method according to any one of claims 34-41, wherein the elution comprises washing the fixed antibody with an elution composition under acidic conditions.

43. The method of claim 42, wherein the elution composition comprises a detergent.

44. The method of claim 43, wherein the detergent is an amphoteric detergent.

45. The method according to any one of claims 34-44, wherein the method comprises neutralizing the eluted protein in a buffer solution prior to evaluation and optionally prior to enzymatic digestion.

46. ​​The method according to any one of claims 33-45, wherein the antibody is immobilized by attaching to a solid surface.

47. The method according to any one of claims 33-46, wherein the evaluation of citrullination comprises measuring a first concentration of citrullinated peptides in the digested peptide and a second concentration of characteristic peptides in the digested peptide, and optionally determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.

48. The method according to any one of claims 33-47, wherein the method comprises denaturing the proteins in the immunoenriched sample prior to the evaluation.

49. The method according to any one of claims 34-48, wherein the method comprises denaturing the eluted protein prior to the evaluation.

50. The method of claim 49, wherein the method comprises denaturing the eluted protein prior to enzymatic digestion of the eluted protein.

51. The method according to any one of claims 33-50, the method comprising diluting a plasma or serum sample prior to the immunoenrichment and performing the immunoenrichment on the diluted sample.

52. The method of claim 51, wherein the method comprises diluting the plasma or serum sample by 2 to 1000 times.

53. The method according to claim 51 or 52, wherein the volume of the diluted sample is at least 5 μl.

54. The method according to any one of claims 1-53, wherein the sequence of said peptide fragment comprises one of the following sequences, wherein said citrullinated sites are indicated by underscores: AITT R SGQTLSK (SEQ ID NO: 247), ESSSHPGIAEFPS R GK (SEQ ID NO: 248), QFTSSTSYN R GDSTFESK (SEQ ID NO: 249), SGQSED R QPVPGQQMTLK (SEQ ID NO: 250), ASHLGLA R SNLDEDIIAEENIVSR (SEQ ID NO: 251), QLGLPGPPDVPDHAAYHPF R R (SEQ ID NO: 252), GPCRAFIQLWAFDAVK (SEQ ID NO: 253), VGFYESDVMG R GHAR (SEQ ID NO: 254), ATAS R GASQAGAPQGR (SEQ ID NO: 255), L R TEGDGVYTLNDK (SEQ ID NO: 256) and DLLF R DDTVCLAK (SEQ ID NO: 257).

55. The method according to any one of claims 1-54, wherein the biological sample comprises whole blood, plasma, serum or blood supernatant.

56. The method according to claim 55, wherein the citrullinated site is: R1391 of proteoglycan 4 (corresponding to R1391 of Q92954), R573 of fibrinogen α chain (corresponding to R573 of P02671), or R591 of fibrinogen α chain (corresponding to R591 of P02671), at R591 or R593 of P02671.

57. The method of claim 56, wherein the peptide fragment comprises the following amino acid sequence: AITT R SGQTLSK (SEQ ID NO: 247), ESSSHPGIAEFPS R GK (SEQ ID NO: 248) or QFTSSTSYN R GDSTFESK (SEQ ID NO: 249), where the underlined R is the citrullinated site.

58. The method according to any one of claims 1-54, wherein the biological sample comprises synovial fluid.

59. The method according to claim 58, wherein the citrullinated site is: R1391 of proteoglycan 4 (corresponding to R1391 of Q92954), R573 of fibrinogen α chain (corresponding to R573 of P02671), R591 of fibrinogen α chain (corresponding to R591 of P02671) at R591 or R593 of P02671, R573 of complement C3 (corresponding to R573 of P01024), R688 of inter-α-trypsin inhibitor heavy chain H4 (corresponding to R688 of Q14624), or R297 of α-1-microglobulin / bisKunitz inhibitor precursor (corresponding to R297 of P02760).

60. The method of claim 59, wherein the sequence of the peptide fragment comprises one of the following sequences, wherein the citrullinated site is indicated by an underscore: AITT R SGQTLSK (SEQ ID NO: 247), ESSSHPGIAEFPS R GK (SEQ ID NO: 248), QFTSSTSYN R GDSTFESK (SEQ ID NO: 249), SGQSED R QPVPGQQMTLK (SEQ ID NO: 250), QLGLPGPPDVPDHAAYHPF R R (SEQ ID NO: 252) and GPC R AFIQLWAFDAVK (SEQ ID NO: 253).

61. The method according to any one of claims 1-60, wherein the method comprises evaluating citrullination of two or more proteins or peptide fragments thereof.

62. The method according to any one of claims 1-60, wherein the method further comprises evaluating the citrullination of at least one additional citrullination site listed in Table A or any one of Tables 3-18.

63. A method, the method comprising: (a) Immunoenriching a biological sample to form an immunoenriched sample by immunoenriching a target protein, wherein the immunoenrichment comprises contacting the sample with an antibody that specifically binds to both citrullinated and uncitrullinated forms of the target protein, or specifically binds to both citrullinated and uncitrullinated forms of a peptide fragment of the target protein, wherein the target protein or the peptide fragment contains a citrullinated site, and (b) Evaluate the citrullination at the citrullination sites in the immunoenriched samples.

64. A method comprising: (a) Immunoenriching a biological sample to form an immunoenriched sample by immunoenriching the target protein, wherein the immunoenrichment comprises contacting the sample with an antibody that binds to both citrullinated and non-citrullinated forms of the target protein, or specifically binds to both citrullinated and non-citrullinated forms of a peptide fragment of the target protein, wherein the target protein or the peptide fragment contains a citrullinated site. (b) Enzymatic digestion of the immunoenriched sample to form peptide fragments of the target protein, wherein portions (a) and (b) can be performed in any order, and (c) Evaluate the citrullination of the peptide fragment at the citrullination site.

65. A method, the method comprising: (a) Immunoenrichment of a biological sample by contacting it with a fixed antibody, the fixed antibody binding to both citrullinated and non-citrullinated forms of a target protein, the target protein containing a citrullinated site. (b) Elute the proteins bound to the fixed antibodies. (d) Enzymatic digestion of the eluted protein to form its peptide fragments, and (e) Evaluate the citrullination of the peptide fragment at the citrullination site.

66. A method, the method comprising (a) Enzymatic digestion of proteins in biological samples to form their peptide fragments. (b) Immunoenrichment of the enzymatically digested biological sample by contacting it with a fixed antibody, wherein the fixed antibody specifically binds to both citrullinated and non-citrullinated forms of a peptide fragment containing a citrullinated site. (c) Elution of peptides bound to the immobilized antibody, and (d) Evaluate the citrullination of the peptide at the citrullination site.

67. The method according to any one of claims 63-66, wherein the sample comprises whole blood, plasma, serum or blood supernatant.

68. A method for evaluating the activity of a PAD4 inhibitor, the method comprising: (a) Obtaining biological samples from the subject after treatment with at least one dose of a PAD4 inhibitor, and (b) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the biological sample.

69. An in vitro method for evaluating the activity of a PAD4 inhibitor, the method comprising: (a) Treating a biological sample from a subject with a PAD4 inhibitor to form a treated biological sample, and following said treatment, (b) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the biological sample.

70. An in vitro method for evaluating the activity of a PAD4 inhibitor, the method comprising: (i) The biological samples obtained from the subjects will be divided into multiple biological samples. (ii) Contact each of the plurality of biological samples with a different dose of the PAD4 inhibitor, and (iii) For each of the plurality of biological samples, assess the citrullination of PAD4-dependent citrullination sites on proteins or peptide fragments present in the biological sample.

71. The method of claim 70, the method further comprising calculating the IC50 of the PAD4 inhibitor based on the outcome of the assessment.

72. The method according to any one of claims 68 or 70-71, the method further comprising selecting or adjusting the dose of the PAD4 inhibitor to be administered to the subject based on the outcome of the assessment.

73. An in vitro method for evaluating citrullinated endogenous PAD4, the method comprising: (a) Incubate whole blood samples from the subjects at 35ºC–40ºC for the incubation period. (b) After the incubation period, plasma or supernatant is separated from the whole blood sample, and (c) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the plasma or supernatant.

74. An in vitro method for assessing changes in citrullination in a subject, the method comprising: (a) Incubate whole blood samples from the subject at 35ºC–40ºC for an incubation period, wherein the samples were obtained from the subject after administration of the PAD4 inhibitor. (b) After the incubation period, plasma or supernatant is separated from the whole blood sample, and (c) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the plasma or supernatant.

75. The method of claim 74, wherein the method further comprises: (d) Incubate a second whole blood sample from the subject at 35ºC–40ºC for an incubation period, wherein the second whole blood sample was obtained from the subject prior to administration of the PAD4 inhibitor. (e) After the incubation period, plasma or supernatant is separated from the second whole blood sample, and (f) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in plasma or supernatant from the second whole blood sample.

76. The method of claim 74, the method further comprising comparing the outcome of the evaluation in step (f) with the outcome of the evaluation in step (c).

77. A method for evaluating the effect of a PAD4 inhibitor, the method comprising: (a) Expose whole blood samples from subjects to PAD4 inhibitors in vitro. (b) Incubate the whole blood sample at 35ºC-40ºC for the incubation period. (c) After the incubation period, plasma or supernatant is separated from the whole blood sample, and (d) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the plasma or supernatant.

78. The method of claim 75, wherein the method further comprises (e) Incubate the control whole blood sample at 35ºC-40ºC for the incubation period. (f) After the incubation period, control plasma or supernatant is separated from the control whole blood sample, and (g) Assess the citrullination of citrullinated sites on proteins or peptide fragments present in the control plasma or supernatant.

79. The method of claim 78, further comprising comparing the outcome of the assessment in step (g) with the outcome of the assessment in step (d).

80. An in vitro method for evaluating PAD4-dependent citrullination, the method comprising: The citrullination of citrullinated sites on proteins or peptide fragments present in plasma or supernatant separated from whole blood samples that have been incubated at 35ºC–40ºC for an incubation period is evaluated.

81. The method of claim 88, wherein the plasma or supernatant has been frozen, and the method includes thawing the plasma or supernatant prior to the evaluation.

82. A method for assessing citrullination at citrullination sites, the method comprising: (i) Measuring a first concentration of citrullinated protein or 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) Measure the second concentration of the corresponding total protein in the biological sample by MS.

83. The method of claim 82, wherein the method comprises (i) measuring a first concentration of a citrullinated peptide from a target protein in a biological sample by means of MS, wherein the citrullinated peptide is citrullinated at a citrullination site, and (ii) measuring a second concentration by means of measuring the concentration of a characteristic peptide from the target protein in the biological sample by means of MS.

84. The method according to claim 82 or 83, the method further comprising calculating the ratio of the first concentration to the second concentration.

85. The method according to any one of claims 82-84, wherein the method comprises measuring the first concentration and the second concentration of each of a plurality of different proteins or peptides.

86. The method of claim 85, wherein the method comprises measuring the first concentration of each of two different peptides, each of the peptides being a non-overlapping fragment of the same protein and containing different citrullinated sites.

87. The method of claim 85 or 86, wherein the method comprises calculating a ratio of the first concentration to the second concentration of each of the plurality of different proteins or peptides.

88. The method according to any one of claims 63-87, wherein the citrullination site is a site listed in Table A or any one of Tables 3-18.

89. The method according to any one of claims 63-88, wherein the citrullinated site is selected from one or more of the following: R1391 of proteoglycan 4 (corresponding to R1391 of Q92954). R573 of the fibrinogen α chain (corresponding to R573 of P02671). R591 of the fibrinogen α chain (corresponding to R591 of P02671). R573 of complement C3 (corresponding to R573 of P01024). R748 of complement C3 (corresponding to R748 of P01024). R688 of the heavy chain H4, an inhibitor of inter-α-trypsin (corresponding to R688 of Q14624). R297 of the α-1-microglobulin / biskunitz inhibitor precursor (corresponding to R297 of P02760). R715 of α-2-macroglobulin (corresponding to R715 of P01023). R32 of gel sol protein (corresponding to R32 of P06396). R59 of haptoglobin (corresponding to R59 of P00738), and R651 of serum transferrin (corresponding to R651 of P02787).

90. The method of claim 89, wherein the method comprises evaluating citrullination at one of the following peptide sequences, wherein the citrullinated site is indicated by an underline: AITT R SGQTLSK (SEQ ID NO: 247), ESSSHPGIAEFPS R GK (SEQ ID NO: 248), QFTSSTSYN R GDSTFESK (SEQ ID NO: 249), SGQSED R QPVPGQQMTLK (SEQ ID NO: 250), ASHLGLA R SNLDEDIIAEENIVSR (SEQ ID NO: 251), QLGLPGPPDVPDHAAYHPF R R (SEQ ID NO: 252), GPCRAFIQLWAFDAVK (SEQ ID NO: 253), VGFYESDVMG R GHAR (SEQ ID NO: 254), ATAS R GASQAGAPQGR (SEQ ID NO: 255), L R TEGDGVYTLNDK (SEQ ID NO: 256) and DLLF R DDTVCLAK (SEQ ID NO: 257).

91. The method according to any one of claims 1-90, wherein the biological sample is obtained from a subject who is ACPA positive.

92. The method according to any one of claims 1-91, wherein the biological sample is obtained from a subject who is positive for endogenous PAD4 antibody.

93. The method of claim 92, wherein the subject is positive for an endogenous PAD4 activation antibody.

94. The method according to any one of claims 1-93, wherein the biological sample is obtained from a subject who has been diagnosed with citrullination-related disease or is at risk of developing citrullination-related disease.

95. The method according to any one of claims 1-94, wherein the method further comprises selecting a subject from whom the biological sample is derived for the treatment of a citrullination-related disease.

96. The method of claim 95, wherein the citrullination-related disease is an autoimmune disorder.

97. The method of claim 96, 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).

98. The method according to any one of claims 1-97, wherein the biological sample is obtained from the subject after the subject has been treated with at least one dose of a PAD4 inhibitor.

99. The method according to any one of claims 1-98, wherein the biological sample is obtained from a subject who is a normal healthy subject.

100. The method according to any one of claims 1-99, wherein the method further comprises administering a PAD4 inhibitor to the subject, and optionally evaluating citrullination of citrullinated sites on proteins or peptide fragments thereof in samples collected from the subject after administration of the PAD4 inhibitor.

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