Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/745—Assays involving non-enzymic blood coagulation factors
  • G01N2333/75—Fibrin; Fibrinogen
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
  • G01N2440/18—Post-translational modifications [PTMs] in chemical analysis of biological material citrullination
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the present application relates to methods involving assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in a biological sample from a subject.
• the methods may be used for, inter alia, assessing the citrullination modifying activity of agents including PALM (peptidyl arginine deiminase 4) modulators, such as small molecules or antibodies, for example.
• PALM peptidyl arginine deiminase 4
• PALM peptidyl arginine deiminase 4
• PAD4 is an enzyme that catalyzes the conversion of arginine to citrulline.
• About 15% of RA patients have antibodies that activate PAD4, and presence of such PAD4-activating antibodies correlates with severe joint erosive disease.
• Compounds capable of modulating the activity of PAD4 may be useful in treating various conditions. For example, PAD4 inhibitors may be useful for treating autoimmune diseases, such as RA and others.
• PAD4 modulators are useful, for example, for monitoring treatment with existing PAD4 modulators and for assessing novel PAD4 modulators in development, among other uses.
• the present disclosure provides methods of assessing citrullination of proteins or peptides, and methods of assessing the activity of PAD4 modulators or other therapeutic agents that can affect citrullination, by assessing citrullination of one or more citrullination sites on a protein or a peptide fragment thereof present in a biological sample.
• the present disclosure relates, inter alia, to embodiments including the following, for example:
• a method of determining citrullination of a protein or peptide fragment thereof comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof in a biological sample from a subject, wherein the biological sample has been exposed to a PAD4 inhibitor, wherein the citrullination site is selected from one or more of the following:
• R297 of alpha- 1-microglobulin/bikunin precursor (corresponding to R297 of P02760), R715 of alpha-2-macroglobulin (corresponding to R715 of PO 1023),
• a method of assessing the activity of a PAD4 inhibitor comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample from a subject, wherein the biological sample has been exposed to the PAD4 inhibitor, wherein the citrullination site is selected from one or more of the following:
• R297 of alpha- 1-microglobulin/bikunin precursor (corresponding to R297 of P02760), R715 of alpha-2-macroglobulin (corresponding to R715 of P01023),
• assessing citrullination of the citrullination site comprises liquid chromatography and mass spectrometry (LC-MS).
• assessing citrullination of the citrullination site comprises measuring, in the biological sample, a first concentration of the citrullinated protein or peptide fragment thereof and a second concentration of corresponding total protein.
• assessing citrullination of the citrullination site comprises determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.
• control biological sample is a biological sample that: (a) has not been exposed to a PAD4 modulator or PAD4 inhibitor, (b) is from the same subject, and/or (c) is from the same subject prior to treatment with the PAD4 inhibitor.
• the assessing comprises determining a difference between the citrullination ratio for the biological sample and the reference citrullination ratio. 16. The method of any one of embodiments 1-15, wherein the method comprises contacting the biological sample with exogenous PAD4.
• a method comprising:
• citrullination site is selected from one or more of the following:
• R715 of alpha-2-macroglobulin (corresponding to R715 of PO 1023),
• immunoenriching comprises contacting the sample with an antibody that binds to both citrullinated and noncitrullinated forms of the protein and/or to both citrullinated and noncitrullinated forms of the peptide fragment thereof comprising the citrullination site,
• the immunoenriching comprises contacting the sample with an immobilized antibody that binds to both citrullinated and noncitrullinated forms of the protein and/or to both citrullinated and noncitrullinated forms of the peptide fragment thereof comprising the citrullination site, and eluting protein and/or peptide bound to the immobilized antibody.
• the method comprises neutralizing eluted protein in a buffer prior to assessing, and optionally prior to enzymatically digesting.
• assessing citrullination comprises measuring a first concentration of citrullinated peptide in the digested peptides and a second concentration of signature peptide in the digested peptides, and optionally determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.
• sequence of the peptide fragment comprises one of the following sequences, wherein the citrullination site is designated by underlining: AITTRSGQTLSK (SEQ ID NO: 247), ESSSHHPGIAEFPSRGK (SEQ ID NO: 248), QFTSSTSYNRGDSTFESK (SEQ ID NO: 249), SGQSEDRQPVPGQQMTLK (SEQ ID NO: 250), ASHLGLARSNLDEDIIAEENIVSR (SEQ ID NO: 251), QLGLPGPPDVPDHAAYHPFRR (SEQ ID NO: 252), GPCRAFIQLWAFDAVK (SEQ ID NO: 253), VGFYESDVMGRGHAR (SEQ ID NO: 254), ATASRGASQAGAPQGR (SEQ ID NO: 255), LRTEGDGVYTLNDK (SEQ ID NO: 256), and DLLFRDDTVCLAK (SEQ ID NO: 257).
• citrullination site is: R1391 of proteoglycan 4 (corresponding to R1391 of Q92954), R573 of fibrinogen alpha chain (corresponding to R573 of P02671), or R591 of fibrinogen alpha chain (corresponding to R591 of P02671), at R591 ofP02671 or R593 ofP02671.
• peptide fragment comprises the amino acid sequence of AITTRSGQTLSK (SEQ ID NO: 247), ESSSHHPGIAEFPSRGK (SEQ ID NO: 248), or QFTSSTSYNRGDSTFESK (SEQ ID NO: 249), wherein the underlined R is the citrullination site.
• citrullination site is: R1391 of proteoglycan 4 (corresponding to R1391 of Q92954), R573 of fibrinogen alpha chain (corresponding to R573 of P02671), R591 of fibrinogen alpha chain (corresponding to R591 of P02671), at R591 ofP02671 or R593 ofP02671, R573 of complement C3 (corresponding to R573 of P01024), R688 of inter-alpha-trypsin inhibitor heavy chain H4 (corresponding to R688 of QI 4624), or R297 of alpha- 1-microglobulin/bikunin precursor (corresponding to R297 of P02760).
• sequence of the peptide fragment comprises one of the following sequences, wherein the citrullination site is designated by underlining: AITTRSGQTLSK (SEQ ID NO: 247), ESSSHHPGIAEFPSRGK (SEQ ID NO: 248), QFTSSTSYNRGDSTFESK (SEQ ID NO: 249), SGQSEDRQPVPGQQMTLK (SEQ ID NO: 250), QLGLPGPPDVPDHAAYHPFRR (SEQ ID NO: 252), and GPCRAFIQLWAFDAVK (SEQ ID NO: 253).
• a method comprising:
• immunoenriching comprises contacting the sample with an antibody that specifically binds to both citrullinated and noncitrullinated forms of the protein of interest or that specifically binds to both citrullinated and noncitrullinated forms of a peptide fragment of the protein of interest, the protein of interest or peptide fragment comprising a citrullination site, and
• a method comprising:
• immunoenriching comprises contacting the sample with an antibody that binds to both citrullinated and noncitrullinated forms of the protein of interest or that specifically binds to both citrullinated and noncitrullinated forms of a peptide fragment of the protein of interest, the protein of interest or the peptide fragment comprising a citrullination site,
• a method comprising (a) immunoenriching a biological sample by contacting the sample with an immobilized antibody that binds to both citrullinated and noncitrullinated forms of a protein of interest, the protein of interest comprising a citrullination site,
• a method of assessing the activity of a PAD4 inhibitor comprising
• An in vitro method of assessing PAD4-dependent citrullination comprising assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in plasma or supernatant that has been separated from a whole blood sample that has been incubated at 35-40°C for an incubation period.
• a method of assessing citrullination at a citrullination site comprising
• citrullination site is selected from one or more of the following:
• R573 of fibrinogen alpha chain (corresponding to R573 of P02671), R591 of fibrinogen alpha chain (corresponding to R591 of P02671), R573 of complement C3 (corresponding to R573 of PO 1024), R748 of complement C3 (corresponding to R748 of PO 1024),
• R297 of alpha- 1-microglobulin/bikunin precursor (corresponding to R297 of P02760), R715 of alpha-2-macroglobulin (corresponding to R715 of PO 1023), R32 of gel solin (corresponding to R32 of P06396),
• the method comprises assessing citrullination at one of the following peptide sequences, wherein the citrullination site is designated by underlining: AITTRSGQTLSK (SEQ ID NO: 247), ESSSHHPGIAEFPSRGK (SEQ ID NO: 248), QFTSSTSYNRGDSTFESK (SEQ ID NO: 249), SGQSEDRQPVPGQQMTLK (SEQ ID NO: 250), ASHLGLARSNLDEDIIAEENIVSR (SEQ ID NO: 251), QLGLPGPPDVPDHAAYHPFRR (SEQ ID NO: 252), GPCRAFIQLWAFDAVK (SEQ ID NO: 253), VGFYESDVMGRGHAR (SEQ ID NO: 254), ATASRGASQAGAPQGR (SEQ ID NO: 255), LRTEGDGVYTLNDK (SEQ ID NO: 256), and DLLFRDDTVCLAK (SEQ ID NO: 257).
• AITTRSGQTLSK SEQ
• 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).
• lupus e.g., systemic lupus erythematosus (SLE)
• lupus nephritis e.g., vasculitis (e.g., ANCA-associated vasculitis)
• thrombosis e.g, venous thrombosis
• IBD inflammatory bowel disease
• a method of assessing the activity of a PAD4 modulator comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample from a subject, wherein the biological sample has been exposed to a PAD4 modulator, wherein the protein is selected from one or more of: alpha-2-HS- glycoprotein, alpha-2-macroglobulin, albumin, antithrombin-III, apolipoprotein A-I, apolipoprotein A-IV, apolipoprotein B-100, apolipoprotein E, apolipoprotein LI, C4b- binding protein alpha chain, ceruloplasmin, clusterin, complement C2, complement C3, complement C4-A, complement C4-B, complement factor B, complement factor H, complement factor I, complement component C9, fibrinogen alpha chain, fibrinogen beta chain, fibrinogen gamma chain, fibronectin, galectin-3 -bind
• citrullination site is selected from one or more citrullination sites shown in Table A or Tables 3-18.
• citrullination site is selected from one or more of the following:
• R297 of alpha- 1-microglobulin/bikunin precursor (corresponding to R297 of P02760), R715 of alpha-2-macroglobulin (corresponding to R715 of PO 1023), R32 of gel solin (corresponding to R32 of P06396),
• R59 of haptoglobin (corresponding to R59 of P00738), and R651 of serotransferrin (corresponding to R651 ofP02787).
• citrullination site is selected from one or more citrullination sites shown in Table 11 and/or wherein the sequence of the peptide fragment comprises a peptide sequence listed in Table 11.
• assessing citrullination of the citrullination site comprises mass spectrometry (MS).
• assessing citrullination of the citrullination site comprises liquid chromatography and mass spectrometry (LC-MS).
• assessing citrullination of the citrullination site comprises measuring, in the biological sample, a first concentration of citrullinated protein or peptide and a second concentration of corresponding total protein.
• assessing citrullination of the citrullination site comprises determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.
• control biological sample is a biological sample that has not been exposed to the PALM modulator.
• a method of assessing the activity of a PALM modulator comprising
• the assessing comprises (i) measuring a first concentration of citrullinated protein or peptide in the sample and (ii) measuring a second concentration of corresponding total protein in the sample, wherein the citrullinated protein or peptide is citrullinated at the citrullination site and the corresponding total protein encompasses modified and unmodified forms of the protein, and optionally (iii) determining a citrullination ratio, which is a ratio of the first concentration to the second concentration.
• control biological sample is from the same subject as the biological sample.
• a method of assessing effects of a PALM modulator comprising
• An in vitro method of assessing PAD4-dependent citrullination comprising assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in plasma or supernatant that has been separated from a whole blood sample that has been incubated at 35-40°C for an incubation period.
• a method comprising:
• immunoenriching a sample for a protein of interest to form an immunoenriched sample, wherein the immunoenriching comprises contacting the sample with an antibody that specifically binds to both citrullinated and noncitrullinated forms of the protein of interest, the protein of interest comprising a citrullination site, and
• a method comprising:
• immunoenriching a sample for a protein or a peptide of interest to form an immunoenriched sample, wherein the immunoenriching comprises contacting the sample with an antibody that binds to both citrullinated and noncitrullinated forms of the protein or peptide of interest, the protein or peptide of interest comprising a citrullination site, (b) enzymatically digesting the immunoenriched sample to form digested peptides from the protein of interest either before or after the immunoenriching, and
• the method comprises neutralizing eluted protein in a buffer prior to assessing, and optionally prior to enzymatically digesting.
• assessing citrullination comprises measuring a first concentration of citrullinated peptide in the digested peptides and a second concentration of signature peptide in the digested peptides, and optionally determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.
• a method comprising
• the method of embodiment 101 or 102, wherein the method comprises preparing the biological sample for assessment prior to the assessing.
• the preparing comprises enzymatically digesting proteins in the biological sample to produce digested peptides and optionally cleaning up the digested peptides.
• the preparing comprises immunoenriching the biological sample for the protein or peptide fragment thereof using an antibody that binds to citrullinated and non-citrullinated forms of the protein or peptide.
• citrullination site is selected from one or more citrullination sites shown in Table A or in one or more of Tables 3- 18.
• citrullination site is selected from one or more of the following: R1391 of proteoglycan 4 (corresponding to R1391 of Q92954), R573 of fibrinogen alpha chain (corresponding to R573 of P02671), R591 of fibrinogen alpha chain (corresponding to R591 of P02671), R573 of complement C3 (corresponding to R573 of PO 1024), R748 of complement C3 (corresponding to R573 of PO 1024),
• R297 of protein AMBP (corresponding to R297 of P02760), R715 of alpha-2-macroglobulin (corresponding to R715 of PO 1023), R32 of gel solin (corresponding to R32 of P06396), R59 of haptoglobin (corresponding to R59 of P00738), and R651 of serotransferrin (corresponding to R651 ofP02787).
• AITTRSGQTLSK SEQ ID NO: 247)
• ESSSHHPGIAEFPSRGK SEQ ID NO: 248
• QFTSSTSYNRGDSTFESK SEQ ID NO: 249
• SGQSEDRQPVPGQQMTLK SEQ ID NO: 250
• ASHLGLARSNLDEDIIAEENIVSR SEQ ID NO: 251
• QLGLPGPPDVPDHAAYHPFRR SEQ ID NO: 252
• GPCRAFIQLWAFDAVK SEQ ID NO: 253
• VGFYESDVMGRGHAR SEQ ID NO: 254
• ATASRGASQAGAPQGR SEQ ID NO: 255
• LRTEGDGVYTLNDK SEQ ID NO: 256
• the method comprises (i) measuring a first concentration of a citrullinated peptide from a target protein in a biological sample by MS, wherein the citrullinated peptide is citrullinated at a citrullination site, and (ii) measuring the second concentration by measuring the concentration of a signature peptide from the target protein in the biological sample by MS.
• citrullinated protein is selected from alpha-2-HS-glycoprotein, alpha-2-macroglobulin, albumin, antithrombin-III, apolipoprotein A-I, apolipoprotein A-IV, apolipoprotein B-100, apolipoprotein E, apolipoprotein LI, C4b-binding protein alpha chain, ceruloplasmin, clusterin, complement C2, complement C3, complement C4-A, complement C4-B, complement factor B, complement factor H, complement factor I, complement component C9, fibrinogen alpha chain, fibrinogen beta chain, fibrinogen gamma chain, fibronectin, galectin-3 -binding protein, gelsolin, haptoglobin,
• citrullinated peptide comprises a sequence selected from the peptide sequences listed in Tables 3-18.
• citrullinated protein is selected from proteoglycan 4, fibrinogen alpha chain, complement C3, inter-alpha-trypsin inhibitor heavy chain H4, protein AMBP, alpha-2 macroglobulin, gelsolin, haptoglobin, and serotransferrin, or (ii) the citrullinated peptide is a peptide fragment of a protein listed in (i).
• R1391 of proteoglycan 4 (corresponding to R1391 of Q92954), R573 of fibrinogen alpha chain (corresponding to R573 of P02671), R591 of fibrinogen alpha chain (corresponding to R591 of P02671), R573 of complement C3 (corresponding to R573 of P01024), R748 of complement C3 (corresponding to R573 of P01024), R688 of inter-alpha-trypsin inhibitor heavy chain H4 (corresponding to R688 of QI 4624),
• R297 of protein AMBP (corresponding to R297 of P02760), R715 of alpha-2-macroglobulin (corresponding to R715 of PO 1023),
• R59 of haptoglobin (corresponding to R59 of P00738), and R651 of serotransferrin (corresponding to R651 ofP02787).
• a citrullinated peptide comprising a peptide sequence selected from: AITTRSGQTLSK (SEQ ID NO: 247), ESSSHHPGIAEFPSRGK (SEQ ID NO: 248), QFTSSTSYNRGDSTFESK (SEQ ID NO: 249), SGQSEDRQPVPGQQMTLK (SEQ ID NO: 250), ASHLGLARSNLDEDIIAEENIVSR (SEQ ID NO: 251), QLGLPGPPDVPDHAAYHPFRR (SEQ ID NO: 252), GPCRAFIQLWAFDAVK (SEQ ID NO: 253), VGFYESDVMGRGHAR (SEQ ID NO: 254), ATASRGASQAGAPQGR (SEQ ID NO: 255), LRTEGDGVYTLNDK (SEQ ID NO: 256), and DLLFRDDTVCLAK (SEQ ID NO: 257), in each case where
• citrullinated protein or citrullinated peptide comprises fibrinogen alpha chain and/or gelsolin or a peptide fragment thereof.
• citrullinated protein or citrullinated peptide is fibrinogen alpha chain or a peptide fragment thereof.
• citrullinated peptide comprises the amino acid sequence of QFTSSTSYNRGDSTFESK (SEQ ID NO: 249), wherein the underlined R is the citrullination site, or wherein the citrullinated peptide comprises the amino acid sequence of ESSSHHPGIAEFPSRGK (SEQ ID NO: 248), wherein the underlined R is the citrullination site.
• citrullinated protein or citrullinated peptide comprises gelsolin or a peptide fragment thereof.
• citrullinated peptide comprises the amino acid sequence of ATASRGASQAGAPQGR (SEQ ID NO: 255), wherein the underlined R is the citrullination site.
• the biological sample is obtained from a subject.
• invention 139, 140 or 141 wherein the method further comprises determining one or more of the following based on the first and second concentrations or a ratio of the first and second concentrations: probability of a clinical outcome, risk for developing a citrullination-related disease, and diagnosis of a citrullination-related disease.
• 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).
• lupus e.g., systemic lupus erythematosus (SLE)
• lupus nephritis e.g., vasculitis (e.g., ANCA-associated vasculitis)
• thrombosis e.g, venous thrombosis
• IBD inflammatory bowel disease
• any of the method embodiments listed above that include a PAD4 modulator may, in further embodiments, also be performed with other therapeutic agents.
• Nonlimiting examples of such other therapeutic agents include agents that may be used for treatment of citrullination-related diseases, and agents that target molecules involved in biological pathways, such as, for example, NETosis or METosis, which may also involve PAD4.
• a PAD4 modulator may be substituted with such another therapeutic agent.
• the PAD4 modulator may, in some cases, be a PAD4 inhibitor, examples of which are provided throughout the disclosure.
• the assessing of citrullination may be performed by MS, including by LC-MS, for example.
• citrullination sites for assessment may be as recited, for instance, in embodiments above.
• biological samples may be any such samples recited herein, including without limitation, biological fluid samples such as blood, plasma, serum, blood supernatant, synovial fluid, lymph, pleural fluid, interstitial fluid, sweat, tears, sputum, or urine, as well as tissue samples.
• a citrullination ratio as described below, and/or a concentration of citrullinated protein or peptide and a concentration of corresponding total protein may be determined as part of the method.
• a biological sample may be prepared prior to assessing, in a variety of ways, such as dilution, freezing and thawing, denaturing of proteins, enzymatic digestion of proteins, separation of a supernatant from the sample for analysis, and the like.
• the above embodiments may also be applied to control samples as well as to biological samples of interest, for instance, to allow for comparisons of citrullination assessments between samples of interest and controls.
• FIG. 1 shows dose-dependent inhibition of human PAD4 with antibody hzl3-5 D31E at four concentrations of recombinant human PAD4 (rhPAD4) ranging from 13.5 nM (1 pg/mL) to 108 nM (8 pg/mL).
• FIGs. 2A-2F show reduced extracellular citrullinated histone 3 in lipopolysaccharide (LPS)-stimulated human CD14+ monocytes treated with the specified concentrations of an indicated antibody or with isotype control antibody.
• FIG. 2A shows citrullinated histone 3 levels in monocytes incubated with hzl3-5.
• FIG. 2B shows citrullinated histone 3 levels in monocytes incubated with hzl3-5 D31E.
• FIG. 2C citrullinated histone 3 levels in monocytes incubated with hz20-2.
• FIG. 2D shows citrullinated histone 3 levels in monocytes incubated with hz20-7.
• FIG. 2E shows citrullinated histone 3 levels in monocytes incubated with hzl3- 3.
• FIG. 2F shows citrullinated histone 3 levels in monocytes incubated with hzl3-12.
• FIGs. 3 A-3E show efficacy of anti-human PAD4 mAbs and humanized derivatives in an LPS ALI PD model.
• FIG. 3 A, FIG. 3B, and FIG. 3C respectively show the effects of clone 20, humanized clone 20-based hz20-2 antibody and humanized clone 20-based hz20-7 antibody at the indicated doses on extracellular citrullinated H3 relative to total extracellular H3; the percent reductions indicate reductions relative to the isotype control condition.
• 3E respectively show the effects of clone 20-based hz20-2 antibody and clone 20-based hz20-7 antibody at the indicated doses on extracellular citrullinated ITIH4 relative to total extracellular ITIH4; the percent reductions indicate reductions relative to the isotype control condition.
• Mpk mg/kg.
• IC Isotype Control antibody.
• Naive not nebulized with LPS.
• FIGs. 4A-4E show efficacy of anti-human PAD4 mAbs clone 13 and humanized derivatives in an LPS ALI PD model.
• FIG. 4 A, FIG. 4B, and FIG. 4C respectively show the effects of clone 13, humanized clone 13 -based hz 13-12 antibody, and humanized clone 13- based hzl3-5 antibody at the indicated doses on extracellular citrullinated H3 relative to total extracellular H3; the percent reductions indicate reductions relative to the isotype control condition.
• FIGs. 5A-5E show experimental design and human PAD4 endpoints of an LPS AJI PD study using Hu-PAD4 knock-in mice.
• FIG. 5A shows a study timetable.
• FIG. 5B shows an overview of experimental procedures including treatment at the knee joint and extraction of citrullinated proteins from explanted tissue.
• Extracellular Cit-PRG4 (FIG. 5C), extracellular Cit-ITIH4 (FIG. 5D) and human PAD4 (FIG. 5E) of patella explant supernatant are shown.
• FIGs. 7A-7C show efficacy of anti-human PAD4 antibodies in an LPS AJI PD study.
• Extracellular Cit-PRG4 of patella explant supernatant is shown.
• the effects of clone 13-based hzl3-12 antibody (FIG. 7 A), clone 13 -based hzl3-5 antibody (FIG. 7B), and clone 20-based hz20-2 antibody (FIG. 7C) at the indicated doses on extracellular citrullinated PRG4 relative to total extracellular PRG4 are shown; the percent reductions indicate reductions relative to the isotype control condition.
• “Mpk” mg/kg.
• IC Isotype Control antibody.
• PBS phosphate buffered saline.
• Naive not injected with LPS.
• FIGs. 8A-8B show efficacy of anti-human PAD4 antibodies hzl3-5 and hzl3-5 D31E in an LPS AJI study.
• the antibodies inhibited citrullination of ITIH4 (FIG. 8 A) and PRG4 (FIG. 8B); results showed dose dependent inhibition by the hzl3-5 D31E antibody.
• Mpk mg/kg.
• IC Isotype Control antibody.
• Naive not injected with LPS.
• FIG. 9A shows the results for the gelsolin (GSN) peptide.
• Figure discloses SEQ ID NO: 405.
• FIG. 9B shows the results for the complement C3 (C3) peptide.
• Figure discloses SEQ ID NO: 406.
• FIG. 9C shows the results for the SerpinCl peptide.
• Figure discloses SEQ ID NO: 407.
• FIG. 9D shows the results for the fibrinogen A (FGA) peptide.
• FIG. 10A-10C show that citrullination of endogenous serum proteins by exogenous PAD4 is inhibited by hzl3-5 D3 IE in a dose dependent manner.
• Samples were analyzed using targeted proteomic approach on nanoLC-TimsTOF platform. Citrullination of the peptides listed in Table 15 was assessed. The % citrullination is shown and was calculated as described for FIGs. 9A-9D.
• FIG. 10A shows inhibition of citrullination of the FGA peptide ESSSHHPGIAEFPSRGK (SEQ ID NO: 248).
• FIG. 10B shows inhibition of citrullination of the FGA peptide QFTSSTSYNRGDSTFESK (SEQ ID NO: 249).
• FIG. 10C shows inhibition of citrullination of the PRG4 peptide.
• FIG. 11 A-l IB show that induction and inhibition of citrullination ex vivo in incubated blood (from 1 st draw) was confirmed using a targeted proteomic approach.
• Whole blood samples were treated with Ab hzl3-5 D31E, PBS (control), or isotype control and incubated for 48 h at 37°C.
• PBS-Oh shows citrullination levels before induction.
• FIG. 11 A shows the results for the GSN peptide.
• FIG. 1 IB shows results for the FGA peptide.
• FIG. 12 shows targeted proteomic analysis of ex vivo citrullinated FGA in TruCulture samples after immunocapture enrichment.
• the FGA peptide QFTSSTSYNRGDSTFESK (SEQ ID NO: 249) was measured on the LC-TripleQuad 7500 system. Representative chromatograms are shown from two different donor samples in which citrullination percentages of 0.5% (left panel) and 2.6% (right panel) were measured. The % citrullination was calculated as described for FIGs. 9A-9D.
• Figure 12 also discloses “QFTSSTSYNRGDSTFES” as SEQ ID NO: 409.
• FIGs. 13 A-13B show that the hzl3-5 D3 IE antibody maintained potency in the presence of endogenous PAD4 antibodies from RA patients.
• FIG. 13 A shows PAD4 autoantibodies measured by OD450 in ELISA from purified IgG of serum samples of 21 RA patients and 10 healthy control subjects (NHV).
• FIG. 13B shows inhibition of H3 citrullination by hzl3-5 D3 IE in the presence of purified IgG from RA (closed squares) or NHV (open squares) serum.
• Dotted lines represent H3 citrullination by PAD4 in the presence of purified IgG from RA donors (lower line) and NHV donors (upper line) without hzl3-5 D31E.
• FIG. 14 shows the citrullinated proteoglycan 4 (Cit-PRG4) plasma concentrations in pM from rheumatoid (RA) patents and normal healthy volunteers (NHV). Error bars represent mean ⁇ SD. 30 RA and 30 NHV samples were analyzed for this dataset.
• the term “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated.
• the term “about” generally refers to a range of numerical values (e.g., +/-5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
• the terms modify all of the values or ranges provided in the list.
• the term about may include numerical values that are rounded to the nearest significant figure.
• a “biological sample” herein refers to a sample taken from a biological source, such as from a subject.
• a “biological sample” may comprise fluid and/or tissue, and thus it may be a “biological fluid sample” or a “tissue sample,” and it may be processed, such as centrifuged or mixed with other agents or the like, in order to facilitate analysis.
• certain biological fluid samples may comprise supernatant following separation of particulate and fluid matter.
• the biological sample comprises, for example, blood or blood components such as whole blood, serum, plasma, or blood supernatant. In some cases, it may comprise synovial fluid.
• the biological sample may be a tissue sample, for instance, a tumor sample or a neoplasia sample.
• polypeptide refers to a polymer of amino acid residues, and is not limited to a minimum length.
• a “protein” may comprise one or more polypeptides.
• Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition.
• the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
• a “polypeptide” or “protein” refers to a polypeptide or protein, respectively, which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site- directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins or errors due to PCR amplification.
• a protein may comprise two or more polypeptides.
• Some amino acids within a protein or its peptide fragment may be “citrullinated.” As used herein, this term means that the protein or its peptide fragment contains at least one amino acid that is converted from one of the 20 naturally occurring amino acids to a citrulline amino acid. For example, certain arginine (Arg, R) residues may be chemically modified to convert their side chains to the side chain of the amino acid citrulline (Cit). Thus, such arginine residues, and the corresponding peptides or proteins on which they are found are referred to as being citrullinated.
• the amino acid residue that is converted to citrulline may also be referred to herein as a “citrullination site.”
• a residue at a particular amino acid position within a protein may be citrullinated.
• that particular amino acid position may be referred to as a “citrullination site.”
• a citrullination site herein may in some cases be identified with reference to an exemplary, reference amino acid sequence for the protein.
• one such sequence may be provided herein as a reference sequence for identifying the residue corresponding to a citrullination site, and that site may be identified in other naturally occurring variants of the protein through sequence alignment with the reference sequence provided herein.
• a given protein may also have one or more such citrullination sites when more than one residue in the protein is found to be citrullinated.
• PAD4 or “protein arginine deiminase 4” or “peptidyl arginine deiminase 4,” as used herein, refers to human PAD4 (huPAD4; UniProt ID: Q9UM07), unless expressly noted otherwise (i.e., murine PAD4, cynomolgus PAD4, or the like).
• Exemplary human PAD4 amino acid sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 and SEQ ID NO: 3.
• a PAD4 modulator may modulate the activity of additional proteins in addition to PAD4, such as, for example, additional PAD enzymes, such as PAD2; while in other cases, the activity modulation may be specific to PAD4 and the PAD4 modulator does not modulate the activity of other proteins such as, for example, other PAD enzymes such as PAD2.
• agonist refers to a compound that causes an increase in at least one activity or function of a molecule to which it binds, or otherwise activates or helps to activate the molecule.
• antagonist refers to a compound that causes a decrease in at least one activity or function of a molecule to which it binds, or that otherwise blocks or inhibits at least one activity or function of the molecule.
• inhibitors more generally refer to a decrease or cessation of any event (such as protein ligand binding) or to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic.
• To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and/or amount as compared to a reference. It is not necessary that the inhibition or reduction be complete.
• by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20% or greater.
• by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater.
• by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.
• a PALM modulator may be a small molecule compound, or it may be a large molecule such as an antibody, or it may be another type of biologic.
• An “anti -PALM antibody” or a “PALM-antibody” or an “antibody that specifically binds to PALM” or an “antibody that binds to PALM” and similar phrases refer to an antibody that specifically binds to PALM as defined herein.
• the term “antibody” herein refers to a molecule comprising at least complementarity-determining region (CDR) 1, CDR2, and CDR3 of a heavy chain and at least CDR1, CDR2, and CDR3 of a light chain, wherein the molecule is capable of binding to antigen.
• the term is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, diabodies, etc.), full length antibodies, single-chain antibodies, antibody conjugates, and antibody fragments, so long as they exhibit the desired PALM-specific binding activity.
• an “isolated” antibody is one that has been separated from a component of its natural environment.
• an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods.
• electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
• chromatographic e.g., ion exchange or reverse phase HPLC
• an “antigen” refers to the target of an antibody, i.e., the molecule to which the antibody specifically binds.
• epitope denotes the site on an antigen, either proteinaceous or non-proteinaceous, to which an antibody binds.
• Epitopes on a protein can be formed both from contiguous amino acid stretches (linear epitope) or comprise noncontiguous amino acids (conformational epitope), e.g., coming in spatial proximity due to the folding of the antigen, i.e., by the tertiary folding of a proteinaceous antigen.
• Linear epitopes are typically still bound by an antibody after exposure of the proteinaceous antigen to denaturing agents, whereas conformational epitopes are typically destroyed upon treatment with denaturing agents.
• heavy chain refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence.
• a heavy chain comprises at least a portion of a heavy chain constant region.
• full-length heavy chain refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.
• light chain refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence.
• a light chain comprises at least a portion of a light chain constant region.
• full-length light chain refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.
• “Framework” or “FR” refers to the residues of the variable region residues that are not part of the complementary determining regions (CDRs).
• the FR of a variable region generally consists of four FRs: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR- L1)-FR2- CDR-H2(CDR-L2)-FR3- CDR-H3(CDR-L3)-FR4.
• variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
• the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). See, e.g., Kindt et al. Kuby Immunology, 6 th ed., W.H. Freeman and Co., page 91 (2007).
• a variable domain may comprise heavy chain (HC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and/or FR4; and light chain (LC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and/or FR4. That is, a variable domain may lack a portion of FR1 and/or FR4 so long as it retains antigen-binding activity.
• a single VH or VL domain may be sufficient to confer antigen-binding specificity.
• antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150 :880-887 (1993) ; Clarkson et al., Nature 352 :624-628 (1991).
• each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant heavy domains (CHI, CH2, and CH3).
• VH variable domain
• CHI variable heavy domain
• CH2 constant heavy domain
• VL variable domain
• CL constant light
• Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
• the term includes native sequence Fc regions and variant Fc regions.
• a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain at Gly446 and Lys447 (EU numbering).
• Antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain.
• an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine and lysine, respectively. Therefore, the C-terminal lysine, or the C-terminal glycine and lysine, of the Fc region may or may not be present.
• “Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.
• the “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
• the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, 8, y, and p, respectively.
• the light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.
• chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
• a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs.
• a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non- human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
• a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
• a “humanized form” of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
• the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
• polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
• each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
• the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
• a “multispecific” antibody is one that binds specifically to more than one target antigen, while a “bispecific” antibody is one that binds specifically to two antigens.
• An “antibody conjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a therapeutic agent or a label.
• Percent (%) amino acid sequence identity and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
• leader sequence refers to a sequence of amino acid residues located at the N terminus of a polypeptide that facilitates secretion of a polypeptide from a mammalian cell.
• a leader sequence may be cleaved upon export of the polypeptide from the mammalian cell, forming a mature protein.
• Leader sequences may be natural or synthetic, and they may be heterologous or homologous to the protein to which they are attached. Nonlimiting exemplary leader sequences also include leader sequences from heterologous proteins.
• an antibody lacks a leader sequence.
• an antibody comprises at least one leader sequence, which may be selected from native antibody leader sequences and heterologous leader sequences.
• nucleic acid molecule or “polynucleotide” includes any compound and/or substance that comprises a polymer of nucleotides.
• Each nucleotide is composed 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.
• cytosine I guanine
• A adenine
• T thymine
• U uracil
• the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule.
• the sequence of bases is typically represented from 5’ to 3’.
• nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules.
• DNA deoxyribonucleic acid
• cDNA complementary DNA
• RNA ribonucleic acid
• mRNA messenger RNA
• the nucleic acid molecule may be linear or circular.
• nucleic acid molecule includes both sense and antisense strands, as well as single stranded and double stranded forms.
• the herein described nucleic acid molecule can contain naturally occurring or non- naturally occurring nucleotides.
• nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and/or in vivo, e.g., in a host or patient.
• DNA e.g., cDNA
• RNA e.g., mRNA
• nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
• An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
• isolated nucleic acid encoding an anti-PAD4 antibody refers to one or more nucleic acid molecules encoding anti-PAD4 antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
• vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.
• host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
• binding or “binding” or “specific binding” and similar terms, when referring to a protein and its ligand or an antibody and its antigen target for example, or some other binding pair, means that the binding affinity between the members of the binding pair is sufficiently strong that the interaction cannot be due to random molecular associations (i.e. “nonspecific binding”).
• nonspecific binding typically requires a dissociation constant (KD) of IpM or less, and may often involve a KD of 100 nM or less.
• Binding affinity refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). Affinity can generally be represented by the dissociation constant (KD). Affinity of an antibody for an antigen can be measured by common methods known in the art, such as surface plasmon resonance (SPR), for instance.
• SPR surface plasmon resonance
• Treatment covers any administration or application of a therapeutic for disease in a human, and includes inhibiting the disease or progression of the disease or one or more disease symptoms, inhibiting or slowing the disease or its progression or one or more of its symptoms, arresting its development, partially or fully relieving the disease or one or more of its symptoms, or preventing a recurrence of one or more symptoms of the disease.
• autoimmune disease or “autoimmune disorder,” as used herein, encompasses a disease characterized by the subject’s immune system attacking its own normal cells and tissues, and also encompasses immune-mediated diseases which may or may not be characterized by presence of auto-antibodies.
• the disclosure provides many nonlimiting examples of autoimmune diseases throughout.
• autoimmune diseases include rheumatoid arthritis (RA), lupus (e.g., systemic lupus erythematosus (SEE)), 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).
• RA rheumatoid arthritis
• lupus e.g., systemic lupus erythematosus (SEE)
• lupus nephritis e.g., vasculitis (e.g., ANCA-associated vasculitis), thrombosis (e.g, venous thrombosis), and inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn’s disease).
• IBD
• an effective amount refers to an amount of a drug effective for treatment of a disease or disorder in a subject, such as to partially or fully relieve one or more symptoms.
• an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
• the present disclosure encompasses several methods of assessing the citrullination modifying activity of a PALM modulator or another therapeutic agent, and also of assessing citrullination of a biological sample from a subject, inter alia.
• the citrullination site or sites can be on proteins or peptides.
• the peptides are peptide fragments of proteins, for instance, digested peptides that arise from enzymatic digestion of proteins.
• the expression “assessing citrullination of a citrullination site” encompasses assessing one citrullination site on one protein or peptide, as well as assessing more than one citrullination site on one protein or peptide, as well as assessing multiple citrullination sites.
• the disclosure herein may recite a citrullination site on a protein or peptide
• the “a” in this expression indicates “one or more” citrullination sites
• citrullination of more than one protein or peptide may also be assessed.
• Assessing multiple citrullination sites can encompass assessing citrullination sites on a plurality of proteins or on a plurality of peptides.
• the plurality of citrullination sites may be a plurality of citrullination sites on one protein or on peptide fragments of such protein, and/or the plurality of citrullination sites may be a plurality of citrullination sites on a plurality of different proteins or peptides.
• the methods of assessing citrullination disclosed herein may be used to assess citrullination at any citrullination site
• the citrullination site e.g., the plurality of citrullination sites
• the methods herein include, inter alia, a method of assessing citrullination at a citrullination site by mass spectrometry (MS).
• MS mass spectrometry
• the methods comprise assessment by liquid chromatography followed by MS (LC-MS).
• liquid chromatography e.g., HPLC, RPLC, nLC, pLC, or the like
• the method comprises measuring, in a biological sample: (i) a first concentration of a citrullinated protein or a citrullinated peptide (which may be from a protein, e.g., a protein that has been enzymatically digested), wherein the citrullinated protein or citrullinated peptide is citrullinated at a citrullination site, and (ii) a second concentration of the corresponding total protein (which includes modified and unmodified forms of the protein) in the sample.
• a first concentration of a citrullinated protein or a citrullinated peptide which may be from a protein, e.g., a protein that has been enzymatically digested
• a second concentration of the corresponding total protein which includes modified and unmodified forms of the protein
• the concentration of corresponding total protein is measured by measuring a concentration of a signature peptide from the protein.
• the signature peptide is a peptide that is present both in modified and unmodified forms of the protein, and the signature peptide is a peptide that is not itself modified. Accordingly, when the concentration of signature peptide present in a sample within a well is measured by mass spectrometry, for example, the concentration of the signature peptide represents the well total concentration of the protein.
• the method of measuring concentrations (i) and (ii) is by MS, such as LC-MS. In some embodiments, the method further comprises calculating a citrullination ratio.
• a “citrullination ratio,” as used herein, refers to a ratio of the first concentration (i) to the second concentration (ii).
• a citrullination ratio may be expressed in a variety of ways, for instance, as a percentage, or as any other fraction that is proportional to the aforementioned ratio of the first concentration to the second concentration.
• the method comprises assessing citrullination of more than one citrullination site. Accordingly, the method can comprise measuring the first concentration and the second concentration for each of a plurality of (meaning two or more) different proteins or peptides that each contain a citrullination site. In some such cases, the method comprises measuring the first concentration and the second concentration for each of the plurality of proteins or peptides. In some such cases, at least two different peptides of the plurality are nonoverlapping fragments of the same protein and contain different citrullination sites; in those cases, the second concentration is the same for the peptides that are fragments of the same protein.
• the method comprises calculating a ratio of the first concentration to the second concentration for each of the plurality of different proteins or peptides. Exemplary citrullination sites, proteins, and peptide fragments thereof are discussed below and provided in Table A and Tables 3-18 herein.
• the biological sample is obtained from a subject. In some cases, the biological sample is a whole blood, plasma, serum, blood supernatant, or synovial fluid sample. In other cases, the biological sample is a tissue sample. In some cases, the method further comprises comparing the first and second concentrations, or a ratio of the first and second concentrations to first and second concentrations or a ratio of first and second concentrations obtained from a control.
• the method further comprises determining one or more of the following based on the first and second concentrations or a ratio of the first and second concentrations: probability of a clinical outcome, risk for developing a citrullination-related disease, diagnosis of a citrullinati on-related disease, and selecting the subject from which the biological sample was derived for a treatment of a citrullination-related disease.
• the subject either has a citrullination-related disease or may be at risk of developing a citrullination-related disease, and an assessment of citrullination in a biological sample, according to methods herein, may be used to confirm a diagnosis of such a disease, or to determine whether or not the subject is at risk of developing such a disease.
• the method may allow for monitoring of citrullination in a subject’s biological samples, in order to determine a probability of clinical outcome, for example, based on comparison to reference subjects whose clinical outcomes are known, or to determine whether a PALM modulator or another therapeutic agent would be useful for treating the subject, or to determine if a PALM modulator or other therapeutic agent being currently administered to the subject is operating on the subject to reduce citrullination.
• the method further comprises determining, based on the first and second concentrations or a ratio of the first and second concentrations, whether the subject should receive treatment, for instance, in some cases, for a citrullination-related disease.
• the method further comprises determining, based on the first and second concentrations or a ratio of the first and second concentrations, whether the subject should receive treatment with a therapeutic agent.
• a therapeutic agent include those that may be used for treatment of citrullination-related diseases, and those that target a biological pathway, such as, for example, NETosis or METosis, which may also involve PALM, such as, for example, an anti-histone antibody (e.g., CIT-013, for example), as well as PALM modulators, such as PALM inhibitors.
• the method comprises determining whether a subject already receiving treatment with a therapeutic agent or other therapy should have the treatment adjusted, such as to increase or decrease a dose of a therapeutic agent being administered, or to start treatment with an agent such as a PAD4 modulator or an agent that targets a molecule involved in a biological pathway that involves PAD4.
• the method further comprises determining whether a subject should receive treatment with a PAD4 modulator, or whether a subject already receiving treatment with a PAD4 modulator should have treatment adjusted, such as to increase or reduce the dose of the PAD4 modulator or to discontinue its use.
• the PAD4 modulator is a PAD4 inhibitor.
• PAD4 modulators include, for example, therapeutic agents that can impact the expression or activity of PAD4, including the citrullination activity of PAD4.
• Such molecules include antibodies such as anti-PAD4 antibodies, as well as small molecules, such as small molecule PAD4 inhibitors, among others described in this disclosure.
• the present disclosure also encompasses a method of assessing the activity of a therapeutic agent, including, without limitation, an agent that may be used for treatment of a citrullination-related disease, an agent that targets a biological pathway involving PAD4, an agent that targets NETosis and/or METosis, an anti-histone antibody, and a PAD4 modulator such as a PAD4 inhibitor.
• a therapeutic agent including, without limitation, an agent that may be used for treatment of a citrullination-related disease, an agent that targets a biological pathway involving PAD4, an agent that targets NETosis and/or METosis, an anti-histone antibody, and a PAD4 modulator such as a PAD4 inhibitor.
• Such therapeutic agents may, in some cases, comprise large molecules such as antibodies or fusion proteins, or may comprise small molecules.
• a method of assessing the activity of a therapeutic agent comprises assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample from a subject.
• the biological sample has been exposed to a PAD4 modulator or other therapeutic agent, either directly by adding it to the sample prior to assessment, or indirectly, by the subject having received at least one dose of the PAD4 modulator or other therapeutic agent prior to the sample being obtained.
• the therapeutic agent being assessed is a PAD4 modulator.
• the PAD4 modulator is a PAD4 inhibitor.
• At least two different citrullination sites are assessed. In some cases, at least three different sites are assessed. In some cases, at least four different sites are assessed. In some cases, citrullination of at least two different proteins is assessed. In some cases, citrullination of at least three different proteins is assessed. In some cases, citrullination of at least four different proteins is assessed. In some cases, citrullination of 2-4 citrullination sites and/or 2-4 different proteins is assessed.
• the protein comprising the citrullination site for assessment is selected from one or more of: alpha-2-HS-glycoprotein, alpha-2- macroglobulin, albumin, antithrombin-III, apolipoprotein A-I, apolipoprotein A-IV, apolipoprotein B-100, apolipoprotein E, apolipoprotein LI, C4b-binding protein alpha chain, ceruloplasmin, clusterin, complement C2, complement C3, complement C4-A, complement C4-B, complement factor B, complement factor H, complement factor I, complement component C9, fibrinogen alpha chain, fibrinogen beta chain, fibrinogen gamma chain, fibronectin, galectin-3 -binding protein, gelsolin, haptoglobin, haptoglobin-related protein, hemopexin, immunoglobulin heavy variable 1-3, immunoglobulin heavy variable 1-8, immunoglobulin heavy variable 1-46, immunoglobulin
• a citrullination site from a protein above, or a peptide fragment thereof is assessed.
• the method herein comprises a method of assessing the activity of a PAD4 modulator or other therapeutic agent, the method comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample from a subject, wherein the biological sample has been exposed to a PALM 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.
• the citrullination site to be assessed is selected from one or more citrullination sites shown in Table A below.
• Table A the amino acid residue corresponding to a citrullination site is noted in the left column, with a reference accession number that may be used to locate the citrullination site in the publicly available UniProt database is listed in the right column.
• the UniProt accession number for each protein listed is provided in the table.
• the public database provides an amino acid and gene sequence corresponding to the accession number (e.g., P01023 for alpha-2-macroglobulin). It is understood that each of the proteins listed in the table below may have more than one naturally occurring amino acid sequence.
• citrullination sites of Table A is assessed, while in other cases, at least two, at least three, at least five, at least ten, at least twenty, or at least fifty of the sites are assessed, such as 2-5, 2-10, 2-20, 2-50, 10-50, 5-50, 10-50, or 5-20 of the sites are assessed.
• citrullination sites of only one protein are assessed, while in other cases, citrullination sites of two or more proteins, such as at least 2, 3, 5, 6, 7, or 8 proteins are assessed.
• citrullination sites of 2-5, 2-10, 5-10, 2-20, 5-10, or 10-20 different proteins are assessed.
• Table A lists exemplary citrullination sites, i.e., a residue of a particular human protein that may be citrullinated in a biological sample, and in the right column, lists the site as located within the protein sequence that is published in the UniProt database (accessible at www.uniprot.org).
• the citrullination site is a site designated by underlining on one or more peptides shown in Tables 3-18.
• the method comprises assessing citrullination of a peptide fragment of a protein, wherein the sequence of the peptide fragment comprises a peptide sequence shown in any one of Tables 3-18.
• the method comprises assessing citrullination of a peptide fragment of a protein, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 3.
• at least two peptide sequences as shown in Table 3 are assessed, such as 2, 3, 4, or all shown in Table 3.
• the method comprises assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 4. In some cases at least two peptide fragment sequences as shown in Table 4 are assessed, such as 2, 3, 4, or all shown in Table 4. In some cases, the method comprises assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 5. In some cases at least two peptide fragment sequences as shown in Table 5 are assessed, such as 2, 3, 4, or all shown in Table 5. In some cases, the method comprises assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 6.
• the method comprises assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 7. In some cases at least two peptide fragment sequences as shown in Table 7 are assessed, such as 2, 3, 4, or all shown in Table 7. In some cases, the method comprises assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 8. In some cases at least two peptide fragment sequences as shown in Table 8 are assessed, such as 2, 3, 4, or all shown in Table 8.
• the method comprises assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 9. In some cases at least two peptide fragment sequences as shown in Table 9 are assessed, such as 2, 3, 4, or all shown in Table 9. In some cases, the method comprises assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 11. In some cases at least two peptide fragment sequences as shown in Table 11 are assessed, such as 2, 3, 4, or all shown in Table 11. In some cases, the method comprises assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 11.
• the method comprises assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 12. In some cases at least two peptide fragment sequences as shown in Table 12 are assessed, such as 2, 3, 4, or all shown in Table 12. In some cases, the method comprises assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 13. In some cases at least two peptide fragment sequences as shown in Table 13 are assessed, such as 2, 3, 4, or all shown in Table 13.
• the method comprises assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 14. In some cases at least two peptide fragment sequences as shown in Table 14 are assessed, such as 2, 3, 4, or all shown in Table 14. In some cases, the method comprises assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 15. In some cases at least two peptide fragment sequences as shown in Table 15 are assessed, such as 2, 3, 4, or all shown in Table 15. In some cases, the method comprises assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 16.
• the method comprises assessing citrullination of a peptide fragment of a protein, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 17. In some cases at least two peptide sequences as shown in Table 17 are assessed, such as 2, 3, 4, or all shown in Table 17. In some cases, the method comprises assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 18. In some cases at least two peptide fragment sequences as shown in Table 18 are assessed, such as 2, 3, 4, or all shown in Table 18.
• Proteins or peptides that are assessed for citrullination may, in some cases, contain chemical modifications other than citrullination, such as carbamidomethylation of cysteines, oxidation of methionines, and modifications of the N-terminus, among others. Such chemical modifications can be detected, for instance, by mass spectrometry.
• the citrullination site is selected from one or more of the following: R1391 of proteoglycan 4 (corresponding to arginine residue R1391 of the protein sequence found in the UniProt database under accession number Q92954), R573 of fibrinogen alpha chain (corresponding to R573 of P02671), R591 of fibrinogen alpha chain (corresponding to R591 of P02671), R573 of complement C3 (corresponding to R573 of P01024), R688 of inter-alpha-trypsin inhibitor heavy chain H4 (corresponding to R688 of Q14624), R297 of protein AMBP (corresponding to R297 of P02760), R715 of alpha-2-macroglobulin (corresponding to R715 of PO 1023), R32 of gel solin (corresponding to R32 of P06396), R59 of haptoglobin (corresponding to R59 of P00738), and R651 of serotransferrin (corresponding to R651 of P0278
• these citrullination sites may be assessed from one or more peptide fragments of a protein, wherein the sequences of the peptide fragments comprise (where the citrullination site is bold and underlined): AITTRSGQTLSK (SEQ ID NO: 247), ESSSHHPGIAEFPSRGK (SEQ ID NO:
• the citrullination site is assessed on one or more peptide fragments of a protein, wherein the sequences of the peptide fragments comprise (where the citrullination site is bold and underlined): AITTRSGQTLSK (SEQ ID NO: 247), ESSSHHPGIAEFPSRGK (SEQ ID NO: 248), QFTSSTSYNRGDSTFESK (SEQ ID NO:
• the citrullination site is one or more of the following: R715 of alpha-2-macroglobulin (corresponding to R715 of P01023), R297 of protein AMBP (corresponding to R297 of P02760), R250 of clusterin (corresponding to R250 of Pl 0909-2), R23 of clusterin (corresponding to R23 of P10909-3), R165 of clusterin (corresponding to R165 of P10909-4), R209 of clusterin (corresponding to R209 of Pl 0909-5), R178 of clusterin (corresponding to R178 of Pl 0909-6), R198 of clusterin (corresponding to R198 of Pl 0909), R32 of gelsolin (corresponding to R32 of P06396), R59 of haptoglobin (corresponding to R59 of P00738-2 or P00738), R60 of haptoglobin-related protein (corresponding to R60 of P00739), R97 of haptoglobin-related protein (corresponding to R97 of P00739-2), or R651 of serotrans
• only one member within of the above groups of citrullination sites is assessed, while in other cases, at least two, at least three, at least five, and in the larger groups above, at least ten, or at least twenty of the sites are assessed, such as from 2-5, or from 2-10, or from 2 to all of the sites are assessed.
• citrullination sites on only one protein are assessed, while in other cases, citrullination sites on two or more proteins, such as 2-5, 2-10, 5-10, 2-20, 5-10, or 10-20 different proteins.
• the biological sample is a whole blood, serum, plasma, blood supernatant, or synovial fluid sample.
• the biological sample is a whole blood, serum, plasma, or blood supernatant sample.
• the citrullination site to be assessed is 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 are assessed, such as 2, 3, 4, or all shown in Table 11.
• at least two peptide fragment sequences as shown in Table 11 are assessed, such as 2, 3, 4, or all shown in Table 11.
• the protein of interest comprises fibrinogen alpha chain and/or gelsolin.
• the citrullination site for assessment may comprise the fibrinogen citrullination site located at R591 of P02671, which in some cases may be assessed via a peptide of sequence QFTSSTSYNRGDSTFESK (SEQ ID NO: 249), wherein the R is the citrullination site.
• the citrullination site for assessment may comprise the gelsolin citrullination site located at R32 of P06396, which in some cases may be assessed via a peptide of sequence ATASRGASQAGAPQGR (SEQ ID NO: 255), wherein the underlined R is the citrullination site.
• the sample is a whole blood, serum, plasma, blood supernatant, or synovial fluid sample. In some such cases, the sample is a whole blood, serum, plasma, or blood supernatant sample.
• the “assessing” in methods herein encompasses a variety of ways of examining citrullination, such as, without limitation, qualitatively determining whether citrullination has occurred at a particular citrullination site on a protein or peptide, and quantitatively determining citrullination of a protein or a peptide.
• the assessing comprises quantitatively determining citrullination at a particular citrullination site (e.g., a citrullination site disclosed herein).
• quantitatively determining citrullination at a particular citrullination site comprises determining a “citrullination ratio” as defined above.
• MS mass spectrometry
• MS can detect differences between the molecular weights of a protein or peptide that is citrullinated and the corresponding protein or peptide that is not citrullinated.
• intensity measurements obtained with MS can be used to measure the concentration of a citrullinated peptide.
• MS can also be used to measure the total concentration of corresponding protein from which a citrullinated peptide is derived. For instance, such total concentration of protein can be measured by measuring the concentration of a signature peptide.
• such a “signature peptide” is a peptide that is not modified and is present in both modified and unmodified forms of the protein; accordingly, the concentration of the signature peptide represents the concentration of the corresponding total protein, including modified and unmodified forms of the protein.
• an exemplary signature peptide is GSESGIFTNTK (SEQ ID NO: 258; amino acids 548-558).
• This peptide is not post-translationally modified in human matrices tested, is in the near vicinity of citrullinated peptides ESSSHHPGIAEFPSRGK (SEQ ID NO: 248; amino acids 559-575) and QFTSSTSYNRGDSTFESK (SEQ ID NO: 249; amino acids 582-600), and accordingly, it accurately represents total FGA protein concentration.
• Selection of signature peptides is known in the art and is described, for instance, in Qiu, X.I. et al. Signature peptide selection workflow for biomarker quantification using LC-MS based targeted proteomics.
• assessing citrullination of the citrullination site comprises performing liquid chromatography to assist in separating protein or peptide species, followed by mass spectrometry (LC-MS).
• liquid chromatography e.g., HPLC, RPLC, nLC, pLC, or the like
• MS mass spectrometry
• a sample may be assessed directly after being obtained, or alternatively, a sample may be first prepared in some fashion prior to assessment of citrullination, for example, in order to improve the detection of the proteins or peptides of interest in the sample.
• a sample may be diluted with a buffer, a sample comprising cells may be treated to lyse the cells or to separate the cells from the sample fluid such as by centrifugation or filtration.
• a protein of interest or peptide fragments thereof may first be enriched by separating them from other components of the starting sample prior to assessment of citrullination.
• one method of enriching a protein or peptide of interest is to isolate it from the sample.
• such a protein or peptide could be exposed to an affinity reagent such as an antibody and thus targeted for purification or removal from the sample.
• an affinity reagent such as an antibody
• such an affinity reagent may be immobilized on a solid surface such as a bead, chip, plate, or well of a plate, in order to facilitate such enrichment.
• the proteins of the sample may also be enzymatically digested to form peptide fragments, may be denatured to remove tertiary structure, and/or a sample may be diluted or treated to remove or separate certain components prior to assessment.
• other components could be degraded or removed from the sample, such as nucleic acids for instance, by enzymatic degradation or by alcohol precipitation.
• a sample may also be treated so as to promote release of endogenous PAD4 in the sample prior to assessment, or may be contacted with exogenous PAD4 to boost the signal from the sample, among other optional treatments.
• the sample may be incubated with exogenous PAD4 prior to assessing citrullination.
• a concentration of from 5 nM to 15 nM PAD4 may be added, such as from 7nM to 14 nM, from 7 nM to 10 nM, from 10 nM to 15 nM, in some cases, a concentration of 10 nM exogenous PAD4 or about 10 nM exogenous PAD4 is added.
• exogenous calcium ion (Ca2+) is also added to the sample.
• those may also be added exogenously, such as PAD2, which could be added, for example, at concentrations of 2-4 nM, such as at 3 nM or about 3 nM.
• the sample is not exposed to exogenous PAD4 or is not exposed to any exogenous PAD proteins.
• all PAD4 activity in a biological sample is from endogenous PAD4 present in the sample.
• the biological sample may be incubated for a period, which in some cases may induce the release of endogenous PAD4 within the sample. Such incubation may be for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48- 96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60-72 hours. In some cases, the incubation is for 48-96 hours.
• such incubation is conducted at 34-40 °C, such as at 36-39 °C, 36-38 °C, or 37 °C.
• the incubation is conducted in a TruCulture® null tube (Rules Based Medicine, Austin, TX), or a similar container comprising culture media for preservation or analysis of biological samples.
• the PAD4 modulator may be added before the incubation of the sample. In other cases, a PAD4 modulator is added after the incubation.
• exogenous PAD4 is added to a sample
• the sample may also be incubated with the exogenous PAD4, such as for 1-3 hours, or 1-2 hours, or 1, 2, or 3 hours, prior to assessing citrullination.
• such incubation is conducted at 34-40 °C, such as at 36-39 °C, 36-38 °C, or 37 °C.
• both exogenous PAD4 and an exogenous PAD4 modulator may be added together.
• a PAD4 modulator may be added after the incubation with exogenous PAD4.
• an incubation is conducted in a CO2 incubator.
• the activity of exogenous PAD4 added to a sample is quenched by adding EDTA to the sample.
• the method comprises adding EDTA to the biological sample after incubation with the exogenous PAD4 in order to quench the activity of the PAD4.
• a sample prior to analysis, a sample could be treated to expose one or more proteins or peptide fragments thereof comprising citrullination sites for later assessment.
• proteins in a sample may be denatured or digested with enzymes.
• proteins may be denatured to remove tertiary structure, for example, by incubation at above physiological temperatures, such as at 60-90°C, such as 60-80°C, 70-85°C, or 80°C, for a period of 5-30 minutes, such as 10-30, 10-20, 15-25, or 20 minutes.
• a chemical denaturant such as urea could be applied.
• a proteolytic enzyme such as trypsin, LysC, rLysC, LysN, GluC, AspN, rAspN, or a combination of proteolytic enzymes, such as more than one of trypsin, LysC, rLysC, LysN, GluC, AspN, and rAspN, could be used for enzymatic digestion, for example, to form peptide fragments of proteins of interest for later assessment.
• a sample is directly incubated with a PAD4 modulator or other therapeutic agent (i.e., an exogenous PAD4 modulator), such as by contacting the biological fluid with the PAD4 modulator or other therapeutic agent ex vivo.
• a PAD4 modulator or other therapeutic agent i.e., an exogenous PAD4 modulator
• the biological sample has been exposed to a PAD4 modulator or other therapeutic agent in vivo in a subject as a result of the PAD4 modulator or other therapeutic agent being administered to the subject.
• a PAD4 modulator or a metabolite thereof may travel through blood, serum, plasma, or synovial fluid or other bodily fluids or tissues after administration to a subject, or may contact cells in the body which in turn travel to such fluids or to certain bodily tissues.
• a biological sample herein may comprise biological fluid (i.e., a biological fluid sample), such as, without limitation, blood, plasma, serum, blood supernatant, synovial fluid, lymph, pleural fluid, interstitial fluid, sweat, tears, sputum, or urine.
• a biological sample may comprise tissue, such as from a joint, a neoplasm, or a tumor.
• the biological sample comprises synovial fluid.
• the biological sample comprises blood or is derived from blood; in some cases the biological sample comprises serum, plasma, or blood supernatant (e.g. a supernatant that forms after centrifugation or filtration of blood).
• the biological sample is fresh, meaning that it has not been frozen and thawed prior to use. In other cases, the sample has been frozen and thawed before use in the method.
• the method comprises freezing and thawing the biological sample before assessing citrullination of the citrullination site.
• the sample may be frozen at a temperature of -70 °C or below, or -80 °C or below.
• the biological sample is prepared for assessment prior to the assessing, such as according to methods provided above. For instance, in some cases the sample is treated by enzymatically digesting proteins, such as with one or more protease enzymes.
• proteins comprising citrullination sites are enriched, in some cases by being removed from the sample, such as with an antibody or affinity reagent, and/or in some cases other proteins or contaminants are removed from the sample.
• an antibody that specifically binds to the protein to be assessed may be used to enrich that protein for later assessment of citrullination.
• the antibody or affinity reagent may be immobilized, such as placed on a matrix such as a bead or chip or well of a plate, other solid surface.
• the biological sample may be diluted before assessment of citrullination.
• proteins in the biological sample may be denatured to remove tertiary structure prior to assessment of citrullination.
• any combination of the above treatments may be conducted on the sample.
• assessing citrullination of a citrullination site comprises measuring a citrullination ratio, as described above.
• the method comprises comparing the citrullination assessment, such as a citrullination ratio or the concentration of a citrullinated protein or peptide fragment thereof, to that of a reference or control.
• the comparison is to a reference citrullination ratio, which may be obtained, for example, from a reference sample, or which may be a value or range of values that represents those obtained from reference samples.
• the method comprises assessing comprises determining a difference between the citrullination ratio for the biological sample and a reference citrullination ratio.
• control when referring to a biological sample, are used interchangeably to refer to a biological sample against which a measurement from a sample at interest is compared.
• a control is a sample that has not been exposed to PAD4 but is otherwise comparable to the sample of interest.
• the control is a “baseline” sample, which is a sample taken from a subject prior to some event of interest, such as prior to treatment with a therapeutic agent, e.g., PAD4 modulator, and which, for instance, may be compared to a sample from the same subject taken after the event, such as after treatment with the therapeutic agent.
• the control is a sample from the subject taken prior to treatment with a PAD4 modulator.
• control is a sample from the subject taken prior to a change in PAD4 modulator treatment. Accordingly, in some cases, one may determine how treatment with a PAD4 modulator impacts a subject by comparing to such a baseline control sample. In other cases, the control biological sample has been exposed to a different treatment than the biological sample, such as to a different PAD4 modulator or different therapeutic agent than the biological sample. In some cases, the control biological sample has been exposed to a different dose of the PAD4 modulator or other therapeutic agent than the biological sample. In some cases, the control is a sample from a healthy subject, which, for instance, could be compared to a sample from a subject with a disorder, or a subject following treatment with PAD4.
• a control biological sample is a sample that has been exposed to a different therapeutic agent or PAD4 modulator, or to a different dose of the same therapeutic agent or PAD4 modulator as the sample of interest.
• citrullination assessments are compared to those of one or more control samples (e.g., baseline samples or samples from other points of time from the same subject, or samples from other subjects), or to values associated with one or more control samples (e.g., from a pool of healthy subjects).
• a citrullination assessment from a biological sample herein may be controlled to a group of control samples representing typical values found in a particular type of subject, or representing values found across a range of subjects.
• the present disclosure includes methods of assessing citrullination in a biological sample of a subject comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the biological sample.
• the subject has not received treatment with any PALM modulator.
• the subject has received treatment with a PALM modulator.
• the subject has not received treatment with any therapeutic agent.
• the subject has received treatment with a therapeutic agent.
• the present disclosure also encompasses methods of assessing the activity of a PAD4 modulator or other therapeutic agent comprising obtaining a biological sample from a subject following treatment of the subject with at least one dose of a PAD4 modulator or other therapeutic agent, and assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the biological sample.
• these methods further comprises comparing the assessment of the citrullination to that of a control biological sample, such as described above.
• the control biological sample is a baseline sample obtained from the subject prior to treatment with the PAD4 modulator or other therapeutic agent.
• the method comprises obtaining a baseline biological sample from the subject prior to treatment with the PAD4 modulator or other therapeutic agent, and assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the baseline biological sample, and optionally comparing the assessed citrullination (e.g. a citrullination ratio) of the biological sample and the baseline biological sample.
• a citrullination assessment is performed in order to determine if a subject should receive treatment with a PAD4 modulator, or to serve as a baseline for later monitoring of citrullination following treatment with a PAD4 modulator or other therapeutic agent.
• the method comprises (a) contacting a biological sample from a subject with exogenous PAD4, and (b) assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the sample.
• 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.
• the biological sample is prepared as described above prior to the assessing.
• the concentration of exogenous PAD4 may be as provided above (e.g., from 5 nM to 15 nM PAD4, such as from 7nM to 14 nM, from 7 nM to 10 nM, from 10 nM to 15 nM).
• another PAD protein e.g., PAD2
• PAD2 PAD2
• Ca2+ calcium ion
• the protein of interest is enriched by immunoenrichment techniques such as described below, for example, which may comprise using affinity reagents to isolate or remove the protein of interest.
• the methods comprise in vitro methods of assessing the activity of a PAD4 modulator or other therapeutic agent, comprising (a) treating a biological sample from a subject with a PAD4 modulator or other therapeutic agent to form a treated biological sample, and after the treating, (b) assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the biological sample.
• such methods further comprise assessing citrullination of the citrullination site on the protein or a peptide fragment in a control biological sample, such as described above.
• Methods herein also comprise, for example, an in vitro method of assessing the activity of a PAD4 modulator or other therapeutic agent, comprising (i) dividing a biological sample obtained from a subject into a plurality of biological samples, (ii) contacting each of the plurality of biological samples with a different dose of the PAD4 modulator or other therapeutic agent, and (iii) assessing, for each of the plurality of biological samples, citrullination of a PAD4- dependent citrullination site on a protein or peptide fragment thereof that is present in the biological sample.
• such a method comprises calculating an IC50 for the PAD4 modulator or other therapeutic agent based on the outcome of the assessing.
• Methods herein can also include, for example, an in vitro method of assessing citrullination by endogenous PAD4, comprising incubating the biological sample (or the plurality of biological samples) from the subject for an incubation period (for example, to trigger release of endogenous PAD4) and assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the sample.
• Methods herein also include, for example, an in vitro method of assessing citrullination by endogenous PAD4, comprising (a) incubating a whole blood sample from a subject at 34-40°C for an incubation period (for example, to trigger release of endogenous PAD4), (b) after the incubation period, separating plasma or supernatant from the whole blood sample, and (c) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant.
• the incubating is in a TruCulture® null tube.
• Such incubation may be for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60- 72 hours.
• the incubation period is 48 to 96 hours.
• the incubating is at a temperature of 35 to 40°C, at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C.
• the sample is prepared for assessment of citrullination, such as to enrich the protein or peptide to be assessed.
• the method comprises, for example, separating the plasma or supernatant from the whole blood sample, such as by centrifuging or filtering the whole blood sample.
• these methods may also comprise (i) incubating the plasma or supernatant with a protein depletion resin, and (ii) recovering depleted plasma or supernatant that has flowed through the resin to obtain depleted flowthrough, optionally wherein the recovering comprises centrifugation.
• They may also comprise enzymatically digesting polypeptides in the depleted flowthrough before the assessing, and optionally cleaning up enzymatically digested peptides, such as with an iST-BCT kit (PreOmics, Planegg/Martinsried, Germany) or a similar kit, for instance.
• the disclosure herein also encompasses an in vitro method of assessing changes in citrullination in a subject, comprising (a) incubating a whole blood sample from a subject at 34-40°C for an incubation period (such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60-72 hours), wherein the sample is obtained from the subject following administration of a PALM modulator to the subject, (b) after the incubation period, separating plasma or supernatant from the whole blood sample, and (c) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant.
• an incubation period such as at a temperature of 36
• the method further comprises (d) incubating a second whole blood sample from the subject at 34-40°C for an incubation period (such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60-72 hours), wherein the second whole blood sample is obtained from the subject before administration of the PAD4 modulator to the subject, € after the incubation period, separating plasma or supernatant from the second whole blood sample, and (f) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant from the second whole blood sample.
• an incubation period such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at
• the method further comprises performing a parallel set of steps on at least one control whole blood sample.
• the method further comprises (e) incubating a control whole blood sample at 34-40°C for an incubation period (such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60-72 hours), (f) after the incubation period, separating control plasma or supernatant from the control whole blood sample, and (g) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the control plasma or supernatant.
• an incubation period such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C for
• an outcome of the assessing in step (g) is compared with an outcome of the assessing in step (d).
• the disclosure herein also encompasses, for example, an in vitro method of assessing PAD4-dependent citrullination, the method comprising assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in plasma or supernatant that has been separated from a whole blood sample that has been incubated at 34-40°C for an incubation period (such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60-72 hours).
• the sample can be a plasma or supernatant from a whole blood sample.
• the plasma or supernatant sample has not been frozen before assessment, while in other cases the sample has been frozen and thawed, and in some instances the method further comprises freezing and thawing the plasma or supernatant after separation from whole blood but prior to the assessing.
• a sample may be contacted with a reagent, such as an affinity reagent, to enrich or purify a protein or a peptide fragment thereof harboring a citrullination site to be analyzed.
• an antibody or other affinity reagent may be immobilized, such as by attachment to a solid surface, such as a bead, chip, plate or well of a plate. Such immobilization, for instance, may assist in separating a protein of interest for assessment of citrullination from other proteins in the sample.
• the reagent used for immunoenriching is an antibody attached to a solid surface, such as a bead, such as a magnetic bead, or a chip, plate, or well of a plate.
• the sample may be a sample that comprises or is whole blood, serum, plasma, synovial fluid, pleural fluid, interstitial fluid, sputum, urine, or another biological sample.
• the sample may be a whole blood, plasma, serum, or synovial fluid sample. In some embodiments, it is a plasma or serum sample.
• the immunoenrichment is conducted prior to enzymatic digestion of the protein of interest. In other cases, the protein of interest is first enzymatically digested, and then one or more peptide fragments thereof are immunoenriched. In some cases, the sample may be diluted to a volume of at least 5 pl, e.g., to a volume of 5 pl- 1 OOpl, 5 pl-50pl, 5 pl-25 pl, 5 pl- 15 pl, e.g., to a volume of lOpl.
• the immunoenriching may comprise incubating the sample with the immobilized antibody for at least 30 minutes, such as for a period of 30-90 minutes. In some cases, the incubation is for 30-60 minutes, 40-80 minutes, 50 to 70 minutes, 55 to 65 minutes, or for 60 minutes. The incubating may be at a temperature of 22-28°C, or may be at room temperature. In some cases, the immunoenriching comprises shaking during the incubating (e.g., at 800-1200 rpm or at 1000 rpm).
• the immunoenriching comprises removing the immobilized antibody from the sample and washing the immobilized antibody prior to the eluting, for example with a wash buffer, e.g., a wash buffer comprising PBS (phosphate buffered saline).
• the eluting comprises washing the immobilized antibody with an elution composition.
• elution compositions may be used, including, for example elution compositions that elute a protein of interest based on a change of pH, such as elution compositions that are acidic, and/or that comprise a detergent such as a zwitterionic detergent.
• the elution buffer may comprise an acidic solution, optionally further comprising a buffer or ion, such as a zwitterion.
• the buffer is exchanged prior to assessing citrullination, and optionally prior to intermediate steps such as enzymatic digestion, if such steps are performed. For example, if an acidic elution composition is used, then the buffer may be altered to a neutral pH range for subsequent steps, for example.
• proteins that have been immunoenriched according to methods herein are denatured and/or enzymatically digested prior to assessment of citrullination.
• proteins may be denatured to remove tertiary structure, for example, by incubation at above physiological temperatures, such as at 60-90°C, such as 60- 80°C, 70-85°C, or 80°C, for a period of 5-30 minutes, such as 10-30, 10-20, 15-25, or 20 minutes.
• a chemical denaturant such as urea could be applied.
• a proteolytic enzyme such as trypsin, LysC, rLysC, LysN, GluC, AspN, rAspN, or a combination of proteolytic enzymes, such as more than one of trypsin, LysC, rLysC, LysN, GluC, AspN, and rAspN, could be used for enzymatic digestion.
• the citrullination site for assessment may comprise the gelsolin citrullination site located at R32 of P06396, which in some cases may be assessed via a peptide of sequence ATASRGASQAGAPQGR (SEQ ID NO: 255), wherein the underlined R is the citrullination site.
• An exemplary antibody that could be used to immunoenrich fibrinogen may be a recombinant anti-fibrinogen antibody, such as Abeam antibody 244646.
• An exemplary antibody that could be used to immunoenrich gelsolin for example, may be a recombinant anti-gelsolin antibody such as antibody 247406.
• immunoenrichment of the sample is conducted prior to enzymatic digestion.
• assessment of citrullination may be conducted by mass spectroscopy (MS), for example, by liquid chromatography followed by mass spectroscopy such as tandem mass spectroscopy (LC-MS or LC-MS/MS).
• MS mass spectroscopy
• LC-MS tandem mass spectroscopy
• PAD4 modulators such as PAD4 inhibitors and PAD4 agonists
• other therapeutic agents such as those used for treatment of citrullination-related diseases, as well as those that target biological pathways that involve PAD4, such as NETosis and/or METosis, and those that otherwise can impact citrullination activity by PAD4 or PAD4 expression.
• they may be used for testing new PAD4 modulators or other agents to determine their effect on citrullination of certain sites.
• they may be used to check the activity of known PAD4 modulators or other agents.
• a biological sample may be used for testing the effect of a PAD4 modulator or other therapeutic agent that has not been administered to a subject, and the PAD4 modulator or other therapeutic agent may be exposed to the biological sample by incubating the sample with the modulator.
• the biological sample may be taken from a subject who has been previously administered the PAD4 modulator or other therapeutic agent, and thus the sample may have been exposed to the PAD4 modulator through administration of the PAD4 modulator or other therapeutic agent to the subject from whom the sample is obtained.
• the PAD4 modulator or other therapeutic agent is an anti -histone antibody, such as CIT-013.
• the PAD4 modulator or other therapeutic agent is a PAD4 modulator.
• it is a PAD4 inhibitor, such as an anti-PAD4 antibody or small molecule PAD4 inhibitor.
• a PAD4 modulator does not significantly affect the activity of any protein species other than PAD4.
• the PAD4 modulator does not significantly affect the activity of PAD2, for example.
• the PAD4 modulator modulates the activity of PAD4 as well as at least one other protein, such as PAD2.
• PAD4 modulators may be administered, for example, to subjects who have a citrullination-related disease such as an autoimmune disease or infectious disease or cancer, for example, or who are at risk for developing such a disease.
• a citrullination-related disease such as an autoimmune disease or infectious disease or cancer, for example, or who are at risk for developing such a disease.
• diseases and related subjects are discussed in more detail in following sections herein.
• the subject has been diagnosed with an autoimmune disorder or is at risk for developing an autoimmune disorder.
• the citrullination-related disease is the citrullination-related disease is rheumatoid arthritis, lupus (e.g., systemic lupus erythematosus (SLE)), lupus nephritis, vasculitis (e.g., ANCA-associated vasculitis), thrombosis (e.g, venous thrombosis), or inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn’s disease).
• lupus e.g., systemic lupus erythematosus (SLE)
• lupus nephritis e.g., vasculitis (e.g., ANCA-associated vasculitis), thrombosis (e.g, venous thrombosis), or inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn’s disease).
• IBD
• the subject has not been diagnosed with an autoimmune disease such as RA, or the subject is not at considered to be at risk of developing such a disease.
• a normal, healthy subject In some cases, the subject is positive for anti-citrullinated protein antibodies (ACPA positive). In some cases, the subject is positive for anti-PAD4 activating antibodies. In cases where a subject is ACPA positive or positive for anti-PAD4 activating antibodies, the subject may also be considered at risk for developing an autoimmune disorder such as RA.
• ACPA positive anti-citrullinated protein antibodies
• the subject may also be considered at risk for developing an autoimmune disorder such as RA.
• Methods may also be used, for example, to assess citrullination levels in subjects not currently on therapeutic treatment, or who are not receiving a PAD4 modulator.
• a normal, healthy subject In some cases, the subject is positive for anti- citrullinated protein antibodies (ACPA positive). In some cases, the subject is considered at risk for developing an autoimmune disorder or citrullination-related disease.
• ACPA positive anti- citrullinated protein antibodies
• Methods as described herein may also be performed, for example, to assess citrullination of sites in laboratory animals such as mice, citrullinated by murine PAD4, for instance, by assessing citrullination of murine citrullination sites and related peptides such as to test murine PAD4 modulators and inhibitors. See for example international Patent Publication No. W02024/020579, describing exemplary murine PAD4 antibodies.
• PAD4 modulators include various small molecule compounds that may increase or reduce PAD4 activity, such as citrullination of amino acid residues.
• small molecule PAD4 modulators include benzimidazole-derived compounds and heteroaryl compounds, as well as peptides, such as peptide macrocycles.
• Exemplary small molecule PAD4 modulators include compounds disclosed in International Patent Publication No. W02014/015905, which describes benzimidazole-derived PAD4 modulators such as PAD4 inhibitors, for instance 2-(azaindole-2-yl)benzimidazoles. Additional small molecule PAD4 modulators such as PAD4 inhibitors, are also described in International Patent Publication Nos.
• WO2016/185279 WO2017/007405, WO2017/100601, WO20 17/100594, W02017/147102, WO2018/022897, WO2018/049296, WO2019/058393, W02020/033490, W02020/033514, W02020/033520, W02020/033488, WO2021/158840, WO2021/163254, WO2022/173722, WO2022/140428, WO2022/221642, and WO2023/083365, for example.
• antibodies may be used as PAD4 modulators.
• Examples of such antibodies include those described in 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.
• anti-PAD4 antibodies is based on a murine anti-human antibody called “clone 13,” which was prepared in its original murine anti-human form, and was then humanized to create a series of antibodies called hzl3-l to hzl3-12, of which hzl3- 5 and hzl3-12 were further modified at position D31 to D3 IE (antibodies hzl3-5 D3 IE and hzl3-12 D3 IE) .
• a PAD4 modulator or PAD4 inhibitor comprises an anti-PAD4 antibody that specifically binds to protein arginine deiminase 4 (PAD4) and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HCDR3 comprising 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) comprising the amino acid sequence of SEQ ID NO: 7, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 9.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region 1
• HCDR2 comprising the amino acid sequence of SEQ ID NO: 5
• an HCDR3 comprising the amino acid sequence of
• the antibody specifically binds to protein arginine deiminase 4 (PAD4) and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 62, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HCDR3 comprising 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) comprising the amino acid sequence of SEQ ID NO: 7, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 9.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region 1
• LCDR1 light chain complementarity determining region 1
• LCDR2 comprising the amino acid sequence of SEQ ID NO: 8
• LCDR3 comprising the amino acid sequence of SEQ ID NO:
• the antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 4 or 62 and an HCDR3 comprising 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) comprising the amino acid sequence of SEQ ID NO: 7.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region 1
• LCDR1 light chain complementarity determining region 1
• antibodies herein comprise the above set of these three CDRs.
• the antibodies comprise a VH comprising a HCDR1 comprising the amino acid sequence of SEQ ID NO: 4 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a VL comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 7.
• the antibodies comprise a VH comprising a HCDR1 comprising the amino acid sequence of SEQ ID NO: 62 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a VL comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 7.
• the antibody comprises a heavy chain variable region (VH) comprising 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) comprising the amino acid sequence of SEQ ID NO: 223.
• VH heavy chain variable region
• VL light chain variable region
• the antibody further comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 224.
• the antibody comprises a VH comprising a glycine at Kabat position 94 (Gly94).
• Gly94 is an amino acid residue in the loop adjacent to the VH CDR3 (i.e., the VH CDR3 loop).
• Gly94 is implicated in the flexibility and/or the geometry of the VH CDR3 loop.
• Gly94 may interact with VH CDR3.
• Gly94 is implicated in the activity of the anti-PAD4 antibody.
• mutation of Gly94 to a different amino acid, such as a threonine results in a decrease in flexibility in the VH CDR3 loop, a change in geometry in the VH CDR3 loop, a decrease in interaction between the amino acid at position 94 and the VH CDR3 loop, a decrease in binding of the anti-PAD4 antibody to PAD4, a decrease in activity of the anti- PAD4 antibody, an increase in binding of the anti-PAD4 antibody to extracellular matrix (ECM) proteins, a change in anti-PAD4 antibody secondary structure, a decrease in the stability of the anti-PAD4 antibody, or any combination thereof.
• ECM extracellular matrix
• the VH of the antibody comprises a glycine at Kabat position 94 (position 98 of SEQ ID NO: 10). (See Fig. IE of priority application No. 63/528,323 and International Patent Publication No. W02024/020579).
• 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 any one of SEQ ID NOs: 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, or 68. In some embodiments, the antibody comprises a VL 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 NOs: 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, or 70.
• the antibody comprises both a VH 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 NOs: 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, or 68; 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 any one of SEQ ID NOs: 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, or 70.
• the antibody comprises a VH comprising the 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.
• the antibody comprises a VL comprising the 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.
• the antibody comprises a both a VH comprising the 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 and a VL comprising the 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.
• the antibody VH and VL further comprise (a) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or 62 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 6 and an LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, (b) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or 62, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 6, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 8 an LCDR3 comprising the amino acid sequence of SEQ ID NO: 9, or (c) the amino acid sequence of SEQ ID NO: 221 or 225 in the VH and the amino acid sequence of SEQ ID NO: 222 in the VL, further optionally with a light chain constant region comprising the amino acid sequence of SEQ ID NO: 224.
• the antibody comprises a VH comprising the 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.
• the antibody comprises a VL comprising the 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.
• the antibody comprises both a VH comprising the 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; and a VL comprising the 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.
• VH comprising the 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
• VL comprising the 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.
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 10 and a VL comprising the amino acid sequence of SEQ ID NO: 12;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 14 and a VL comprising the amino acid sequence of SEQ ID NO: 16;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: 20;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 22 and a VL comprising the amino acid sequence of SEQ ID NO: 24;
• An isolated antibody that specifically binds to protein arginine deiminase 4 wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 28;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 30 and a VL comprising the amino acid sequence of SEQ ID NO: 32;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 36;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 38 and a VL comprising the amino acid sequence of SEQ ID NO: 40;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 42 and a VL comprising the amino acid sequence of SEQ ID NO: 44;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 46 and a VL comprising the amino acid sequence of SEQ ID NO: 48;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 50 and a VL comprising the amino acid sequence of SEQ ID NO: 52;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 54 and a VL comprising the amino acid sequence of SEQ ID NO: 56;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 58 and a VL comprising the amino acid sequence of SEQ ID NO: 60;
• PAD4 protein arginine deiminase 4
• the antibody binds to an epitope on PAD4 comprising SEQ ID NO: 217 and SEQ ID NO: 218.
• the anti-PAD4 antibody is an IgA, IgG, or IgM antibody.
• the antibody is an IgG antibody, such as a human IgGl, IgG2, IgG3, or IgG4 antibody or a murine IgGl or IgG2 antibody.
• the antibody comprises a wild-type, human IgGl, IgG2, or IgG4 heavy chain constant region.
• the antibody comprises a full length heavy chain and/or a full length light chain. In other cases, the antibody lacks a C-terminal lysine at the end of the heavy chain constant region.
• the antibody lacks a C-terminal glycine-lysine at the end of the heavy chain constant region.
• the antibody is an antibody fragment, such as an Fv, singlechain Fv (scFv), Fab, Fab’, or (Fab’)2.
• the anti-PAD4 antibody is a bispecific or multispecific antibody, or which is conjugated covalently or noncovalently to at least one other molecule.
• the antibody is conjugated covalently or noncovalently to at least one other molecule, wherein the at least one other molecule comprises a detection label and/or a drug.
• the antibody comprises a human IgGl heavy chain constant region comprising an amino acid sequence 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 Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.
• the antibody comprises a light chain constant region comprising an amino acid sequence at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises both a human IgGl heavy chain constant region comprising an amino acid sequence 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 Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; and a light chain constant region comprising an amino acid sequence at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises a heavy chain constant region comprising an amino acid sequence modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.
• the antibody comprises a light chain constant region comprising an amino acid sequence modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises both a heavy chain constant region comprising an amino acid sequence modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; and a light chain constant region comprising an amino acid sequence modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises a human IgGl heavy chain constant region comprising amino acid sequence of any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.
• the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises both a human IgGl heavy chain constant region comprising the amino acid sequence of any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 194.
• anti-PAD4 antibodies derived from a second mouse anti -human antibody, clone 20, and its humanized variants hz20-l to hz20-14, as also described in International Patent Publication W02024/020579, as well as in the instant priority application, US Provisional Appl. No. 63/528,323, filed July 21, 2023, the contents of both being incorporated herein by reference.
• an anti-PAD4 antibody comprises 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) comprising the amino acid sequence of SEQ ID NO: 72, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 73, and an HCDR3 comprising 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) comprising the amino acid sequence of SEQ ID NO: 75, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 76, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 77.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region 1
• HCDR1 heavy chain complementarity determining region 1
• LCDR3 comprising the amino acid sequence of S
• 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 any one of SEQ ID NOs: 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134.
• the antibody comprises a VL 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 NOs: 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136.
• the antibody comprises a VH comprising the 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 a VL comprising the 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.
• the antibody VH further comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 72, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 73, or an HCDR3 comprising the amino acid sequence of SEQ ID NO: 74.
• the antibody VL further comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 75, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 76, or an LCDR3 comprising the amino acid sequence of SEQ ID NO: 77.
• the antibody comprises a VH comprising the 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.
• the antibody comprises a VL comprising the 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.
• the antibody comprises a VH comprising the 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; and comprises a VL comprising the 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.
• the following exemplary antibodies are within the scope of this disclosure:
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 78 and a VL comprising the amino acid sequence of SEQ ID NO: 80;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 90 and a VL comprising the amino acid sequence of SEQ ID NO: 92;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 98 and a VL comprising the amino acid sequence of SEQ ID NO: 100;
• PAD4 protein arginine deiminase 4
• PAD4 protein arginine deiminase 4
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 118 and a VL comprising the amino acid sequence of SEQ ID NO: 120;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 122 and a VL comprising the amino acid sequence of SEQ ID NO: 124;
• An isolated antibody that specifically binds to protein arginine deiminase 4 (PAD4), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 126 and a VL comprising the amino acid sequence of SEQ ID NO: 128;
• An isolated antibody that specifically binds to protein arginine deiminase 4 wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 130 and a VL comprising the amino acid sequence of SEQ ID NO: 132;
• PAD4 protein arginine deiminase 4
• the antibody binds to an epitope on PAD4 comprising SEQ ID NO: 219 and SEQ ID NO: 220.
• the antibody is an IgA, IgG, or IgM antibody.
• the antibody is an IgG antibody, such as a human IgGl, IgG2, IgG3, or IgG4 antibody or a murine IgGl or IgG2 antibody.
• the antibody comprises a wild-type, human IgGl, IgG2, or IgG4 heavy chain constant region.
• the antibody is of the human IgGl isotype.
• the antibody is of the human IgGl isotype with a P329G, L234A and L235A (LALAPG; EU numbering) mutation to reduce Fc-region effector function.
• the antibody is of the human IgG2 isotype.
• the antibody is of the IgG4 isotype with an S228P mutation (EU numbering) in the hinge region to improve stability of IgG4 antibody.
• the antibody e.g., a non-humanized antibody
• the antibody comprises a full length heavy chain and/or a full length light chain. In other cases, the antibody lacks a C-terminal lysine at the end of the heavy chain constant region. In yet other cases, the antibody lacks a C-terminal glycine-lysine at the end of the heavy chain constant region. In some cases, the antibody is an antibody fragment, such as an Fv, singlechain Fv (scFv), Fab, Fab’, or (Fab’)2.
• the antibody is a bispecific or multispecific antibody, or which is conjugated covalently or noncovalently to at least one other molecule. In some embodiments, the antibody is conjugated covalently or noncovalently to at least one other molecule, wherein the at least one other molecule comprises a detection label and/or a drug.
• the antibody comprises a human IgGl heavy chain constant region comprising an amino acid sequence 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 Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.
• the antibody comprises a light chain constant region comprising an amino acid sequence at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises both a human IgGl heavy chain constant region comprising an amino acid sequence 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 Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; and a light chain constant region comprising an amino acid sequence at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises a heavy chain constant region comprising an amino acid sequence modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.
• the antibody comprises a light chain constant region comprising an amino acid sequence modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises both a heavy chain constant region comprising an amino acid sequence modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; and a light chain constant region comprising an amino acid sequence modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises a human IgGl heavy chain constant region comprising amino acid sequence of any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.
• the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises both a human IgGl heavy chain constant region comprising the amino acid sequence of any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises a VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35, an HCDR2 comprising the amino acid sequence of positions 50-66, and an HCDR3 comprising the amino acid sequence of positions 99-108 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.
• the antibody further comprises a VL comprising an LCDR1 comprising the amino acid sequence of residues 24- 38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• the antibody further comprises a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172.
• the antibody comprises:
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 138, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 138, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 138, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 140, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 140, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 140, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 142, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 142, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 142, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 144, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 144, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 144, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 146, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 146, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 146, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 148, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 148, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 148, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 150, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 150, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 150, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 152, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 152, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 152, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 154, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 154, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 154, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 156, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 156, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 156, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 158, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 158, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 158, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 160, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 160, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 160, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170;
• - a VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 162, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 162, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 162, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170; - a VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 164, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 164, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 164, and a VL comprising
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 166, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 166, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 166, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170; or
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 168, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 168, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 168, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• the antibody comprises:
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 138, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 138, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 138, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 140, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 140, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 140, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 142, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 142, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 142, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 144, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 144, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 144, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 146, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 146, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 146, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 148, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 148, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 148, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 150, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 150, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 150, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 152, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 152, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 152, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 154, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 154, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 154, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 156, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 156, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 156, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172;
• - a VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 158, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 158, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 158, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172; - a VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 160, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 160, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 160, and a VL comprising
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 162, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 162, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 162, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 164, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 164, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 164, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172;
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 166, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 166, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 166, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172; or
• VH comprising an HCDR1 comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 168, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 168, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 168, and a VL comprising an LCDR1 comprising the amino acid sequence of residues 24-38 of SEQ ID NO: 172, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 172, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 172.
• the antibody comprises a VH comprising an amino acid sequence that is at least 90% identical to, at least 95% identical to, 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.
• the antibody comprises a VL comprising an amino acid sequence that is at least 90% identical to, at least 95% identical to, or at least 97% identical to the amino acid sequence of SEQ ID NO: 170. In other embodiments, the antibody comprises a VL comprising an amino acid sequence that is at least 90% identical to, at least 95% identical to, 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.
• the antibody comprises a VH comprising 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.
• the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 170.
• the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 172.
• the antibody comprises a VH comprising 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 also a VL comprising the amino acid sequence of SEQ ID NO: 170.
• the antibody comprises a VH comprising 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 also a VL comprising the amino acid sequence of SEQ ID NO: 172.
• the 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) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 138, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 138, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 138; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region
• 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: 138. In some embodiments, the antibody comprises 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 both 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: 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.
• the antibody comprises a VH comprising 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 a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises 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.
• the antibody comprises both 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: 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.
• the antibody comprises a VH comprising 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.
• the antibody comprises a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 138. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence 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: 138; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 138 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 138. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence of SEQ ID No: 172. 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: 138; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 138 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the 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) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 140, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 140, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 140; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region 1
• 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: 140. In some embodiments, the antibody comprises 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 both 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: 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.
• the antibody comprises a VH comprising 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 a VL comprising 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 a VH comprising 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 a VL comprising 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.
• the antibody comprises 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 both 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: 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 VH comprising 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.
• the antibody comprises a VL comprising 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 a VH comprising 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 a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 140. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence 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: 140; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 140 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 140. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence of SEQ ID No: 172. 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: 140; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 140 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the 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) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 142, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 142, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 142; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region
• 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: 142. In some embodiments, the antibody comprises 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 both 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: 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.
• the antibody comprises a VH comprising 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 a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises 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.
• the antibody comprises both 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: 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.
• the antibody comprises a VH comprising 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.
• the antibody comprises a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 142. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence 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: 142; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 142 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 142. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence of SEQ ID No: 172. 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: 142; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 142 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the 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) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 144, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 144, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 144; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region
• 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: 144. In some embodiments, the antibody comprises 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 both 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: 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.
• the antibody comprises a VH comprising 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 a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises 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.
• the antibody comprises both 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: 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.
• the antibody comprises a VH comprising 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.
• the antibody comprises a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 144. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence 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: 144; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 144 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 144. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence of SEQ ID No: 172. 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: 144; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 144 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the 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) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 146, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 146, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 146; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region
• 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: 146. In some embodiments, the antibody comprises 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 both 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: 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.
• the antibody comprises a VH comprising 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 a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises 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.
• the antibody comprises both 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: 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.
• the antibody comprises a VH comprising 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.
• the antibody comprises a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 146. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence 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: 146; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 146 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 146. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence of SEQ ID No: 172. 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: 146; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 146 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the 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) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 148, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 148, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 148; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region
• 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: 148. In some embodiments, the antibody comprises 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 both 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: 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.
• the antibody comprises a VH comprising 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 a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises 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.
• the antibody comprises both 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: 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.
• the antibody comprises a VH comprising 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.
• the antibody comprises a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 148. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence 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: 148; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 148 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 148. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence of SEQ ID No: 172. 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: 148; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 148 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the 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) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 150, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 150, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 150; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region 1
• 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: 150. In some embodiments, the antibody comprises 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 both 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: 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.
• the antibody comprises a VH comprising 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 a VL comprising 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 a VH comprising 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 a VL comprising 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.
• the antibody comprises 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 both 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: 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 VH comprising 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.
• the antibody comprises a VL comprising 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 a VH comprising 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 a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 150. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence 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: 150; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 150 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 150. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence of SEQ ID No: 172. 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: 150; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 150 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the methods can be used to assess an antibody that specifically binds to protein arginine deiminase 4 (PAD4), and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 152, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 152, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 152; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region 1
• 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: 152. In some embodiments, the antibody comprises 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 both 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: 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.
• the antibody comprises a VH comprising 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 a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises 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.
• the antibody comprises both 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: 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.
• the antibody comprises a VH comprising 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.
• the antibody comprises a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 152. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence 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: 152; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 152 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 152. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence of SEQ ID No: 172. 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: 152; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 152 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 154, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 154, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 154; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region 1
• HCDR1 heavy chain complementarity determining region 1
• HCDR1 heavy chain complementarity determining region 1
• 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 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 both 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; 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.
• the antibody comprises a VH comprising 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 a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises 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.
• the antibody comprises both 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; 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.
• the antibody comprises a VH comprising 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.
• the antibody comprises a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 154.
• the antibody comprises a VH comprising 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.
• the antibody comprises a VL comprising the amino acid sequence 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; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 154 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 154. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence of SEQ ID No: 172. 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; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 154 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the methods may be used to assess an 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) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 156, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 156, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 156; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining
• 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: 156. In some embodiments, the antibody comprises 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 both 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: 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.
• the antibody comprises a VH comprising 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 a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises 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.
• the antibody comprises both 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: 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.
• the antibody comprises a VH comprising 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.
• the antibody comprises a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 156. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence 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: 156; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 156 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 156. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence of SEQ ID No: 172. 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: 156; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 156 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the antibody 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) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 158, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 158, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 158; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region 1
• HCDR1
• 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: 158. In some embodiments, the antibody comprises 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 both 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: 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.
• the antibody comprises a VH comprising 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 a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises 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.
• the antibody comprises both 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: 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.
• the antibody comprises a VH comprising 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.
• the antibody comprises a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 158. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence 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: 158; and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 158 and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 158. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence of SEQ ID No: 172.
• 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: 158; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 158 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the antibody 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) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 160, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 160, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 160; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region 1
• HCDR1 heavy chain complement
• 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: 160. In some embodiments, the antibody comprises 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 both 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: 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.
• the antibody comprises a VH comprising 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 a VL comprising 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 a VH comprising 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 a VL comprising 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.
• the antibody comprises 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 both 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: 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 VH comprising 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.
• the antibody comprises a VL comprising 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 a VH comprising 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 a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 160. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence 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: 160; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 160 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 160. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence of SEQ ID No: 172. 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: 160; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 160 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the antibody 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) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 162, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 162, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 162; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region 1
• HCDR1 comprising
• 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: 162. In some embodiments, the antibody comprises 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 both 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: 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.
• the antibody comprises a VH comprising 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 a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises 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.
• the antibody comprises both 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: 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.
• the antibody comprises a VH comprising 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.
• the antibody comprises a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 162. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence 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: 162; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 162 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 162. In some embodiments, the antibody comprises a VH comprising 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 comprising the amino acid sequence of SEQ ID No: 172. 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: 162; and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 162 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the antibody 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) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 164, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 164, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 164; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 compris
• VH heavy chain variable
• 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: 164. In some embodiments, the antibody comprises 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 both 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: 164; 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.
• the antibody comprises a VH comprising 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 a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises 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.
• the antibody comprises both 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: 164; 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.
• the antibody comprises a VH comprising 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.
• the antibody comprises a VL comprising 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.
• the antibody comprises both a VH comprising 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 a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 164. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 164; 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 comprising the amino acid sequence 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: 164; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 164 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 164. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 164; 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 comprising the amino acid sequence of SEQ ID No: 172. 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: 164; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 164 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the antibody 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) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 166, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 166, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 166; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region 1
• HCDR1
• 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: 166. In some embodiments, the antibody comprises 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 both 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: 166; 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 166 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises a VL comprising 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.
• the antibody comprises both a VH comprising the amino acid sequence of SEQ ID No: 166 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions and a VL comprising 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.
• the antibody comprises 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.
• the antibody comprises both a VH comprising the amino acid sequence of SEQ ID No: 166 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions and a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 166. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 166; 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 comprising the amino acid sequence 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: 166; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 166 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 166. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 166; 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 comprising the amino acid sequence of SEQ ID No: 172. 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: 166; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 166 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the antibody 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) comprising the amino acid sequence of positions 26-35 of SEQ ID NO: 168, an HCDR2 comprising the amino acid sequence of positions 50-66 of SEQ ID NO: 168, and an HCDR3 comprising the amino acid sequence of positions 99-108 of SEQ ID NO: 168; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence residues 24-38 of SEQ ID NO: 170, an LCDR2 comprising the amino acid sequence of residues 54-60 of SEQ ID NO: 170, and an LCDR3 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170.
• VH heavy chain variable region
• HCDR1 heavy chain complementarity determining region 1
• HCDR1
• 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: 168. In some embodiments, the antibody comprises 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 both 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: 168; 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 168 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions. In some embodiments, the antibody comprises a VL comprising 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.
• the antibody comprises both a VH comprising the amino acid sequence of SEQ ID No: 168 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions and a VL comprising 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.
• the antibody comprises 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.
• the antibody comprises both 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: 168; 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 168 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions.
• the antibody comprises a VL comprising 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.
• the antibody comprises both a VH comprising the amino acid sequence of SEQ ID No: 168 modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions and a VL comprising 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.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 168. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 168; 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 comprising the amino acid sequence 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: 168; and a VL comprising the amino acid sequence of SEQ ID No: 170. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 168 and a VL comprising the amino acid sequence of SEQ ID No: 170.
• the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 168. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 168; 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 comprising the amino acid sequence of SEQ ID No: 172. 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: 168; and a VL comprising the amino acid sequence of SEQ ID No: 172. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID No: 168 and a VL comprising the amino acid sequence of SEQ ID No: 172.
• the anti-PAD4 antibody is an IgG antibody, such as a human IgGl, IgG2, IgG3, or IgG4 antibody or a murine IgGl or IgG2 antibody.
• the antibody comprises a wild-type, human IgGl, IgG2, or IgG4 heavy chain constant region.
• the antibody comprises a full length heavy chain and/or a full length light chain.
• the antibody lacks a C-terminal lysine at the end of the heavy chain constant region.
• the antibody lacks a C-terminal glycine-lysine at the end of the heavy chain constant region.
• the antibody is an antibody fragment, such as an Fv, single-chain Fv (scFv), Fab, Fab’, or (Fab’)2.
• the antibody is a bispecific or multispecific antibody, or which is conjugated covalently or noncovalently to at least one other molecule. In some embodiments, the antibody is conjugated covalently or noncovalently to at least one other molecule, wherein the at least one other molecule comprises a detection label and/or a drug.
• the antibody comprises a human IgGl heavy chain constant region comprising an amino acid sequence 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 Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.
• the antibody comprises a light chain constant region comprising an amino acid sequence at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises both a human IgGl heavy chain constant region comprising an amino acid sequence 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 Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; and a light chain constant region comprising an amino acid sequence at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises a heavy chain constant region comprising an amino acid sequence modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.
• the antibody comprises a light chain constant region comprising an amino acid sequence modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises both a heavy chain constant region comprising an amino acid sequence modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions compared to the amino acid sequence of any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; and a light chain constant region comprising an amino acid sequence modified by 1-10 amino acid substitutions, 1-5 amino acid substitutions, or 1-3 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises a human IgGl heavy chain constant region comprising amino acid sequence of any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.
• the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 194.
• the antibody comprises both a human IgGl heavy chain constant region comprising the amino acid sequence of any one of SEQ ID Nos: 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192; and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 194.
• an antibody PAD4 modulator may further incorporate any of the features, singly or in combination, as described in the sections that follow.
• an antibody provided herein is an antibody fragment.
• Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and scFv fragments, and other fragments described below.
• Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and scFv fragments and other fragments described below.
• Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and scFv fragments and other fragments described below.
• scFv fragments see, e.g., Pluckthiin, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Ver
• Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).
• Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
• a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).
• Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.
• recombinant host cells e.g., E. coli or phage
• an antibody provided herein is a multispecific antibody, for example, a bispecific antibody.
• Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites.
• one of the binding specificities is TREM2 and the other is for any other antigen.
• bispecific antibodies may bind to two different epitopes of TREM2.
• Bispecific antibodies may also be used to localize drugs such as cytotoxic agents or to localize detection labels to cells that express TREM2.
• the multispecific antibody e.g., bispecific antibody comprises a first variable domain comprising the CDRs or variable regions as described herein.
• Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
• Multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob -in-hole” engineering (see, e.g., U.S. Patent No. 5,731, 168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., US Patent No.
• an antibody provided herein is a chimeric antibody.
• Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
• a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.
• a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
• a chimeric antibody is a humanized antibody.
• a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
• a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
• HVRs e.g., CDRs, (or portions thereof) are derived from a non-human antibody
• FRs or portions thereof
• a humanized antibody optionally will also comprise at least a portion of a human constant region.
• some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
• a non-human antibody e.g., the antibody from which the HVR residues are derived
• Human framework regions that may be used for humanization include but are not limited to framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151 :2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carte r et al. Pro c. Natl. Acad. Set. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci.
• the humanized antibodies may comprise a human IgGl, IgG2, IgG3, or IgG4 heavy chain constant region.
• the glycosylation of an antibody is modified.
• an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation).
• Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen.
• carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
• one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
• Such aglycosylation can increase the affinity of the antibody for antigen.
• Glycosylation of the constant region on N297 can be prevented by mutating the N297 residue to another residue, e.g., N297A, and/or by mutating an adjacent amino acid, e.g., 298 to thereby reduce glycosylation on N297.
• an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
• altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
• carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies described herein to thereby produce an antibody with altered glycosylation.
• PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Led 3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al. (2002) J Biol. Chem. 277:26733-26740).
• PCT Publication WO 99/54342 by Umana et al.
• glycoprotein-modifying glycosyl transferases ⁇ e.g., beta(l,4)-N- acetylglucosaminyltransferase III (GnTIII)
• GnTIII glycoprotein-modifying glycosyl transferases
• An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody.
• the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
• PEG polyethylene glycol
• the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
• polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (CI-CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
• the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies described herein. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.
• an antibody described herein comprises one or more human constant regions.
• the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD.
• the human light chain constant region is of an isotype selected from K and A.
• an antibody described herein comprises a human IgG constant region, such as an IgGl, IgG2, IgG3, or IgG4.
• an antibody described herein comprises a human IgG4 heavy chain constant region.
• an antibody described herein comprises an S241P mutation in the human IgG4 constant region.
• an antibody described herein comprises a human IgG4 constant region and a human K light chain.
• the choice of heavy chain constant region can determine whether or not an antibody will have effector function in vivo.
• effector function includes antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), and can result in killing of the cell to which the antibody is bound.
• cell killing may be desirable, for example, when the antibody binds to a cell that supports the maintenance or growth of the tumor.
• Exemplary cells that may support the maintenance or growth of a tumor include, but are not limited to, tumor cells themselves, cells that aid in the recruitment of vasculature to the tumor, and cells that provide ligands, growth factors, or counter-receptors that support or promote tumor growth or tumor survival.
• an antibody comprising a human IgGl heavy chain or a human IgG3 heavy chain is selected.
• an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated.
• Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
• the carbohydrate attached thereto may be altered.
• Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997).
• the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
• modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibodies with certain improved properties.
• an antibody may be afucosylated, for example, by mutating residues such as Asn297 that are normally glycosylated with fucose-containing glycosylations, or through other means.
• antibodies herein may comprise an afucosylated human IgGl constant region.
• Antibodies are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibodies may have reduced fucosylation and/or improved ADCC function. Examples of such antibodies are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibodies with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibodies may have improved CDC function. Such antibodies are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
• Antibodies are also provided with amino-terminal leader extensions.
• one or more amino acid residues of the amino-terminal leader sequence are present at the aminoterminus of any one or more heavy or light chains of an antibody.
• An exemplary aminoterminal leader extension comprises or consists of three amino acid residues, VHS, present on one or both light chains of an antibody.
• human FcRn high affinity binding polypeptides can be assayed, e.g., in transgenic mice, in humans, or in non-human primates to which the polypeptides with a variant Fc region are administered. See also, e.g., Petkova et al. International Immunology 18(12): 1759-1769 (2006).
• an afucosylated antibody mediates ADCC in the presence of human effector cells more effectively than a parent antibody that comprises fucose
• ADCC activity may be determined using the in vitro ADCC assay as herein disclosed, but other assays or methods for determining ADCC activity, e.g. in an animal model etc., are contemplated.
• the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody.
• one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, 322, 330, and/or 331 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
• the effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.
• one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
• CDC complement dependent cytotoxicity
• one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement.
• This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.
• the Fc region can be modified to decrease antibody dependent cellular cytotoxicity (ADCC) and/or to decrease the affinity for an Fey receptor by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241 , 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280,
• ADCC antibody dependent cellular cytotoxicity
• Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E.
• Exemplary variants include 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F7324T (EU numbering).
• Other Fc modifications that can be made to Fes are those for reducing or ablating binding to FcyR and/or complement proteins, thereby reducing or ablating Fc- mediated effector functions such as ADCC, ADCP, and CDC.
• Exemplary modifications include but are not limited substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, 328, 330, and/or 331 (e.g., 330 and 331), wherein numbering is according to the EU index.
• Exemplary substitutions include but are not limited to 234A, 235E, 236R, 237A, 267R, 269R, 325L, 328R, 330S, and 33 IS (e.g., 330S, and 33 IS), wherein numbering is according to the EU index.
• An Fc variant can comprise 236R/328R.
• FcyR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331 S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removal of the glycosylation at position 297 by mutational or enzymatic means or by production in organisms such as bacteria that do not glycosylate proteins.
• substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331 S, 220S, 226S, 229S, 238S, 233P, and 234V are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.
• the human IgG1.3 Fc constant region contains L234A, L235E, and G237A substitutions.
• the IgGlfa.P238K (or IgGl.P238K) contains a P238K substitution.
• the IgGl. lf comprises L234A, L235E, G237A, A330S, and P331S substitutions. (All numbering under the EU index.)
• Fc variants that enhance affinity for an inhibitory receptor FcyRIIb can also be used. Such variants can provide an Fc fusion protein with immunomodulatory activities related to FcyRIIb cells, including for example B cells and monocytes. In one embodiment, the Fc variants provide selectively enhanced affinity to FcyRIIb relative to one or more activating receptors. Modifications for altering binding to FcyRIIb include one or more modifications at a position selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, 330, 331, and 332, according to the EU index.
• Exemplary substitutions for enhancing FcyRIIb affinity include but are not limited to 234 A, 234D, 234E, 234F, 234W, 235D, 235E, 235F, 235R, 235Y, 236D, 236N, 237A, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, 330S, 33 IS, and 332E.
• Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y.
• Fc variants for enhancing binding to FcyRIIb include 235Y/267E, 236D/267E, 239D/268D, 239D/267E, 267E/268D, 267E/268E, and 267E/328F. (All numbering under the EU index.)
• Fc modifications that increase binding to an Fey receptor include amino acid modifications at any one or more of amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 338, 340, 360, 373, 376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Patent Publication No. WO 00/42072.
• the Fc region can comprise a non-naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCX Patent Publications WO 00/42072; WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO 05/040217, WO 05/092925 and WO 06/0201 14).
• the affinities and binding properties of an Fc region for its ligand can be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art including but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration).
• in vitro assay methods biochemical or immunological based assays
• equilibrium methods e.g., enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay (RIA)
• kinetics e.g., BIACORE analysis
• indirect binding assays e.g., competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophore
• the antibody is modified to increase its biological half-life.
• this can be done by increasing the binding affinity of the Fc region for FcRn,
• one or more of more of following residues can be mutated: 252, 254, 256, 433, 435, 436, as described in U.S. Pat. No. 6,277,375.
• Specific exemplary substitutions include one or more of the following: T252L, T254S, and/or T256F.
• the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos.
• exemplary variants that increase binding to FcRn and/or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including for example 2591, 308F, 428L, 428M, 434S, 4341 1. 434F, 434Y, and 434X1.
• Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428 L, 428F, 250Q/428L (Hinton et al. 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al.
• hybrid IgG isotypes with particular biological characteristics can be used.
• an IgGl/IgG3 hybrid variant can be constructed by substituting IgGl positions in the CH2 and/or CH3 region with the amino acids from IgG3 at positions where the two isotypes differ.
• a hybrid variant IgG antibody can be constructed that comprises one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F.
• an IgGl/IgG2 hybrid variant can be constructed by substituting IgG2 positions in the CH2 and/or CH3 region with amino acids from IgGl at positions where the two isotypes differ.
• a hybrid variant IgG antibody can be constructed that comprises one or more substitutions, e.g., one or more of the following amino acid substitutions: 233E, 234L, 235L, +236G (referring to an insertion of a glycine at position 236), and 327A.
• IgGl variants with strongly enhanced binding to FcyRIIIa have been identified, including variants with S239D/I332E and S239D/I332E/A330L mutations which showed the greatest increase in affinity for FcyRIIIa, a decrease in FcyRIIb binding, and strong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006).
• IgGl mutants containing L235V, F243L, R292P, Y300L and P396L mutations which exhibited enhanced binding to FcyRIIIa and concomitantly enhanced ADCC activity in transgenic mice expressing human FcyRIIIa in models of B cell malignancies and breast cancer have been identified (Stavenhagen et al., 2007; Nordstrom et aL, 2011).
• Other Fc mutants that can be used include: S298A/E333A/L334A, S239D/I332E, S239D/I332E/A330L, L235V/F243L/R292P/Y300L/ P396L, and M428L/N434S.
• an Fc is chosen that has reduced binding to FcyRs.
• An exemplary Fc, e.g., IgGl Fc, with reduced FcyR binding comprises the following three amino acid substitutions: L234A, L235E and G237A.
• an Fc is chosen that has reduced complement fixation.
• An exemplary Fc e.g., IgGl Fc, with reduced complement fixation has the following two amino acid substitutions: A330S and P331S.
• an Fc is chosen that has essentially no effector function, i.e., it has reduced binding to FcyRs and reduced complement fixation.
• An exemplary Fc, e.g., IgGl Fc, that is effectorless comprises the following five mutations: L234A, L235E, G237A, A330S and P331S.
• IgG4 constant domain When using an IgG4 constant domain, it can include the substitution S228P, which mimics the hinge sequence in IgGl and thereby stabilizes IgG4 molecules. Fc modifications described in WO 2017/087678 or WO2016081746 may also be used.
• the glycosylation of an antibody is modified.
• an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation).
• Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen.
• carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
• one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
• Such aglycosylation can increase the affinity of the antibody for antigen.
• Glycosylation of the constant region on N297 can be prevented by mutating the N297 residue to another residue, e.g., N297A, and/or by mutating an adjacent amino acid, e.g., 298 to thereby reduce glycosylation on N297.
• an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
• altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
• carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies described herein to thereby produce an antibody with altered glycosylation.
• PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Led 3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740).
• PCT Publication WO 99/54342 by Umana et al.
• glycoprotein-modifying glycosyl transferases ⁇ e.g., beta(l,4)-N- acetylglucosaminyltransferase III (GnTIII)
• GnTIII glycoprotein-modifying glycosyl transferases
• An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody.
• the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
• PEG polyethylene glycol
• the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
• polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (CI-CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
• the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies described herein. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.
• the methods herein may in some embodiments be conducted in connection with a method of treating a subject with a PAD4 modulator or another therapeutic agent, such as those described in the preceding sections.
• the subject from which the biological sample is taken may be a subject that is undergoing treatment with a PAD4 modulator or other therapeutic agent.
• the methods may be used to evaluate such treatment, for example, by assessing the activity of the PAD4 modulator in a biological sample from the subject at one or more time points during treatment with the PAD4 modulator.
• the terms “disease” and “disorder” are used interchangeably herein in the context of an indication to be treated.
• the subject may have a biological sample assessed for citrullination in the absence of being treated with any PAD4 modulator, or in some cases prior to any treatment.
• a sample might be assessed for a purpose such as determining a likely clinical outcome, determining risk for developing a citrullination-related disease, diagnosing a citrullination-related disease, and selecting the subject from which the biological sample was derived for a treatment of a citrullination-related disease.
• a “citrullination-related disease” refers to a disease characterized by presence of increased citrullination of polypeptides in biological samples from disease subjects, and/or by one or more of presence of NETosis, presence of METosis, presence of anti-citrullinated protein antibodies (ACPA), and increased PAD4 expression.
• the subject may have a citrullination-related disease or be at risk of developing a citrullinati on-associated disease, or a subject’s sample may be tested to determine if they have a citrullination-related disease or are at risk of developing a citrullination-related disease.
• a citrullination-related disease is associated with one or more of NETosis, METosis, presence of anti-citrullinated protein antibodies (ACPA), increased PALM expression, and increased citrullination of polypeptides in a biological sample.
• the citrullination-related disease is an autoimmune disorder or an infectious disease or cancer.
• citrullination by PALM is a stress response and may serve as a signal for removal of stressed cells.
• Proteins citrullinated by PAD4 become antigenic substrates and are targets for both cellular (i.e., T cell) and humoral (i.e., B cell-derived antibody) adaptive immune responses.
• T cell i.e., T cell
• B cell-derived antibody i.e., B cell-derived antibody
• PALM activity may lead to generation of anti-citrullinated protein antibodies (ACPA).
• PALM neutrophil extracellular traps
• NET neutrophil extracellular traps
• METosis neutrophil extracellular traps
• the disorder is an autoimmune disorder.
• the autoimmune disorder comprises or is rheumatoid arthritis (RA).
• the RA is juvenile-onset RA, juvenile idiopathic arthritis (JIA), or juvenile rheumatoid arthritis (JRA).
• the subject has rheumatoid synovitis or significant systemic involvement secondary to RA (including but not limited to vasculitis, pulmonary fibrosis or Felty's syndrome).
• the subject is positive for ACPA.
• the subject is positive for anti -PALM autoantibodies. Typically, such anti-PAD4 autoantibodies activate PALM.
• PALM plays a role in autoimmune diseases such as rheumatoid arthritis (RA), lupus (including systemic lupus erythematosus (SLE), lupus nephritis, vasculitis (including anti-neutrophilic cytoplasmic antibody (ANCA)-associated vasculitis, inflammatory bowel disease (IBD) (including ulcerative colitis and Crohn’s disease), thrombosis (e.g., venous thrombosis), antiphospholipid antibody syndrome, and cystic fibrosis.
• RA rheumatoid arthritis
• SLE systemic lupus erythematosus
• vasculitis including anti-neutrophilic cytoplasmic antibody (ANCA)-associated vasculitis
• IBD inflammatory bowel disease
• thrombosis e.g., venous thrombosis
• antiphospholipid antibody syndrome e.g., Curran et al.; Yad
• the autoimmune disorder comprises or is rheumatoid arthritis (RA).
• the disorder is RA, or the subject to be treated has been diagnosed with RA.
• the subject is considered at risk of developing RA.
• the RA is juvenile-onset RA, juvenile idiopathic arthritis (JIA), or juvenile rheumatoid arthritis (JRA).
• the subject has rheumatoid synovitis or significant systemic involvement secondary to RA (including but not limited to vasculitis, pulmonary fibrosis or Felty's syndrome).
• the subject is positive for anti-citrullinated protein antibodies (ACPA).
• the subject is positive for anti-PAD4 autoantibodies. Typically, such anti-PAD4 autoantibodies activate PAD4.
• Rheumatoid arthritis is a major autoimmune disease the pathobiology of which commonly includes the presence of auto-antibodies including anti-citrullinated protein antibodies (ACPA).
• ACPA anti-citrullinated protein antibodies
• PAD4 a post-translational modification enzyme, citrullinates proteins that serve as neo-auto-antigens. These neo-auto-antigens, when presented, result in the production of ACPAs and are recognized by ACPAs to form immune complexes, thus leading to the initiation and progression of the disease.
• PALM The role of PALM in the pathogenesis of RA in an ACPA-independent fashion, for example, is reviewed in Curran AM, Naik P, Giles JT, Darrah E. Nat Rev Rheumatol.
• the PADI gene is identified as a risk locus for RA, as reviewed in Curran AM, Naik P, Giles JT, Darrah E. Nat Rev Rheumatol. 2020 Jun;16(6):301-315.
• Single-nucleotide polymorphorphisms in the PADI4 gene that encodes PAD4 have been identified that contribute to a susceptibility haplotype for RA, for example.
• anti-PAD4 antibodies found in RA subjects are antibodies that activate PAD4 in RA subjects.
• As Curran et al. reviews anti-PAD4 auto-antibodies have been detected in up to 45% patients with RA and shown to be associated with disease activity. Preclinically, deficiency of PAD4 has been shown to ameliorate experimental inflammatory arthritis mouse models (Weri Y. et al., Sci Rep.
• a subject whose biological sample is assessed for citrullination herein is at risk for developing RA or is found to be at risk for developing RA at least in part on the basis of the assessment results herein.
• the subject at risk for developing RA has a first-degree relative with RA (i.e., a parent or sibling) and/or presence of anti-citrullinated protein antibodies (ACPA) in serum and/or presence of rheumatoid factor (RF) in serum.
• APA anti-citrullinated protein antibodies
• RF rheumatoid factor
• presence of anti-citrullinated protein antibodies may be determined in some cases using an anti-CCP test, such as an ELISA test.
• anti- CCP antibody test positivity i.e., presence of ACPA
• anti-CCP testing performed, such as due to joint pain or lung disease.
• Those 46% went on to meet the classification for RA within the subsequent 5 years.
• anti-CCP test results may be positive up to 10 years prior to onset of RA symptoms.
• PAD4 has been found in synovial fluid and synovial biopsies of RA subjects, along with citrullinated proteins, and it has also been found in NETs generated from neutrophils of RA subjects. It is thought that citrullination of these target proteins such as fibrinogen, vimentin, and histones, is promoted in the subclinical phase of RA development, and may be triggered by factors such as cigarette smoking (which is known to increase PAD expression in lung tissue) and periodontal disease (via PAD activity of the oral microbe P. gingivalis). (Curran et al.; Chang et al., Arthitis Res. Ther.
• a subject at risk for developing RA has a history of smoking (e.g., cigarettes, cigars) or of using tobacco products (e.g., chewing tobacco), and/or has periodontal disease.
• a subject at risk for developing RA does not have clinical symptoms of arthritis.
• the subject shows subclinical symptoms of arthritis, such as joint inflammation visible by imaging, such as ultrasound or magnetic resonance imaging (MRI), presence of ACPA via an anti-CCP test, presence of rheumatoid factor (RF), or an SNP or other genetic alteration in the PADI4 gene or its promoter region characteristic of subjects with RA.
• the subject has such subclinical symptoms along with one or both of a first-degree relative with RA and serum ACPA or serum rheumatoid factor (RF).
• the subject has been diagnosed with arthralgia or undifferentiated arthritis.
• arthralgia herein refers to symptoms of pain or aching in at least one joint, such as an ankle, toe, shoulder, elbow, wrist, knee, hip, or one or more joints of the hand, fingers, or spine.
• a subject with arthralgia may also have tenderness, redness, warmth, loss of mobility, stiffness, weakness, numbness and/or tingling in one or more joints.
• Undifferentiated arthritis refers to diagnosis of arthritis in a subject for which the type of arthritis, such as RA or osteoarthritis, is not specified or cannot be determined.
• the subject may also have one or more of a first-degree relative with RA, ACPA in serum, RF in serum, and subclinical joint inflammation (e.g., by ultrasound or MRI).
• the treating may comprise, for example, lessening effects of one or more present clinical symptoms and/or one or more present sub-clinical symptoms.
• an RA subject or a subject at risk of developing RA has a comorbidity.
• the comorbidity is a lung disorder such as interstitial lung disease (ILD), pleural effusion, cricoarytenoiditis, constrictive or follicular bronchiolitis bronchiectasis, pulmonary vasculitis, or pulmonary hypertension.
• ILD interstitial lung disease
• pleural effusion cricoarytenoiditis
• constrictive or follicular bronchiolitis bronchiectasis constrictive or follicular bronchiolitis bronchiectasis
• pulmonary vasculitis or pulmonary hypertension.
• the lung disorder is a parenchymal lung disease (e.g., pneumonia), an airway disease (e.g., cricoarytenoiditis), or a pleural disease (e.g., pleural effusion).
• a parenchymal lung disease e.g., pneumonia
• an airway disease e.g., cricoarytenoiditis
• a pleural disease e.g., pleural effusion.
• the comorbidity is a lung disorder characterized by inflammation and/or scarring (fibrosis) of the lung, such as interstitial lung disease (ILD), also known as pulmonary fibrosis.
• ILD interstitial lung disease
• the subject has not been diagnosed with RA, but has a lung disorder, such as a disorder characterized by inflammation and/or scarring of the lung, such as interstitial lung disease (ILD), also known as pulmonary fibrosis, or has a parenchymal lung disease (e.g., pneumonia), an airway disease (e.g., cricoarytenoiditis), or a pleural disease (e.g., pleural effusion), or has interstitial lung disease (ILD), pleural effusion, cricoarytenoiditis, constrictive or follicular bronchiolitis bronchiectasis, pulmonary vasculitis, or pulmonary hypertension.
• a lung disorder such as a disorder characterized by inflammation and/or scarring of the lung, such as interstitial lung disease (ILD), also known as pulmonary fibrosis, or has a parenchymal lung disease (e.g., pneumonia), an airway disease (e.g., cricoary
• ILD can also be a comorbidity with other autoimmune diseases such as scleroderma, dermatomyositis and polymyositis, mixed connective tissue disease, Sjogren’s syndrome, and sarcoidosis, as well as result from certain infectious diseases such as pneumonia, or exposure to certain drugs or harmful substances such as asbestos, or can result from uncontrolled gastroesophageal reflux.
• autoimmune diseases such as scleroderma, dermatomyositis and polymyositis, mixed connective tissue disease, Sjogren’s syndrome, and sarcoidosis
• the autoimmune disorder comprises or is a rheumatic autoimmune disease other than RA.
• PAD4 gene polymorphism for example, is not only associated with RA, but is also associated with lupus, such as systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, and lupus nephritis.
• SLE systemic lupus erythematosus
• Padi4 -/- individuals have been found to display decreased autoantibodies, type I IFN responses, immune cell activation, vascular dysfunction, and NET immunogenicity.
• Human T cells express both PAD4 and PAD2, and when exposed to either PAD2 or PAD4 inhibitors, display abrogation of Thl polarization.
• Padi4 knock-out mice showed significant improvements in proteinuria progression compared with wild-type mice, decreased neutrophil infiltration in kidneys, and reduced phosphorylation of p38 MAPK and lower expression of JNK-associated leucine zipper protein (JLP), a p38 MAPK scaffold protein.
• JLP JNK-associated leucine zipper protein
• NETosis is associated with the pathophysiology of lupus and other autoimmune and renal diseases, including, for instance systemic lupus erythematosus, vasculitis (e.g., ANCA- associated vasculitis), antiphospholipid antibody syndrome, type 1 diabetes mellitus, and renal inflammatory diseases (gomerulophritides, e.g., proliferative glomerulonephritis and non-proliferative gromerulonephritis), and is also associated with the pathophysiology of cancer.
• vasculitis e.g., ANCA- associated vasculitis
• antiphospholipid antibody syndrome e.g., type 1 diabetes mellitus
• type 1 diabetes mellitus e.g., type 1 diabetes mellitus
• renal inflammatory diseases e.g., proliferative glomerulonephritis and non-proliferative gromerulonephritis
• NETs are extracellular web-like structures composed of chromatin backbone and various peptides and proteins that are formed by neutrophils in response to various stimuli in a process called NETosis.
• NETosis has been found to involve the citrullination of histones, such as histone H3, which requires PALM activity.
• NETosis is a key driver of disease.
• the disease is cancer (e.g., a cancer disclosed herein), or an autoimmune disease, such as, e.g., lupus (e.g., systemic lupus erythematosus), vasculitis (e.g., ANCA-associated vasculitis), antiphospholipid antibody syndrome, type 1 diabetes mellitus, inflammatory bowel disease (IBD) (e.g., ulcerative colitis and Crohn’s disease), cystic fibrosis, or a renal disease such as renal inflammatory disease (e.g., proliferative glomerulonephritis and non-proliferative gromerulonephritis).
• lupus e.g., systemic lupus erythematosus
• vasculitis e.g., ANCA-associated vasculitis
• IBD inflammatory bowel disease
• cystic fibrosis e.g., cystic fibrosis
• renal disease e.g.,
• abnormal PAD4 activity is known to be associated with autoimmune disorders in addition to RA, such as multiple sclerosis (MS), autoimmune encephalomyelitis, obstructive nepropathy, Alzheimer’s disease (AD), and inflammatory bowel disease (IBD) (e.g., ulcerative colitis and Crohn’s disease), as well as ankylosing spondylitis, osteoarthritis, glaucoma, Scrapie, and HIV/AIDS.
• MS multiple sclerosis
• autoimmune encephalomyelitis e.g., obstructive nepropathy, Alzheimer’s disease (AD), and inflammatory bowel disease (IBD) (e.g., ulcerative colitis and Crohn’s disease)
• IBD inflammatory bowel disease
• elevated levels of PAD enzymes and/or citrullinated proteins have been found in all of those conditions.
• the autoimmune disorder comprises IBD.
• the autoimmune disorder comprises colitis, such as ulcerative colitis, Crohn's disease, gluten-sensitive enteropathy, or Whipple’s disease.
• the autoimmune disorder comprises lupus, such as systemic lupus erythematosus, cutaneous lupus erythematosus, or lupus nephritis. In some embodiments, the autoimmune disorder comprises vasculitis.
• vasculitis examples include Bechet’s Disease, Buerger’s Disease (Thromboangiitis Obliterans), eosinophilic granulomatosis with polyangiitis (EGPA; formerly known as Churg Strauss), cryoglobulinemia, giant cell arteritis (temporal arteritis), Henoch-Schbnlein purpura (HSP; IgA vasculitis), microscopic polyangiitis, polyarteritis nodosa, polymyalgia rheumatica, rheumatoid vasculitis, Takayasu’s arteritis, granulomatosis with polyangiitis (GPA; formerly known as Wegener’s), ANCA-associated vasculitis (such as PR3-ANCA associated vasculitis or MPO-ANCA associated vasculitis), hypersensitivity vasculitis, isolated aortitis, central nervous system vasculitis, primary angi
• the autoimmune disorder comprises thrombosis.
• the autoimmune disorder comprises arthritis, such as, e.g., acute arthritis, chronic arthritis, gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, septic arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, menopausal arthritis, estrogen-depletion arthritis, ankylosing spondylitis, or rheumatoid spondylitis.
• arthritis such as, e.g., acute arthritis, chronic arthritis, gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, septic arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral
• the autoimmune disorder comprises multiple sclerosis (MS), such may include: primary progressive multiple sclerosis (PPMS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), and progressive relapsing multiple sclerosis (PRMS).
• MS multiple sclerosis
• PPMS primary progressive multiple sclerosis
• RRMS relapsing-remitting multiple sclerosis
• SPMS secondary progressive multiple sclerosis
• PRMS progressive relapsing multiple sclerosis
• the autoimmune disorder comprises systemic sclerosis (scleroderma), idiopathic inflammatory myopathy (such as, e.g., dermatomyositis, polymyositis, necrotizing autoimmune myopathy, or sporadic inclusion body myositis), Sjogren’s syndrome, sarcoidosis, autoimmune hemolytic anemia, immune pancytopenia, paroxysmal nocturnal hemoglobinuria, autoimmune thrombocytopenia (such as, e.g., idiopathic thrombocytopenic purpura, immune- mediated thrombocytopenia, acute thrombocytopenic purpura, chronic thrombocytopenic purpura), thyroiditis (such as, e.g., Grave's disease, Hashimoto’s thyroiditisjuvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephritis, tubulointer) s
• the autoimmune disorder comprises a methotrexate-resistant autoimmune disorder, such as methotrexate-resistant RA, lupus, vasculitis, thrombosis, MS, or the like.
• the autoimmune disorder comprises a renal disease, such as a renal inflammatory disease such as, e.g., kidney fibrosis, chronic kidney disease, proliferative glomerulonephritis, or nonproliferative gromerulonephritis.
• the subject has a disorder comprising: acid-induced lung injury, acne (PAPA), acute lymphocytic leukemia, acute respiratory distress syndrome, Addison’s disease, adrenal hyperplasia, adrenocortical insufficiency, ageing, AIDS, alcoholic hepatitis, alcoholic liver disease, allergen induced asthma, allergic bronchopulmonary, aspergillosis, allergic conjunctivitis, alopecia, Alzheimer’s disease, amyloidosis, amyotrophic lateral sclerosis, weight loss, angina pectoris, angioedema, anhidrotic ecodermal dysplasia-ID, ankylosing spondylitis, anterior segment, inflammation, antiphospholipid syndrome, aphthous stomatitis, appendicitis, arthritis, asthma, atherosclerosis, atopic dermatitis, autoimmune diseases, autoimmune hepatitis, bee sting-induced inflammation, Bechet’s disease, Bechet’
• the disorder is cancer.
• neutrophil inflammation, neutrophil extracellular traps (NET), and/or monocyte extracellular traps (MET) have been identified in cancers, and are associated with poorer prognosis.
• NET neutrophil extracellular traps
• MET monocyte extracellular traps
• studies have shown that PAD4- catalyzed NET formation is upregulated in multiple tumors, and that PALM is overexpressed in a variety of cancers.
• a small molecule PALM inhibitor was also shown to inhibit tumor growth and to inhibit histone H3 citrullination in a cancer model.
• the cancer is a cancer that is typically responsive to immunotherapy. In some embodiments, the cancer is a cancer that is not typically responsive to immunotherapy. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a blood malignancy (liquid tumor).
• the cancer is carcinoma, lymphoma, blastoma, sarcoma, or leukemia.
• the cancer is squamous cell cancer, small-cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma squamous cell carcinoma, small-cell lung cancer (SCLC), non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell renal carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., clear cell renal carcinoma),
• the disease is an infectious disease.
• occurrence of NETosis has also been found in various infections.
• Infectious diseases may include, for example, viral diseases (including AIDS (HIV infection), hepatitis (A, B, C, D, and E), and herpes), bacterial infections, fungal infections, protozoal infections and parasitic infections.
• pathogenic infections include, but are not limited to, HIV, Hepatitis (A, B, & C), Influenza, Herpes (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, Epstein Barr virus), Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa, adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci,
• the infectious disease is caused by a viral pathogen. In other cases, it is caused by a bacterial pathogen.
• a PALM modulator may be administered alone or with other modes of treatment. Such other modes of treatment may be provided before, substantially contemporaneous with, or after administration of a PALM modulator. Thus, some subjects herein receiving treatment with a PALM modulator, such as a PAD4 inhibitor, or being considered for treatment with a PALM modulator or PALM inhibitor, may also be receiving treatment with another therapy.
• a subject may be treated with therapeutic agents, for example, such as a disease-modifying anti-rheumatic drug (DMARD) such as methotrexate (Trexall® or Otrexup®), adalimumab (Humira®), etanercept (Enbrel®), infliximab (Remicade®), hydroxychloroquine (Plaquenil®), sulfasalazine (Azulfidine®), leflunomide (Arava®), abatacept (Orencia®), anakinra (Kineret®), Certolizumab (Cimzia®), golimumab (Simponi®), rituximab (Rituxan®), sarilumab (Kevzara®), tocilizumab (Actemra®), baricitinib (Olumiant®), tofacitin
• DMARD disease-modifying anti-rheumatic drug
• an anti-PAD4 antibody may be administered with one or more of: anti-TNF agents (e.g., anti-TNF antibodies) such as infliximab (Remicade®), adalimumab (Humira®), golimumab (Simponi®), certolizumab (Cimzia®), and etanercept (Enbrel®); glucocorticoids such as prednisone or methylprednisolone; leflunomide (Arava®); azathioprine (Imuran® or Azasan®); JAK inhibitors such as CP 590690; SYK inhibitors such as R788; TYK2 inhibitors such as deucravacitinib (Sotyktu®), anti-IL-6 antibodies; anti-IL-6R antibodies; anti-CD-20 antibodies; anti-CD19 antibodies; anti-GM-CSF antibodies; and anti-GM-CSF-R antibodies.
• anti-TNF agents e.g
• anti-PAD4 antibodies may be administered with other therapeutic agents, for example, interferon alpha; interferon beta; anti-Type I interferon receptor antibodies such as anifrolumab (Saphnelo®); prednisone; anti- alpha4 integrin antibodies such as Tysabri®; anti-BAFF/BLyS antibodies such as belimumab (Benlysta®); anti-CD20 antibodies such as Rituxan® (rituximab); calcineurin inhibitors such as cyclosporin or voclosporin (Lupkynis®); complement inhibitors such as eculizumab (Soliris®) or avacopan (Tavneos®); mycophenolate mofetil (CellCept®) or mycophenolate sodium (MyFortic®); cyclophosphamide (Cytoxan®); FTY720 (fmgolimod, e.g., Gilenya®); and Cladribine®
• a subject may be treated with one or more therapeutic agents such as cyclosporine, tacrolimus, cyclophosphamide, azathioprine (Imuran®), mycophenolate (CellCept®), rituximab (Rituxan®), and Belimumab (Benlysta®), steroids (e.g., prednisone or prednisolone), blood pressure medication (e.g., antiotensin- convertin enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs)).
• therapeutic agents such as cyclosporine, tacrolimus, cyclophosphamide, azathioprine (Imuran®), mycophenolate (CellCept®), rituximab (Rituxan®), and Belimumab (Benlysta®), steroids (e.g., prednisone or prednisolone), blood pressure medication (e.g., antiotensin-
• ⁇ for the treatment of vasculitis, includes administration of therapeutic agents such as steroids (e.g., prednisone, prednisolone, methylprednisolone, or dexamethasone), methotrexate (Trexall®), azathioprine (Imuran®, Azasan®), mycophenolate (CellCept®), cyclophosphamide, tocilizumab (Actemra®), rituximab (Rituxan®), Avacopan, plasma exchange, mycophenolate mofetil (MMF), azathioprine (AZA), leflunomide (LEF), belimumab, meprolizumab, and omalizumab.
• steroids e.g., prednisone, prednisolone, methylprednisolone, or dexamethasone
• Trexall® methotrexate
• azathioprine Imuran®,
• a subject may be treated with anti-cancer agents, such as an immune checkpoint inhibitor, a chemotherapeutic agent, growth inhibitory agent, radiotoxic agent, immunosuppressive agent, anti-cancer vaccine such as a gene therapy vaccine, anti-angiogenesis agent and/or anti-neoplastic composition.
• anti-cancer agents such as an immune checkpoint inhibitor, a chemotherapeutic agent, growth inhibitory agent, radiotoxic agent, immunosuppressive agent, anti-cancer vaccine such as a gene therapy vaccine, anti-angiogenesis agent and/or anti-neoplastic composition.
• Immune checkpoint inhibitors include molecules that inhibit particular signaling pathways that regulate the immune system. See e.g., Weber (2010) Semin. Oncol. 37:430; Pardoll (2012) Nat. Rev. Cancer 12:252.
• Immune checkpoint inhibitors in some embodiments, comprise an antagonist of PD-1, PD-L1, CTLA4, LAG-3, Galectin 1, Galectin 9, CEACAM-1, BTLA, CD25, CD69, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM1, TIM3, TIM4, ILT4, IL-6, IL-10, TGFp, VEGF, KIR, LAG-3, adenosine A2A receptor, PI3Kdelta, or IDO.
• an immune checkpoint inhibitor comprises an agonist of B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, 0X40, OX40L, GITR, GITRL, CD27, CD40, CD40L, DR3, CD28H, IL-2, IL-7, IL-12, IL-15, IL-21, IFNa, STING, or a Toll-like receptor agonist such as a TLR2/4 agonist.
• an immune checkpoint inhibitor comprises an agent that binds to a member of the B7 family of membrane-bound proteins such as B7-1, B7-2, B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6.
• an immune checkpoint inhibitor binds to a member of the TNF receptor family or a co-stimulatory or co- inhibitory molecule binding to a member of the TNF receptor family such as CD40, CD40L, 0X40, OX40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL/ Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fnl4, TWEAK, BAFFR, ED AR, XEDAR, EDAI, EDA2, TACI, APRIL, BCMA, LTPR, LIGHT, DeR3, HVEM, VEGL/TL1A, TRAMP/DR3, TNFR1, TNFP, TNFR2, TNFa, ip2, FAS, FASL, RELT, DR6, TROY, or N
• an immune checkpoint inhibitor antagonizes or inhibits a cytokine that inhibits T cell activation such as IL-6, IL- 10, TGFP, VEGF.
• an immune checkpoint inhibitor comprises an agonist of a cytokine that stimulates T cell activation such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNa.
• the at least one immune stimulating agent comprises an antagonist of a chemokine, such as CXCR2, CXCR4, CCR2, or CCR4.
• an immune checkpoint inhibitor comprises an antibody.
• an immune checkpoint inhibitor comprises a vaccine, such as a mesothelin-targeting vaccine or attenuated listeria cancer vaccine such as CRS-207.
• Exemplary non-limiting example targets of immune checkpoint inhibitors are CTLA- 4, PD-1, and PD-L1.
• Non-limiting examples of such immune checkpoint inhibitors include anti-CTLA4, anti-PD-1, and anti-PD-Ll antibodies, such as, e.g., pembrolizumab (Keytruda®), ipilimumab (Yervoy®), nivolumab (Opdivo®), atezolizumab (Tecentriq®), avelumab (Bavencio®), dostarlimab (Jemperli®), cemiplimab (Libtayo®), and durvalumab (Imfinzi®).
• pembrolizumab Keytruda®
• ipilimumab Yervoy®
• nivolumab Opdivo®
• atezolizumab Tecentriq®
• avelumab Bavencio®
• Non-limiting examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and Cytoxan®/Neosar® cyclosphosphamide; lenalidomide (Revlimid®); bortezomib (Velcade®); bendamustine (Treanda®); rituximab (Rituxan®); alemtuzumab (Campath®); ofatumumab (Kesimpta®); everolimus (Afinitor®, Zortress®); carfilzomib (KyprolisTM); ifosamade; dexamethasone; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines
• dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esor
• chemotherapeutic agents include anti-hormonal agents that act to regulate or inhibit hormone action on cancers such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and Fareston® toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, Megase® megestrol acetate, Aromasin® exemestane, formestanie, fadrozole, Rivisor® vorozole, Femara® letrozole, and Arimidex® anastrozole; and anti-androgens such as flutamide,
• an anti-angiogenesis agent may be administered.
• an anti-angiogenesis agent can include an antibody or other antagonist to an angiogenic agent, e.g., antibodies to VEGF-A (e.g., bevacizumab (Avastin®)) or to the VEGF-A receptor (e.g., KDR receptor or Fit- 1 receptor), anti-PDGFR inhibitors such as Gleevec® (Imatinib Mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668, Sutent®/SUl 1248 (sunitinib malate), AMG706, or those described in, e.g., international patent application WO 2004/113304).
• an antibody or other antagonist to an angiogenic agent e.g., antibodies to VEGF-A (e.g., bevacizumab (Avastin®)) or to the VEGF-A receptor (e.g., KDR
• Anti-angiogensis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D’Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 (e.g., Table 3 therein listing anti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12): 1359-1364; Tonini etal. (2003) Oncogene 22:6549-6556 (e.g., Table 2 therein listing known anti -angiogenic factors); and Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., Table 1 therein listing anti -angiogenic agents used in clinical trials).
• native angiogenesis inhibitors e.g., angiostatin, endostatin, etc. See, e.g
• a tumor growth inhibitory agent may be administered.
• growth inhibitory agents include, but are not limited to, agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest.
• Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
• DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
• DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
• DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
• Docetaxel (Taxotere®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (Taxol®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
• an anti-inflammatory drug may be administered.
• the antiinflammatory drug can be, e.g., a steroid or a non-steroidal anti-inflammatory drug (NSAID).
• NSAID non-steroidal anti-inflammatory drug
• hormones and steroids including synthetic analogs, such as 17a-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone, Megestrolacetate, Methylprednisolone, Methyl-testosterone, Prednisolone, Triamcinolsone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide, To
• a subject with a disease may receive a vaccination protocol.
• Many experimental strategies for vaccination against infectious diseases and tumors have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition).
• Examples are cell-based therapies such as dendritic cells, or vaccine-like particles (VLPs).
• a subject may be administered other therapies, such as radiation therapy in the case of a tumor, surgical interventions, or the like.
• any of the above agents may, in some cases, be administered with a PAD4 modulator, such as a PAD4 inhibitor, to a subject, or alternatively, a subject already receiving one of the above therapies or agents may have a biological sample assessed by methods herein in order to determine if the subject should receive a PAD4 modulator or PAD4 inhibitor either in combination with or as an alternative to a current treatment for the subject.
• a PAD4 modulator such as a PAD4 inhibitor
• Microtiter plates (96 well Nunc MaxiSorpTM ELISA plates; Thermo Fisher Scientific Cat# 44-2404-21) were coated with 1 pg/ml arginine-containing linear peptide synthesized by GenScript® (SHQESTRGKSKGKAAAAA; SEQ ID NO: 232) in PBS and incubated overnight at 4°C. Plates were washed three times with ELSA washing buffer (Cayman Cat# 400062, Cat# 400035).
• rhPAD4 recombinant human PAD4 (Cayman Chemical #10500, Ann Arbor, MI) was preincubated with at concentrations of 13.5 nM (1 pg/mL), 27 nM, 54 nM, and 108 nM (8 pg/mL).
• Each preincubated solution was then mixed with an equal volume of anti-PAD4 antibody hzl3-5 D3 IE or isotype control antibody hlgGl ,3f, which was serial-diluted in the assay buffer to achieve antibody concentrations ranging from 0.13 nM to 66.7 nM.
• the recombinant PAD4 and antibody mixtures were incubated for 60 min at 4°C. 100 pl of each reaction was added into wells of peptide-coated microtiter plates. Plates were incubated at 37°C overnight, and then washed three times with a washing buffer, and blocked for 1 hour with blocking buffer (Invitrogen Cat# DS98200).
• HRP horse radish peroxidase conjugated anti-citrulline monoclonal antibody Clone 1D9
• TMB peroxidase substrate
• the HRP conjugated anti-citrulline monoclonal antibody, clonelD9 was made using HRP conjugation kit (Abeam Cat# abl02890) by following manufacture’s protocol. lOul of Modifier reagent was added into 90ul of anti-citrulline antibody. The mixture was added directly onto the lyophilized HRP mix. Vials were left standing for 3 hrs in the dark at room temperature. After incubation, lOul of Quencher reagent was added and the solution mixed gently. The conjugates were used after 30 minutes without further purification.
• IC50 values were calculated using GraphPad® Prism 9.4.0 (GraphPad Software, San Diego, CA). IC50s were determined by nonlinear regression curve fit with One site - Fit logIC50.
• Antibody hzl3-5 D31E inhibited PAD4 activity in a dose-dependent manner, as determined by citrulline ELISA.
• the IC50s proportionally increased with increased concentration of rhPAD4.
• Representative curves with 13.5 nM (1 pg/ml) and 108 nM (8 pg/ml) of rhPAD4 are shown in FIG. 1.
• the isotype control antibody showed no effect against rhPAD4 up to 66.7nM for concentrations of rhPAD4 at 13.5nM, 27nM and 54nM; and up to 133nM for concentration of rhPAD4 at 108nM. The experiment was repeated three times.
• Table 1 shows the IC50 for each run of the experiment, followed by the average and standard deviation for the three runs, showing that hzl3-5 D3 IE inhibits citrullination of arginine in the tested peptide with IC50 values from about 0.5 to 5.0 nM, depending on the concentration of rhPAD4.
• Anti-PAD4 antibodies were analyzed for ability to reduce extracellular citrullinated H3 and secretion of cytokines in human blood monocytes stimulated by lipopolysaccharide (LPS).
• LPS lipopolysaccharide
• IgG1.3f constant region
• the antibodies were as follows: (1) hzl3-5, (2) hzl3-5 D31E, (3) hzl3-3, (4) hzl3-12, (5) hz20-2, (6) hz20-7, (7) Isotype control hIgG1.3f.
• Human monocytes (CD14+CD16-) were isolated from fresh human PBMCs by immunomagnetic negative selection using EasySepTM Human Monocyte Isolation Kit (StemCell, Cat. #19359). The isolated human CD14+ monocytes were washed and cultured in Assay medium IMDM (Gibco, Cat. #31980-030), and 10% fetal bovine serum (FBS).
• Assay medium IMDM Gibco, Cat. #31980-030
• FBS fetal bovine serum
• the supernatant was collected for detection of extracellular citrullinated histone H3 (Cit-H3) by ELISA kit (Cayman Chemical, Cat. #501620), and for detection of secreted GM- CSF and other cytokines by Alphalisa (Perkin Elmer, Cat. #AL216).
• the cell lysates were used to isolate mRNA for gene expression by qRT-PCR. Data was analyzed using Excel and GraphPad Prism software.
• the antibodies reduced the amount of extracellular Cit-H3 in a dose-dependent fashion (FIG. 2A-FIG. 2F).
• mice C57B1/6 human-PAD4 knock-in mice were used as well as WT mice. These mice were treated with antibodies shown in Table 2 below at 0.24 mg/kg, 1.2 mg/kg, 6 mg/kg, 3 mg/kg, 10 mg/kg, 30 mg/kg, and/or 100 mg/kg. Control mice were treated with Isotype control murine antibody (IC mAb) at 100 mg/kg.
• IC mAb Isotype control murine antibody
• Anti-human PAD4 mAbs mAbl3 (clone 13) and its humanized derivatives hzl3-5 and hzl3-12, and mAb20 (clone 20) and its humanized derivatives hz20-2 and hz20-7 were tested for their ability to inhibit PAD4 function in the LPS ALI PD model.
• the activity of the antibodies was assessed based on their inhibition of the level of extracellular citrullinated H3 (relative to total extracellular H3) and the level of extracellular citrullinated ITIH4 (relative to total extracellular ITIH4). Compared with isotype control, all of these antibodies reduced extracellular Cit-H3 and extracellular Cit-ITIH4 in the Hu-PAD4 KI mice.
• mAb20 reduced extracellular Cit H3 by 41% in Hu-PAD4 KI mice at the lowest dose tested (30 mg/kg) (FIG. 3A). This activity was specific to human PALM since even at 100 mg/kg, mAb20 did not reduce extracellular Cit-H3 in WT mice.
• hz20-2 and hz20-7 also reduced extracellular Cit-ITIH4. At 30 mg/kg, hz20-2 (FIG. 3D) and hz20-7 (FIG. 3E) reduced extracellular Cit-ITIH4 by -80% and 75%, respectively.
• mAbl3 reduced extracellular Cit-H3 in Hu-PAD4 KI mice but not in WT mice (FIG. 4A).
• mAbl3 reduced extracellular Cit-H3 by 65% and 70% at 30 mg/kg and 100 mg/kg (i.e., at maximum inhibition), respectively (FIG. 4A).
• Table 2 below shows calculated Kd (nM) of each tested antibody, as an indicator of in vivo potency. Table 2 also summarizes % reduction of extracellular Cit-H3 and Cit-ITIH4, as shown in FIG. 3 A- FIG. 3E and FIG. 4A-FIG. 4E.
• mice were treated with these Abs subcutaneously at 1 mg/kg, 3 mg/kg, 10 mg/kg, 30 mg/kg, or 100 mg/kg.
• Control mice were treated with an isotype control murine antibody (IC mAb) at 100 mg/kg.
• IC mAb isotype control murine antibody
• humanized antibody hzl3- 5 D31 E was tested and hz!3-5 was tested at the lowest dose administered.
• Mice were treated with hzl3-5 D3 IE subcutaneously at 0.1 mg/kg, 1 mg/kg, or 30 mg/kg, or with hzl3-5 at 0.1 mg/kg.
• mice were treated with LPS without antibodies to determine the ideal time frame for measuring expression levels of citrullinated proteins.
• Peak expression levels of Cit- PRG4, Cit-ITIH4, and hPAD4 (FIG. 5C- FIG. 5E) were observed at days 2-3 after ia injection of LPS. The expression levels declined by day 6 post ia injection. Hence, day 2 was determined as ideal time to determine Cit-PRG4 and Cit-ITIH4 protein expression.
• Example 5 Citrullination in human serum induced in vitro by exogenous PAD4 and inhibited by the anti-PAD4 antibody hz!3-5 D31E
• Inhibition curves To produce inhibition curves, 7.5 pl of Ab hzl3-5 D31E serial dilutions in PBS (final concentrations ranging from 0.0038 nM to 100 nM) or isotype control (final concentration 100 nM) were added to 80 pl of serum in plates, mixed and stored overnight at -80°C. Serum plates were thawed and 12.2 pl of PAD4 enzyme reaction mix with or without 2 mM CaCh (7 nM or 13.5 nM PAD4, 100 mM Tris, 2 mM DDT final) was added and incubated at 37°C in CO2 incubator for 2 hours. Reactions were stopped with 10 pl of 0.5 M EDTA. Plates were sealed and stored at -80°C.
• the serum mixture (serum + enzyme + antibody, prepared as described above) was thawed and diluted (1 part serum mixture to 10 parts water). Then 5 pl of the resulting diluted serum mixture was combined with 45 pl iST-BCT LYSE buffer (PreOmics, cat# P.O.00116) and heated for 10 minutes at 95°C. Samples were enzymatically digested with addition of trypsin and LysC, and resulting peptides cleaned up according to manufacturer’s protocol using iST-BCT kit (PreOmics, cat# P.O.00116).
• citrullination ratio (CitRatio) of each citrullinated peptide calculated by dividing the intensity of the citrullinated peptide by the total protein intensity (summarized based maxLFQ algorithm).
• CitRatio was fitted against doses in a four-parameters (upper bound, lower bound, ec50 and hill) non-linear model.
• the non-linear model was then compared against a null linear regression model using chi-square statistics.
• the null model fits the mean (CitRatio) against doses. This process generates a p. value (DRC.p.value) that represents goodness of fit and effect size of the dose dependency for each individual citrullination site.
• DRC.p.value p. value
• the peptides in the tables below also in some cases were found to also include chemical modifications at other residues, such as oxidation of methionine (M), carb amidomethylation at certain cysteine (C) residues, or modifications at the N-terminus of the peptide, which are noted by underlining those residues, or in the case of a modification at the N-terminus, underlining the first residue in the peptide sequence.
• M methionine
• C cysteine
• modifications at the N-terminus of the peptide which are noted by underlining those residues, or in the case of a modification at the N-terminus, underlining the first residue in the peptide sequence.
• Each row of the tables provides the protein of which the peptide is a fragment, by name and abbreviation, as well as a UniProt accession number for the UniProt database (www.uniprot.org) providing the complete protein sequence.
• the amino acid residue of the UniProt complete protein sequence corresponding to the citrullination site shown in the peptide fragment of the table is also provided (i.e., in the first row of Table 3, the R residue at position 4 of the peptide for clusturin (CLU) corresponds to R250 of the amino acid sequence having UniProt Accession No. Pl 0909-2, to R23 of the amino acid sequence having UniProt Accession No. P10909-3, and so on.

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