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WO2025024265A1 - Methods of assessing citrullination and activity of pad4 modulators - Google Patents

Methods of assessing citrullination and activity of pad4 modulators Download PDF

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Publication number
WO2025024265A1
WO2025024265A1 PCT/US2024/038719 US2024038719W WO2025024265A1 WO 2025024265 A1 WO2025024265 A1 WO 2025024265A1 US 2024038719 W US2024038719 W US 2024038719W WO 2025024265 A1 WO2025024265 A1 WO 2025024265A1
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Prior art keywords
citrullination
seq
amino acid
protein
assessing
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French (fr)
Inventor
Rasa Santockyte
Laurence MENARD
Qihong Zhao
Qing Xiao
Mary Adams
Sium HABTE
Tai WANG
Kofi Mensah
Shailesh Prabhakarrao DUDHGAONKAR
Ashok Dongre
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Bristol Myers Squibb Co
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Bristol Myers Squibb Co
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    • 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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Abstract

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

Description

METHODS OF ASSESSING CITRULLINATION AND ACTIVITY OF PAD4 MODULATORS
FIELD
The present application relates to methods involving assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in a biological sample from a subject. The methods may be used for, inter alia, assessing the citrullination modifying activity of agents including PALM (peptidyl arginine deiminase 4) modulators, such as small molecules or antibodies, for example.
BACKGROUND
PALM (peptidyl arginine deiminase 4) is the dominant PAD enzyme found in synovial tissues of subjects with rheumatoid arthritis (RA), and is also associated with other autoimmune diseases, such as lupus, lupus nephritis, vasculitis, thrombosis (e.g., venous thrombosis), inflammatory bowel disease (IBD), and others. 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.
Accordingly, methods for assessing the activity of PAD4 modulators are useful, for example, for monitoring treatment with existing PAD4 modulators and for assessing novel PAD4 modulators in development, among other uses.
SUMMARY
The present disclosure provides methods of assessing citrullination of proteins or peptides, and methods of assessing the activity of 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:
1. A method of determining citrullination of a protein or peptide fragment thereof, comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof in a biological sample from a subject, wherein the biological sample has been exposed to a PAD4 inhibitor, wherein the 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 R748 of PO 1024),
R688 of inter-alpha-trypsin inhibitor heavy chain H4 (corresponding to R688 of
QI 4624),
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).
2. A method of assessing the activity of a PAD4 inhibitor, the method comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample from a subject, wherein the biological sample has been exposed to the PAD4 inhibitor, wherein the 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 R748 of PO 1024),
R688 of inter-alpha-trypsin inhibitor heavy chain H4 (corresponding to R688 of
QI 4624),
R297 of alpha- 1-microglobulin/bikunin precursor (corresponding to R297 of P02760), R715 of alpha-2-macroglobulin (corresponding to R715 of P01023),
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).
3. The method of embodiment 2, wherein the PAD4 inhibitor reduces citrullination at the citrullination site in a dose-dependent manner. 4. The method of embodiment 1, 2, or 3, wherein the method comprises assessing citrullination of a peptide fragment of the protein, wherein the peptide fragment comprises the citrullination site.
5. The method of any one of embodiments 1-4, wherein the biological sample has been exposed to a PAD inhibitor in vivo in a subject as a result of the PAD inhibitor being administered to the subject.
6. The method of any one of embodiments 1-4, wherein the biological sample has been exposed to the PAD inhibitor by contacting the biological sample with the PAD inhibitor ex vivo.
7. The method of any one of embodiments 1-6, wherein the assessing citrullination of the citrullination site comprises mass spectrometry (MS).
8. The method of any one of embodiments 1-6, wherein the assessing citrullination of the citrullination site comprises liquid chromatography and mass spectrometry (LC-MS).
9. The method of any one of embodiments 1-6, wherein the assessing citrullination comprises selective reaction monitoring and chromatographic separation.
10. The method of any one of embodiments 1-9, wherein assessing citrullination of the citrullination site comprises measuring, in the biological sample, a first concentration of the citrullinated protein or peptide fragment thereof and a second concentration of corresponding total protein.
11. The method of embodiment 10, wherein assessing citrullination of the citrullination site comprises determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.
12. The method of embodiment 11, wherein the method comprises comparing the citrullination ratio to a reference citrullination ratio.
13. The method of embodiment 12, wherein the reference citrullination ratio is a citrullination ratio determined for a control biological sample.
14. The method of embodiment 13, wherein the 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.
15. The method of any one of embodiments 12-14, wherein 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.
17. A method comprising:
(i) contacting a biological sample from a subject with exogenous PAD4, and
(ii) assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample; wherein the citrullination site is selected from one or more of the following:
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 R748 of PO 1024),
R688 of inter-alpha-trypsin inhibitor heavy chain H4 (corresponding to R688 of
QI 4624),
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).
18. The method of embodiment 16 or 17, wherein the method comprises incubating the exogenous PALM with the biological sample.
19. The method of embodiment 18, wherein the method comprises incubating the exogenous PALM with the biological sample at a temperature of 36-38 °C.
20. The method of embodiment 18 or 19, wherein the incubating is for 1-3 hours.
21. The method of any one of embodiments 18-20, wherein the method comprises adding EDTA to the biological sample after the incubation with exogenous PALM.
22. The method of any one of embodiments 1-21, wherein the method comprises incubating the biological sample for an incubation period before assessing citrullination.
23. The method of embodiment 22, wherein the incubating is performed at 35-40°C.
24. The method of embodiment 22 or 23, wherein the incubation period is 48 to 96 hours.
25. The method of any one of embodiments 1-24, comprising preparing the biological sample for assessment prior to the assessing. 26. The method of embodiment 25, wherein the preparing comprises enzymatically digesting proteins in the biological sample to form peptide fragments thereof.
27. The method of embodiment 25 or 26, wherein the preparing comprises depleting proteins or peptide fragments thereof from the biological sample that are not targeted by the assessing.
28. The method of any one of embodiments 25-27, wherein the preparing comprises diluting the biological sample.
29. The method of any one of embodiments 25-28, wherein the preparing comprises denaturing proteins or peptide fragments thereof in the biological sample.
30. The method of any one of embodiments 25-29, wherein the preparing comprises enriching the biological sample for a protein or peptide fragment thereof that comprises the citrullination site to be assessed.
31. The method of embodiment 30, wherein the preparing comprises contacting the biological sample with an antibody that specifically binds to citrullinated and noncitrullinated forms of the protein or peptide fragment thereof that comprises the citrullination site to be assessed.
32. The method of any one of embodiments 1-31, wherein the method comprises freezing and thawing the biological sample before assessing citrullination of the citrullination site.
33. The method of any one of embodiments 1-32, comprising:
(a) immunoenriching the sample for the protein or peptide fragment thereof to form an immunoenriched sample, wherein the immunoenriching comprises contacting the sample with an antibody that binds to both citrullinated and noncitrullinated forms of the protein and/or to both citrullinated and noncitrullinated forms of the peptide fragment thereof comprising the citrullination site,
(b) enzymatically digesting proteins in the sample either before or after the immunoenriching to form the peptide fragment, and
(c) assessing citrullination of the peptide fragment at the citrullination site.
34. The method of embodiment 33, wherein the immunoenriching comprises contacting the sample with an immobilized antibody that binds to both citrullinated and noncitrullinated forms of the protein and/or to both citrullinated and noncitrullinated forms of the peptide fragment thereof comprising the citrullination site, and eluting protein and/or peptide bound to the immobilized antibody.
35. The method of embodiment 33 or 34, wherein the enzymatically digesting is conducted before the immunoenriching. 36. The method of embodiment 33 or 34, wherein the enzymatically digesting is conducted after the immunoenriching.
37. The method of any one of embodiments 33-36, wherein the immunoenriching comprises incubating the sample with the immobilized antibody for at least 30 minutes.
38. The method of embodiment 37, wherein the incubating is at a temperature of 22-28°C.
39. The method of embodiment 37 or 38, wherein the incubating is for a period of 30 to 90 minutes.
40. The method of any one of embodiments 33-38, wherein the immunoenriching comprises shaking during the incubating.
41. The method of any one of embodiments 34-40, wherein the immunoenriching comprises removing the immobilized antibody from the sample and washing the immobilized antibody prior to the eluting.
42. The method of any one of embodiments 34-41, wherein the eluting comprises washing the immobilized antibody with an elution composition under acidic conditions.
43. The method of embodiment 42, wherein the elution composition comprises a detergent.
44. The method of embodiment 43, wherein the detergent is a zwitterionic detergent.
45. The method of any one of embodiments 34-44, the method comprises neutralizing eluted protein in a buffer prior to assessing, and optionally prior to enzymatically digesting.
46. The method of any one of embodiments 33-45, wherein the antibody is immobilized by attachment to a solid surface.
47. The method of any one of embodiments 33-46, wherein the assessing citrullination comprises measuring a first concentration of citrullinated peptide in the digested peptides and a second concentration of signature peptide in the digested peptides, and optionally determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.
48. The method of any one of embodiments 33-47, comprising denaturing proteins in the immunoenriched sample prior to the assessing.
49. The method of any one of embodiments 34-48, comprising denaturing the eluted protein prior to the assessing.
50. The method of embodiment 49, comprising denaturing the eluted protein prior to enzymatically digesting the eluted protein. 51. The method of any one of embodiments 33-50, comprising diluting the plasma or serum sample prior to the immunoenriching and performing the immunoenriching on the sample that has been diluted.
52. The method of embodiment 51, comprising diluting the plasma or serum sample by 2 to 1000 fold.
53. The method of embodiment 51 or 52, wherein the sample that has been diluted has a volume of at least 5 pl.
54. The method of any one of embodiments 1-53, wherein the 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).
55. The method of any one of embodiments 1-54, wherein the biological sample comprises whole blood, plasma, serum, or blood supernatant.
56. The method of embodiment 55, wherein the 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.
57. The method of embodiment 56, wherein the 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.
58. The method of any one of embodiments 1-54, wherein the biological sample comprises synovial fluid.
59. The method of embodiment 58, wherein the 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).
60. The method of embodiment 59, wherein the 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).
61. The method of any one of embodiments 1-60, wherein the method comprises assessing citrullination of two or more proteins or peptide fragments thereof.
62. The method of any one of embodiments 1-60, wherein the method further comprises assessing citrullination of at least one additional citrullination site listed in Table A or in any one of Tables 3-18.
63. A method comprising:
(a) immunoenriching a biological 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 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
(b) assessing citrullination in the immunoenriched sample at the citrullination site.
64. A method comprising:
(a) immunoenriching a biological sample for a protein 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 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,
(b) enzymatically digesting the immunoenriched sample to form peptide fragments of the protein of interest, wherein parts (a) and (b) may be performed in any order, and
(c) assessing citrullination of the peptide fragments at the citrullination site.
65. 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,
(b) eluting protein bound to the immobilized antibody,
(d) enzymatically digesting the eluted protein to form peptide fragments thereof, and
(e) assessing citrullination of the peptide fragments at the citrullination site.
66. A method comprising
(a) enzymatically digesting proteins in a biological sample to form peptide fragments thereof,
(b) immunoenriching the enzymatically digested biological sample by contacting the sample with an immobilized antibody that specifically binds to both citrullinated and noncitrullinated forms of a peptide fragment of a protein of interest, the peptide fragment comprising a citrullination site,
(c) eluting peptides bound to the immobilized antibody, and
(d) assessing citrullination of the peptides at the citrullination site.
67. The method of any one of embodiments 63-66, wherein the sample comprises whole blood, plasma, serum, or blood supernatant.
68. A method of assessing the activity of a PAD4 inhibitor, the method comprising
(a) obtaining a biological sample from a subject following treatment of the subject with at least one dose of a PAD4 inhibitor, and
(b) assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the biological sample.
69. An in vitro method of assessing the activity of a PAD4 inhibitor, the method comprising
(a) treating a biological sample from a subject with a PAD4 inhibitor to form a treated biological sample, and 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.
70. An in vitro method of assessing the activity of a PAD4 inhibitor, the method comprising
(i) dividing a biological sample obtained from a subject into a plurality of biological samples,
(ii) contacting each of the plurality of biological samples with a different dose of the PAD4 inhibitor, 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.
71. The method of embodiment 70, further comprising calculating an IC50 for the PAD4 inhibitor based on the outcome of the assessing.
72. The method of any one of embodiments 68 or 70-71, further comprising selecting or adjusting a dose of a PALM inhibitor to be administered to the subject based on the outcome of the assessing.
73. An in vitro method of assessing citrullination by endogenous PALM, the method comprising
(a) incubating a whole blood sample from a subject at 35-40°C for an incubation period,
(b) after the incubation period, separating plasma or supernatant from the whole blood sample, and
(c) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant.
74. An in vitro method of assessing changes in citrullination in a subject, the method comprising
(a) incubating a whole blood sample from a subject at 35-40°C for an incubation period, wherein the sample is obtained from the subject following administration of a PALM inhibitor to the subj ect,
(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.
75. The method of embodiment 74, further comprising
(d) incubating a second whole blood sample from the subject at 35-40°C for an incubation period, wherein the second whole blood sample is obtained from the subject before administration of the PALM inhibitor to the subject,
(e) after the incubation period, separating plasma or supernatant from the second whole blood sample, and
(f) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant from the second whole blood sample.
76. The method of embodiment 74, further comprising comparing an outcome of the assessing in step (f) with an outcome of the assessing in step (c). 77. A method of assessing effects of a PAD4 inhibitor, the method comprising
(a) exposing a whole blood sample from a subject to a PAD4 inhibitor in vitro,
(b) incubating the whole blood sample at 35-40°C for an incubation period,
(c) after the incubation period, separating plasma or supernatant from the whole blood sample, and
(d) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant.
78. The method of embodiment 75, further comprising
(e) incubating a control whole blood sample at 35-40°C for an incubation period,
(f) after the incubation period, separating control plasma or supernatant from the control whole blood sample, and
(g) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the control plasma or supernatant.
79. The method of embodiment 78, further comprising comparing an outcome of the assessing in step (g) with an outcome of the assessing in step (d).
80. 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 35-40°C for an incubation period.
81. The method of embodiment 88, wherein the plasma or supernatant has been frozen and the method comprises thawing the plasma or supernatant prior to the assessing.
82. A method of assessing citrullination at a citrullination site, the method comprising
(i) measuring a first concentration of a citrullinated protein or a citrullinated peptide in a biological sample by mass spectrometry (MS), wherein the citrullinated protein or citrullinated peptide is citrullinated at a citrullination site, and
(ii) measuring a second concentration of corresponding total protein in the biological sample by MS.
83. The method of embodiment 82, wherein the method comprises (i) measuring a first concentration of a citrullinated peptide from a target protein in a biological sample by MS, wherein the citrullinated peptide is citrullinated at a citrullination site, and (ii) measuring the second concentration by measuring the concentration of a signature peptide from the target protein in the biological sample by MS. 84. The method of embodiment 82 or 83, further comprising calculating a ratio of the first concentration to the second concentration
85. The method of any one of embodiments 82-84, wherein the method comprises measuring the first concentration and the second concentration for each of a plurality of different proteins or peptides.
86. The method of embodiment 85, wherein the method comprises measuring the first concentration for each of two different peptides, each of which are nonoverlapping fragments of the same protein and contain different citrullination sites.
87. The method of embodiment 85 or 86, wherein the method comprises calculating a ratio of the first concentration to the second concentration for each of the plurality of different proteins or peptides.
88. The method of any one of embodiments 63-87, wherein the citrullination site a site listed in Table A or in any one of Tables 3-18.
89. The method of any one of embodiments 63-88, wherein the 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 R748 of PO 1024),
R688 of inter-alpha-trypsin inhibitor heavy chain H4 (corresponding to R688 of QI 4624),
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).
90. The method of embodiment 89, wherein 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).
91. The method of any one of embodiments 1-90, wherein the biological sample is obtained from a subject that is ACPA positive.
92 The method of any one of embodiments 1-91, wherein the biological sample is obtained from a subject that is positive for endogenous PAD4 antibodies.
93. The method of embodiment 92, wherein the subject is positive for endogenous PAD4 activating antibodies.
94. The method of any one of embodiments 1-93, wherein the biological sample is obtained from a subject that has been diagnosed with a citrullinati on-related disease or is at risk for developing a citrullination-related disease.
95. The method of any one of embodiments 1-94, wherein the method further comprises selecting the subject from which the biological sample was derived for treatment of a citrullination-related disease based on the outcome of the assessing.
96. The method of embodiment 95, wherein the citrullination-related disease is an autoimmune disorder.
97. The method of embodiment 96, wherein the citrullination-related disease is rheumatoid arthritis, lupus (e.g., systemic lupus erythematosus (SLE)), lupus nephritis, vasculitis (e.g., ANCA-associated vasculitis), thrombosis (e.g, venous thrombosis), or inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn’s disease).
98. The method of any one of embodiments 1-97, wherein the biological sample is obtained from a subject following treatment of the subject with at least one dose of a PAD4 inhibitor.
99. The method of any one of embodiments 1-98, wherein the biological sample is obtained from a subject that is a normal, healthy subject.
100. The method of any one of claims 1-99, wherein the method further comprises administering a PAD4 inhibitor to the subject, and optionally assessing citrullinati on of a citrullinati on site on a protein or peptide fragment thereof in a sample from the subject collected following administering of the PAD4 inhibitor
Additional exemplary embodiments of the disclosure include the following:
1. A method of assessing the activity of a PAD4 modulator, the method comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample from a subject, wherein the biological sample has been exposed to 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 -binding protein, gelsolin, haptoglobin, haptoglobin-related protein, hemopexin, immunoglobulin heavy variable 1-3, immunoglobulin heavy variable 1-8, immunoglobulin heavy variable 1-46, immunoglobulin heavy variable 1-69, immunoglobulin heavy variable 1-69D, inter-alpha-trypsin inhibitor heavy chain H4, immunoglobulin lambda-like polypeptide 1, immunoglobulin heavy constant mu, inter-alpha-trypsin inhibitor heavy chain H2, isoform 2 of complement C4-A, plasminogen, plasminogen-like protein A, alpha- 1-microglobulin/bikunin precursor (protein AMBP), prothrombin, proteoglycan 4, serotransferrin, serum amyloid A-l protein, serum amyloid A-2 protein, serum amyloid A-4 protein, stromelysin- 1, vitamin D-binding protein, and vitronectin.
2. The method of embodiment 1, wherein the PAD4 modulator is a PAD4 inhibitor.
3. The method of embodiment 1 or 2, wherein the citrullination site is selected from one or more citrullination sites shown in Table A or Tables 3-18.
4. The method of embodiment 3, the method comprising assessing citrullination of a peptide fragment, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Tables 3-18.
5. The method of embodiment 1 or 2, wherein the 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), R688 of inter-alpha-trypsin inhibitor heavy chain H4 (corresponding to R688 of QI 4624),
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).
6. The method of any one of embodiments 1 to 4, the method comprising assessing citrullination of a peptide fragment, wherein the 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).
7. The method of any one of embodiments 1 to 6, wherein the biological sample has been exposed to a PAD4 modulator in vivo in a subject as a result of the PAD4 modulator being administered to the subject.
8. The method of any one of embodiments 1 to 7, wherein the biological sample comprises synovial fluid.
9. The method of embodiment 8, wherein the 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.
10. The method of embodiment 8, wherein the citrullination site selected from one or more citrullination sites shown in Table 11 or 12, or wherein the 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).
11. The method of any one of embodiments 1 to 7, wherein the biological sample comprises blood or is derived from blood.
12. The method of any one of embodiments 1 to 7, wherein the biological sample comprises serum. 13. the method of any one of embodiments 1 to 7, wherein the biological sample comprises plasma.
14. The method of any one of embodiments 1 to 11, wherein the biological sample comprises blood supernatant.
15. The method of any one of embodiments 1 to 6 or 8 to 14, wherein the biological sample has been exposed to the PALM modulator by contacting the biological sample with the PALM modulator ex vivo.
16. The method of any one of embodiments 1 to 15, wherein the method comprises freezing and thawing the biological sample before assessing citrullination of the citrullination site.
17. The method of any one of embodiments 1 to 16, wherein assessing citrullination of the citrullination site comprises mass spectrometry (MS).
18. The method of any one of embodiments 1 to 16, wherein assessing citrullination of the citrullination site comprises liquid chromatography and mass spectrometry (LC-MS).
19. The method of any one of embodiments 1-18, comprising preparing the biological sample for assessment prior to the assessing.
20. The method of embodiment 19, wherein the preparing comprises enzymatically digesting proteins in the biological sample.
21. The method of embodiment 19 or 20, wherein the preparing comprises depleting proteins from the biological sample that are not targeted by the assessing.
22. The method of any one of embodiments 19 to 21, wherein the preparing comprises diluting the biological sample.
23. The method of any one of embodiments 19 to 22, wherein the preparing comprises denaturing proteins in the biological sample.
24. The method of any one of embodiments 19 to 23, wherein the preparing comprises enriching the biological sample for a protein or peptide fragment thereof that comprises the citrullination site to be assessed.
25. The method of embodiment 24, wherein the preparing comprises contacting the biological sample with an antibody that specifically binds to citrullinated and noncitrullinated forms of the protein or peptide that comprises the citrullination site to be assessed.
26. The method of any one of embodiments 1 to 25, wherein the method comprises contacting the biological sample with exogenous PALM.
27. The method of embodiment 26, wherein the method comprises incubating the exogenous PALM with the biological sample. 28. The method of embodiment 27, wherein the method comprises incubating the exogenous PAD4 with the biological sample at a temperature of 36-38 °C.
29. The method of embodiment 27 or 28, wherein the incubating is for 1-3 hours.
30. The method of any one of embodiments 27 to 29, wherein the method comprises adding EDTA to the biological sample after the incubation with exogenous PALM.
31. The method of any one of embodiments 1 to 30, wherein the method comprises incubating the biological sample for an incubation period before assessing citrullination.
32. The method of embodiment 31, wherein the incubating is performed at 35-40°C.
33. The method of embodiment 31 or 32, wherein the incubation period is 48 to 96 hours.
34. The method of any one of embodiments 1 to 33, wherein 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.
35. The method of embodiment 34, wherein assessing citrullination of the citrullination site comprises determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.
36. The method of embodiment 35, wherein the method comprises comparing the citrullination ratio to a reference citrullination ratio.
37. The method of embodiment 36, wherein the reference citrullination ratio is a citrullination ratio determined for a control biological sample.
38. The method of embodiment 37, wherein the control biological sample is a biological sample that has not been exposed to the PALM modulator.
39. The method of any one of embodiments 36-38, wherein the assessing comprises determining a difference between the citrullination ratio for the biological sample and the reference citrullination ratio.
40. A method of assessing the activity of a PALM modulator, the method comprising
(a) obtaining a biological sample from a subject following treatment of the subject with at least one dose of a PALM modulator, and
(b) assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the biological sample.
41. An in vitro method of assessing the activity of a PALM modulator, the method comprising
(a) treating a biological sample from a subject with a PALM modulator 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.
42. The method of embodiment 40 or 41, further comprising assessing citrullination of the citrullination site on the protein or peptide fragment in a control biological sample.
43. The method of embodiment 42, further comprising comparing an outcome of the assessment of citrullination in the biological sample with a corresponding outcome of the assessment of citrullination in the control biological sample.
44. The method of any one of embodiments 40-43, wherein the assessing comprises (i) measuring a first concentration of citrullinated protein or peptide in the sample and (ii) measuring a second concentration of corresponding total protein in the sample, wherein the citrullinated protein or peptide is citrullinated at the citrullination site and the corresponding total protein encompasses modified and unmodified forms of the protein, and optionally (iii) determining a citrullination ratio, which is a ratio of the first concentration to the second concentration.
45. The method of embodiment 44, wherein the method comprises comparing the citrullination ratio of the biological sample to the citrullination ratio of a control biological sample.
46. The method of any one of embodiments 42-43 or 45, wherein the control biological sample is from the same subject as the biological sample.
47. The method of embodiment 46, wherein the control biological sample is a biological sample obtained from the subject prior to treatment with a PALM modulator.
48. The method of any one of embodiments 42-43 or 45-46, wherein the control biological sample has not been exposed to the PALM modulator.
49. The method of any one of embodiments 42-43 or 45-46, wherein the control biological sample has been exposed to a different treatment than the biological sample.
50. The method of any one of embodiments 40-49, wherein the PALM modulator is a PALM inhibitor.
51. The method of any one of embodiments 40 or 42-50, further comprising selecting or adjusting a dose of the PALM modulator based on the outcome of the assessing.
52. An in vitro method of assessing the activity of a PALM modulator, the method comprising
(i) dividing a biological sample obtained from a subject into a plurality of biological samples,
(ii) contacting each of the plurality of biological samples with a different dose of the PALM modulator, 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.
53. The method of embodiment 52, wherein the PAD4 modulator is a PAD4 inhibitor.
54. The method of embodiment 52 or 53, further comprising calculating an IC50 for the PALM modulator based on the outcome of the assessing.
55. The method of any one of embodiments 52-54, further comprising selecting or adjusting a dose of a PALM modulator to be administered to the subject based on the outcome of the assessing.
56. An in vitro method of assessing citrullination by endogenous PALM, the method comprising
(a) incubating a whole blood sample from a subject at 35-40°C for an incubation period,
(b) after the incubation period, separating plasma or supernatant from the whole blood sample, and
(c) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant.
57. The method of embodiment 56, wherein the incubation period is 48 to 96 hours.
58. The method of any one of embodiments 56 or 57, wherein the incubating is at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C.
59. The method of any one of embodiments 56-58, comprising enzymatically digesting polypeptides in the plasma or supernatant before the assessing.
60. The method of any one of embodiments 56-59, further comprising (i) incubating the plasma or supernatant with a protein depletion resin, and (ii) recovering depleted plasma or supernatant that has flowed through the resin to obtain depleted flowthrough, optionally wherein the recovering comprises centrifugation.
61. The method of embodiment 60, comprising enzymatically digesting polypeptides in the depleted flowthrough before the assessing.
62. The method of embodiment 60 or 61, further comprising cleaning up enzymatically digested plasma or supernatant before the assessing.
63. The method of any one of embodiments 56-62, wherein the whole blood sample and the plasma or supernatant separated therefrom have not been frozen and thawed. 64. The method of any one of embodiments 56-62, wherein the whole blood sample and/or the plasma or supernatant separated therefrom has been frozen and thawed.
65. The method of embodiment 64, wherein the method comprises freezing the plasma or supernatant after the separating and subsequently thawing the plasma or supernatant before the assessing.
66. The method of any one of embodiments 56-65, wherein the whole blood sample has been exposed to a PALM modulator.
67. The method of embodiment 66, wherein the whole blood sample has been exposed to a PALM modulator in vitro.
68. The method of embodiment 66, wherein the whole blood sample has been exposed to a PALM modulator in vivo in the subject as a result of the PALM modulator being administered to the subj ect.
69. An in vitro method of assessing changes in citrullination in a subject, the method comprising
(a) incubating a whole blood sample from a subject at 35-40°C for an incubation period, wherein the sample is obtained from the subject following administration of a 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.
70. The method of embodiment 69, further comprising
(d) incubating a second whole blood sample from the subject at 35-40°C for an incubation period, wherein the second whole blood sample is obtained from the subject before administration of the PALM modulator to the subject,
(e) after the incubation period, separating plasma or supernatant from the second whole blood sample, and
(f) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant from the second whole blood sample.
71. The method of embodiment 70, further comprising comparing an outcome of the assessing in step (f) with an outcome of the assessing in step (c).
72. A method of assessing effects of a PALM modulator, the method comprising
(a) exposing a whole blood sample from a subject to a PALM modulator in vitro, (b) incubating the whole blood sample at 35-40°C for an incubation period,
(c) after the incubation period, separating plasma or supernatant from the whole blood sample, and
(d) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant.
73. The method of embodiment 72, further comprising
(e) incubating a control whole blood sample at 35-40°C for an incubation period,
(f) after the incubation period, separating control plasma or supernatant from the control whole blood sample, and
(g) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the control plasma or supernatant.
74. The method of embodiment 73, further comprising comparing an outcome of the assessing in step (g) with an outcome of the assessing in step (d).
75. 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 35-40°C for an incubation period.
76. The method of embodiment 75, wherein the plasma or supernatant has been frozen and the method comprises thawing the plasma or supernatant prior to the assessing.
77. A method comprising:
(a) 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
(b) assessing citrullination in the immunoenriched sample at the citrullination site, optionally wherein the sample is a serum or plasma sample.
78. A method comprising:
(a) 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
(c) assessing citrullination of the digested peptides at the citrullination site, optionally wherein the sample is a serum or plasma sample.
79. A method comprising
(a) immunoenriching a sample by contacting the sample with an immobilized antibody that binds to both citrullinated and noncitrullinated forms of a protein of interest, the protein of interest comprising a citrullination site,
(b) eluting protein bound to the immobilized antibody,
(d) enzymatically digesting the eluted protein to form digested peptides, and
(e) assessing citrullination of the digested peptides at the citrullination site, optionally wherein the sample is a serum or plasma sample.
80. The method of embodiment 77, 78, or 79, wherein the immunoenriching comprises incubating the sample with the immobilized antibody for at least 30 minutes.
81. The method of embodiment 80, wherein the incubating is at a temperature of 22-28°C.
82. The method of embodiment 80-81, wherein the incubating is for a period of 30 to 90 minutes.
83. The method of any one of embodiments 77-82, wherein the immunoenriching comprises shaking during the incubating.
84. The method of any one of embodiments 77-83, wherein the immunoenriching comprises removing the immobilized antibody from the sample and washing the immobilized antibody prior to the eluting.
85. The method of any one of embodiments 79-84, wherein the eluting comprises washing the immobilized antibody with an elution composition under acidic conditions.
86. The method of embodiment 85, wherein the elution composition comprises a detergent.
87. The method of embodiment 86, wherein the detergent is a zwitterionic detergent.
88. The method of any one of embodiments 85-87, the method comprises neutralizing eluted protein in a buffer prior to assessing, and optionally prior to enzymatically digesting.
89. The method of any one of embodiments 77-88, wherein the antibody is immobilized by attachment to a solid surface.
90. The method of any one of embodiments 77-89, wherein the assessing citrullination comprises measuring a first concentration of citrullinated peptide in the digested peptides and a second concentration of signature peptide in the digested peptides, and optionally determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.
91. The method of any one of embodiments 77-90, comprising denaturing proteins in the immunoenriched sample prior to the assessing.
92. The method of any one of embodiments 79-90, comprising denaturing the eluted protein prior to the assessing.
93. The method of embodiment 92, comprising denaturing the eluted protein prior to enzymatically digesting the eluted protein.
94. The method of any one of embodiments 77-93, comprising diluting the sample prior to the immunoenriching and performing the immunoenriching on the sample that has been diluted.
95. The method of embodiment 94, comprising diluting the sample by 2 to 1000 fold.
96. The method of embodiment 94 or 95, wherein the sample that has been diluted has a volume of at least 5 pl.
97. The method of any one of embodiments 40 to 96, wherein the assessing comprises mass spectrometry (MS).
98. The method of embodiment 97, wherein the assessing comprises liquid chromatography and mass spectrometry (LC-MS).
99. The method of embodiment 98, wherein the assessing comprises selective reaction monitoring and chromatographic separation.
100. The method of any one of embodiments 77-99, wherein the method comprises assessing citrullination of two or more proteins of interest and the antibody comprises antibodies that specifically bind citrullinated and noncitrullinated forms of each of the two or more proteins of interest.
101. A method comprising
(i) contacting a biological sample from a subject with exogenous PALM and
(ii) assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample.
102. The method of embodiment 101, wherein the biological sample comprises plasma or serum.
103. The method of embodiment 101 or 102, wherein the method comprises preparing the biological sample for assessment prior to the assessing. 104. The method of embodiment 103, wherein the preparing comprises enzymatically digesting proteins in the biological sample to produce digested peptides and optionally cleaning up the digested peptides.
105. The method of embodiment 103 or 104, wherein the preparing comprises depleting proteins from the biological sample that are not targeted by the assessing.
106. The method of any one of embodiment 105, wherein depleting comprises depletion of human serum albumin.
107. The method of any one of embodiments 101-106, wherein the preparing comprises immunoenriching the biological sample for the protein or peptide fragment thereof using an antibody that binds to citrullinated and non-citrullinated forms of the protein or peptide.
108. The method of any one of embodiments 40 to 107, wherein the protein or peptide fragment thereof or the protein of interest 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 heavy variable 1-69, immunoglobulin heavy variable 1-69D, inter-alpha-trypsin inhibitor heavy chain H4, immunoglobulin lambda-like polypeptide 1, immunoglobulin heavy constant mu, inter-alpha-trypsin inhibitor heavy chain H2, isoform 2 of complement C4-A, plasminogen, plasminogen-like protein A, alpha- 1-microglobulin/bikunin precursor (protein AMBP), prothrombin, proteoglycan 4, serotransferrin, serum amyloid A-l protein, serum amyloid A-2 protein, serum amyloid A-4 protein, stromelysin- 1, vitamin D-binding protein, and vitronectin.
109. The method of any one of embodiments 40-107, wherein the citrullination site is selected from one or more citrullination sites shown in Table A or in one or more of Tables 3- 18.
111. The method of any one of embodiments 40-107, wherein the 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),
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), 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).
112. The method of any one of embodiments 40-107, the method comprising assessing citrullination of a peptide fragment, wherein the 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).
113. The method of any one of embodiments 40-107, wherein the protein or peptide fragment thereof or the protein of interest comprises fibrinogen alpha chain or a peptide fragment thereof and/or gelsolin or a peptide fragment thereof.
114. The method of embodiment 113, wherein the protein or peptide fragment thereof or the protein of interest is fibrinogen alpha chain or a peptide fragment thereof.
115. The method of embodiment 114, wherein the citrullination site is at R591 of P02671 or R593 ofP02671.
116. The method of embodiment 114 or 115, wherein the method comprises (a) assessing citrullination of a peptide comprising the amino acid sequence of QFTSSTSYNRGDSTFESK (SEQ ID NO: 249), wherein the underlined R is the citrullination site or (b) assessing citrullination of a peptide comprising the amino acid sequence of ESSSHHPGIAEFPSRGK (SEQ ID NO: 248), wherein the underlined R is the citrullination site.
117. The method of any one of embodiments 40-107, wherein the protein or peptide fragment thereof or the protein of interest comprises gelsolin or a peptide fragment thereof.
118. The method of embodiment 117, wherein the citrullination site is at R32 of P06396.
119. The method of embodiment 117 or 118, wherein the method comprises assessing citrullination of a peptide comprising the amino acid sequence of ATASRGASQAGAPQGR (SEQ ID NO: 255), wherein the underlined R is the citrullination site.
120. A method of assessing citrullination at a citrullination site, the method comprising
(i) measuring a first concentration of a citrullinated protein or a citrullinated peptide in a biological sample by mass spectrometry (MS), wherein the citrullinated protein or citrullinated peptide is citrullinated at a citrullination site, and
(ii) measuring a second concentration of corresponding total protein in the biological sample by MS.
121. The method of embodiment 120, wherein the method comprises (i) measuring a first concentration of a citrullinated peptide from a target protein in a biological sample by MS, wherein the citrullinated peptide is citrullinated at a citrullination site, and (ii) measuring the second concentration by measuring the concentration of a signature peptide from the target protein in the biological sample by MS.
122. The method of embodiment 120 or 121, further comprising calculating a ratio of the first concentration to the second concentration
123. The method of any one of embodiments 120 to 122, wherein the method comprises measuring the first concentration and the second concentration for each of a plurality of different proteins or peptides.
124. The method of embodiment 123, wherein the method comprises measuring the first concentration for each of two different peptides, each of which are nonoverlapping fragments of the same protein and contain different citrullination sites.
125. The method of embodiment 123 or 124, wherein the method comprises calculating a ratio of the first concentration to the second concentration for each of the plurality of different proteins or peptides.
126. The method of any one of embodiments 120 to 125, wherein the citrullination site is a site listed in Table A or in one or more of Tables 3-18. 127. The method of any one of embodiments 120 to 126, wherein (i) the 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, haptoglobin-related protein, hemopexin, immunoglobulin heavy variable 1-3, immunoglobulin heavy variable 1-8, immunoglobulin heavy variable 1-46, immunoglobulin heavy variable 1-69, immunoglobulin heavy variable 1-69D, inter-alphatrypsin inhibitor heavy chain H4, immunoglobulin lambda-like polypeptide 1, immunoglobulin heavy constant mu, inter-alpha-trypsin inhibitor heavy chain H2, isoform 2 of complement C4-A, plasminogen, plasminogen-like protein A, alpha- 1- microglobulin/bikunin precursor, prothrombin, proteoglycan 4, serotransferrin, serum amyloid A-l protein, serum amyloid A-2 protein, serum amyloid A-4 protein, stromelysin- 1, vitamin D-binding protein, and vitronectin; or (ii) the citrullinated peptide is a peptide fragment of a protein listed in (i).
128. The method of any one of embodiments 120 to 127, wherein the citrullinated peptide comprises a sequence selected from the peptide sequences listed in Tables 3-18.
129. The method of any one of embodiments 120 to 128, wherein (i) the 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).
130. The method of any one of embodiments 120 to 129, wherein the citrullination site is selected from
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),
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).
131. The method of any one of embodiments 120 to 130, wherein the method comprises measuring the amount of 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 wherein the underlined R is the citrullination site.
132. The method of any one of embodiments 120-131, wherein the citrullinated protein or citrullinated peptide comprises fibrinogen alpha chain and/or gelsolin or a peptide fragment thereof.
133. The method of embodiment 132, wherein the citrullinated protein or citrullinated peptide is fibrinogen alpha chain or a peptide fragment thereof.
134. The method of embodiment 133, wherein the citrullination site is at R591 of P02671 or R593 ofP02671.
135. The method of embodiment 133 or 134, wherein the 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.
136. The method of embodiment 132, wherein the citrullinated protein or citrullinated peptide comprises gelsolin or a peptide fragment thereof.
137. The method of embodiment 136, wherein the citrullination site is at R32 of P06396.
138. The method of embodiment 136 or 137, wherein the citrullinated peptide comprises the amino acid sequence of ATASRGASQAGAPQGR (SEQ ID NO: 255), wherein the underlined R is the citrullination site. 139. The method of any one of embodiments 120-138, wherein the biological sample is obtained from a subject.
140. The method of embodiment 139, wherein the biological sample is a whole blood, plasma, serum, blood supernatant, or synovial fluid sample.
141. The method of embodiment 139 or 140, wherein the method further comprises comparing the first and second concentrations to first and second concentrations obtained from a control biological sample.
142. The method of embodiment 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.
143. The method of embodiment 142, wherein the method further comprises selecting the subject from which the biological sample was derived for treatment of a citrullination-related disease.
144. The method of embodiment 143, wherein the PALM modulator is a PALM inhibitor.
145. The method of any one of embodiments 1-144, wherein the biological sample is obtained from a subject that is ACPA positive.
146. The method of any one of embodiments 1-145, wherein the biological sample is obtained from a subject that is positive for endogenous PALM antibodies.
147. The method of embodiment 146, wherein the subject is positive for endogenous PALM activating antibodies.
148. The method of any one of embodiments 1-147, wherein the biological sample is obtained from a subject that has been diagnosed with a citrullination-related disease or is at risk for developing a citrullination-related disease.
149. The method of embodiment 148, wherein the citrullination-related disease is an autoimmune disorder.
150. The method of embodiment 148, wherein the citrullination-related disease is rheumatoid arthritis, lupus (e.g., systemic lupus erythematosus (SLE)), lupus nephritis, vasculitis (e.g., ANCA-associated vasculitis), thrombosis (e.g, venous thrombosis), or inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn’s disease).
151. The method of any one of embodiments 1-144, wherein the biological sample is obtained from a subject that is a normal, healthy subject. 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. Thus, in the above such methods, a PAD4 modulator may be substituted with such another therapeutic agent. In any of the above embodiments, where a PAD4 modulator is included in the method, the PAD4 modulator may, in some cases, be a PAD4 inhibitor, examples of which are provided throughout the disclosure.
In any of the above embodiments, in some cases the assessing of citrullination may be performed by MS, including by LC-MS, for example. In any of the above embodiments, unless particular citrullination sites or proteins or peptides are recited, citrullination sites for assessment may be as recited, for instance, in embodiments above. In any of the methods herein, unless stated otherwise 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. As also indicated above, in any of the methods herein, a citrullination ratio, as described below, and/or a concentration of citrullinated protein or peptide and a concentration of corresponding total protein may be determined as part of the method. In addition, in any of the methods, as indicated above, a biological sample may be prepared prior to assessing, in a variety of ways, such as dilution, freezing and thawing, denaturing of proteins, enzymatic digestion of proteins, separation of a supernatant from the sample for analysis, and the like. The above embodiments may also be applied to control samples as well as to biological samples of interest, for instance, to allow for comparisons of citrullination assessments between samples of interest and controls.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims. References cited herein are incorporated by reference in their entirety, and the amino acid sequences of proteins obtainable at www.uniprot.org through their UniProt accession numbers provided herein are also incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 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. FIG. 3D and FIG. 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. FIG. 4D and FIG. 4E respectively show the effects of clone 13 -based hzl3-12 antibody and clone 13 -based hzl3-5 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. 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. 6A-6C show efficacy of anti-human PAD4 antibodies in an LPS AJI PD study. The effects of clone 13 -based hzl3-12 antibody (FIG. 6 A), clone 13 -based hzl3-5 antibody (FIG. 6B), and clone 20-based hz20-2 antibody (FIG. 6C) at the indicated doses on extracellular citrullinated ITIH4 relative to total extracellular ITIH4 are shown; the percent reductions indicate reductions relative to the isotype control condition. “Mpk” = mg/kg. “IC” = Isotype Control antibody. “Naive”= not injected with pristane. “PBS” = naive, injected with phosphate buffered saline in place of LPS.
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-9D show induction of citrullination and inhibition of citrullination ex vivo in incubated blood. Whole blood samples were treated with Ab hzl3-5 D3 IE, PBS (control), or isotype control and incubated for 48 h at 37°C for induction of citrullination. PBS-Oh shows citrullination levels before induction. Samples were analyzed using global proteomics. Citrullination of the peptides listed in Table 10 was assessed. The average % citrullination for each peptide, calculated as the average ratio of citrullinated peptide intensity to total protein intensity *100%, wherein the total protein intensity was measured by measuring the intensity of a signature peptide, is shown for 6 donors. Black bar: 1st blood draw; gray bar: repeat blood draw 1 week later. 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. Figure discloses SEQ ID NO: 408.
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 1st draw) was confirmed using a targeted proteomic approach. Whole blood samples were treated with Ab hzl3-5 D31E, PBS (control), or isotype control and incubated for 48 h at 37°C. PBS-Oh shows citrullination levels before induction. Samples were analyzed with targeted proteomics. Citrullination of GSN and FGA peptides, sequences of which are provided in Table 16, was assessed. The average % citrullination was calculated as described for FIGs. 9A-9D (N=6 donors). Error bars are standard errors of the mean. 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.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
I. Definitions
Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
In this application, the use of “or” means “and/or” unless stated otherwise. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim in the alternative only. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.
Exemplary techniques used in connection with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), enzymatic reactions, and purification techniques are described, e.g., in Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), among other places.
As used herein, the term “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values (e.g., +/-5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.
The terms “subject” and “patient” are used interchangeably herein to refer to a human unless expressly indicated otherwise (i.e., a murine subject or the like). A “biological sample” herein refers to a sample taken from a biological source, such as from a subject. A “biological sample” may comprise fluid and/or tissue, and thus it may be a “biological fluid sample” or a “tissue sample,” and it may be processed, such as centrifuged or mixed with other agents or the like, in order to facilitate analysis. For example, certain biological fluid samples may comprise supernatant following separation of particulate and fluid matter. In some cases, the biological sample comprises, for example, blood or blood components such as whole blood, serum, plasma, or blood supernatant. In some cases, it may comprise synovial fluid. In other cases, the biological sample may be a tissue sample, for instance, a tumor sample or a neoplasia sample.
The term “polypeptide” refers to a polymer of amino acid residues, and is not limited to a minimum length. A “protein” may comprise one or more polypeptides. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a “polypeptide” or “protein” refers to a polypeptide or protein, respectively, which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site- directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins or errors due to PCR amplification. A protein may comprise two or more polypeptides.
The term “peptide fragment” or “peptide” of a longer polypeptide or protein (for example, a “protein or peptide fragment thereof’) refers to a portion of the polypeptide or protein. In some cases, analysis of a chemical modification of an amino acid residue in a polypeptide, for example, may be performed by analyzing a peptide fragment thereof that includes the amino acid residue in question.
Some amino acids within a protein or its peptide fragment may be “citrullinated.” As used herein, this term means that the protein or its peptide fragment contains at least one amino acid that is converted from one of the 20 naturally occurring amino acids to a citrulline amino acid. For example, certain arginine (Arg, R) residues may be chemically modified to convert their side chains to the side chain of the amino acid citrulline (Cit). Thus, such arginine residues, and the corresponding peptides or proteins on which they are found are referred to as being citrullinated.
The amino acid residue that is converted to citrulline may also be referred to herein as a “citrullination site.” For example, a residue at a particular amino acid position within a protein may be citrullinated. In such a case, that particular amino acid position may be referred to as a “citrullination site.” A citrullination site herein may in some cases be identified with reference to an exemplary, reference amino acid sequence for the protein. In the case where a particular protein has more than one naturally occurring amino acid sequence, one such sequence may be provided herein as a reference sequence for identifying the residue corresponding to a citrullination site, and that site may be identified in other naturally occurring variants of the protein through sequence alignment with the reference sequence provided herein. A given protein may also have one or more such citrullination sites when more than one residue in the protein is found to be citrullinated.
A “PAD” or “protein arginine deiminase” refers to an enzyme that catalyzes the conversion of arginine to citrulline in a protein or peptide under certain conditions. Examples of PADs include PAD4 and PAD2, for example.
“PAD4” or “protein arginine deiminase 4” or “peptidyl arginine deiminase 4,” as used herein, refers to human PAD4 (huPAD4; UniProt ID: Q9UM07), unless expressly noted otherwise (i.e., murine PAD4, cynomolgus PAD4, or the like). Exemplary human PAD4 amino acid sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 and SEQ ID NO: 3.
As used herein, a “PAD4 modulator” refers to a compound (e.g, a small or large molecule or biologic) that alters (i.e., modulates) at least one activity or function of PAD4. In some cases, a “PAD4 modulator” is a “PAD4 inhibitor,” which is a compound that reduces at least one activity of PAD4, i.e., a PAD4 antagonist. In other cases, a “PAD4 modulator” is a compound that increases the activity of PAD4, i.e., a PAD4 agonist. The modulation of the activity of PAD4 may be by any mechanism and may be observed in vitro and/or in vivo. In some cases, a PAD4 modulator may modulate the activity of additional proteins in addition to PAD4, such as, for example, additional PAD enzymes, such as PAD2; while in other cases, the activity modulation may be specific to PAD4 and the PAD4 modulator does not modulate the activity of other proteins such as, for example, other PAD enzymes such as PAD2.
The term “agonist” as used herein refers to a compound that causes an increase in at least one activity or function of a molecule to which it binds, or otherwise activates or helps to activate the molecule. The term “antagonist” or “inhibitor” as used herein refers to a compound that causes a decrease in at least one activity or function of a molecule to which it binds, or that otherwise blocks or inhibits at least one activity or function of the molecule.
The terms “inhibition” or “inhibit” more generally refer to a decrease or cessation of any event (such as protein ligand binding) or to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and/or amount as compared to a reference. It is not necessary that the inhibition or reduction be complete. For example, in certain embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20% or greater. In another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater. In yet another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.
In some cases, a 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. In some aspects, 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. For a review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).
An “antigen” refers to the target of an antibody, i.e., the molecule to which the antibody specifically binds. The term “epitope” denotes the site on an antigen, either proteinaceous or non-proteinaceous, to which an antibody binds. Epitopes on a protein can be formed both from contiguous amino acid stretches (linear epitope) or comprise noncontiguous amino acids (conformational epitope), e.g., coming in spatial proximity due to the folding of the antigen, i.e., by the tertiary folding of a proteinaceous antigen. Linear epitopes are typically still bound by an antibody after exposure of the proteinaceous antigen to denaturing agents, whereas conformational epitopes are typically destroyed upon treatment with denaturing agents.
The term “heavy chain” refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term “full-length heavy chain” refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.
The term “light chain” refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term “full-length light chain” refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.
The term “complementarity determining regions” (“CDRs”) as used herein refers to each of the regions of an antibody variable region which are hypervariable in sequence and which determine antigen binding specificity. Generally, antibodies comprise six CDRs: three in the VH (CDR-H1 or heavy chain CDR1, CDR-H2, CDR-H3), and three in the VL (CDR- Ll, CDR-L2, CDR-L3). Unless otherwise indicated, the CDRs are determined according to the sequence table herein.
“Framework” or “FR” refers to the residues of the variable region residues that are not part of the complementary determining regions (CDRs). The FR of a variable region generally consists of four FRs: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR- L1)-FR2- CDR-H2(CDR-L2)-FR3- CDR-H3(CDR-L3)-FR4.
The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A variable domain may comprise heavy chain (HC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and/or FR4; and light chain (LC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and/or FR4. That is, a variable domain may lack a portion of FR1 and/or FR4 so long as it retains antigen-binding activity. A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150 :880-887 (1993) ; Clarkson et al., Nature 352 :624-628 (1991).
The light chain and heavy chain “constant regions” of an antibody refer to additional sequence portions outside of the FRs and CDRs and variable regions. Certain antibody fragments may lack all or some of the constant regions. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant heavy domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain.
The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, 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. Therefore, 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. Thus, a “full-length heavy chain constant region” or a “full length antibody” for example, which is a human IgGl antibody, includes an IgGl with both a C-terminal glycine and lysine, without the C-terminal lysine, or without both the C-terminal glycine and lysine. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
“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. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. 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.
An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen (i.e., PAD4) to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005).
The terms “full length antibody”, “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or, in the case of an IgG antibody, having heavy chains that contain an Fc region as defined herein above.
The term “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. In certain aspects, 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. In contrast to polyclonal antibody preparations, which 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. Thus, 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.
The term “signal sequence” or “leader sequence” refers to a sequence of amino acid residues located at the N terminus of a polypeptide that facilitates secretion of a polypeptide from a mammalian cell. A leader sequence may be cleaved upon export of the polypeptide from the mammalian cell, forming a mature protein. Leader sequences may be natural or synthetic, and they may be heterologous or homologous to the protein to which they are attached. Nonlimiting exemplary leader sequences also include leader sequences from heterologous proteins. In some embodiments, an antibody lacks a leader sequence. In some embodiments, an antibody comprises at least one leader sequence, which may be selected from native antibody leader sequences and heterologous leader sequences.
The term “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. Often, 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’. Herein, the term 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. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non- naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. 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. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can be unmodified or modified.
An “isolated” 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. The term “vector”, as used herein, 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”.
The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
In this disclosure, “binds” or “binding” or “specific binding” and similar terms, when referring to a protein and its ligand or an antibody and its antigen target for example, or some other binding pair, means that the binding affinity between the members of the binding pair is sufficiently strong that the interaction cannot be due to random molecular associations (i.e. “nonspecific binding”). Such binding typically requires a dissociation constant (KD) of IpM or less, and may often involve a KD of 100 nM or less.
“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). Affinity can generally be represented by the dissociation constant (KD). Affinity of an antibody for an antigen can be measured by common methods known in the art, such as surface plasmon resonance (SPR), for instance.
“Treatment” as used herein, covers any administration or application of a therapeutic for disease in a human, and includes inhibiting the disease or progression of the disease or one or more disease symptoms, inhibiting or slowing the disease or its progression or one or more of its symptoms, arresting its development, partially or fully relieving the disease or one or more of its symptoms, or preventing a recurrence of one or more symptoms of the disease.
An “autoimmune disease” or “autoimmune disorder,” as used herein, encompasses a disease characterized by the subject’s immune system attacking its own normal cells and tissues, and also encompasses immune-mediated diseases which may or may not be characterized by presence of auto-antibodies. The disclosure provides many nonlimiting examples of autoimmune diseases throughout. Some nonlimiting examples of autoimmune diseases include rheumatoid arthritis (RA), lupus (e.g., systemic lupus erythematosus (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). The terms “disease” and “disorder” are used interchangeably herein. In some embodiments, the autoimmune disease is characterized by the presence of auto-antibodies. The term “effective amount” or “therapeutically effective amount” refers to an amount of a drug effective for treatment of a disease or disorder in a subject, such as to partially or fully relieve one or more symptoms. In some embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
Additional definitions of terms or phrases used herein are included in the sections that follow.
II. Methods of Assessing Citrullination
The present disclosure encompasses several methods of assessing the citrullination 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. In some cases, the peptides are peptide fragments of proteins, for instance, digested peptides that arise from enzymatic digestion of proteins. Thus, the expression “assessing citrullination of a citrullination site” encompasses assessing one citrullination site on one protein or peptide, as well as assessing more than one citrullination site on one protein or peptide, as well as assessing multiple citrullination sites. And accordingly, while the disclosure herein may recite a citrullination site on a protein or peptide, the “a” in this expression indicates “one or more” citrullination sites, and citrullination of more than one protein or peptide may also be assessed. Assessing multiple citrullination sites can encompass assessing citrullination sites on a plurality of proteins or on a plurality of peptides. For instance, the plurality of citrullination sites may be a plurality of citrullination sites on one protein or on peptide fragments of such protein, and/or the plurality of citrullination sites may be a plurality of citrullination sites on a plurality of different proteins or peptides. Although the methods of assessing citrullination disclosed herein may be used to assess citrullination at any citrullination site, in some embodiments, the citrullination site (e.g., the plurality of citrullination sites) is a citrullination site disclosed herein. Any and all such combinations are contemplated herein. Among the methods herein include, inter alia, a method of assessing citrullination at a citrullination site by mass spectrometry (MS). In some cases, the methods comprise assessment by liquid chromatography followed by MS (LC-MS). For example, liquid chromatography (e.g., HPLC, RPLC, nLC, pLC, or the like) may be used to separate peptides or proteins for analysis by MS. In some cases, the method comprises measuring, in a biological sample: (i) a first concentration of a citrullinated protein or a citrullinated peptide (which may be from a protein, e.g., a protein that has been enzymatically digested), wherein the citrullinated protein or citrullinated peptide is citrullinated at a citrullination site, and (ii) a second concentration of the corresponding total protein (which includes modified and unmodified forms of the protein) in the sample. In some embodiments, the concentration of corresponding total protein is measured by measuring a concentration of a signature peptide from the protein. The signature peptide is a peptide that is present both in modified and unmodified forms of the protein, and the signature peptide is a peptide that is not itself modified. Accordingly, when the concentration of signature peptide present in a sample within a well is measured by mass spectrometry, for example, the concentration of the signature peptide represents the well total concentration of the protein. In some embodiments, the method of measuring concentrations (i) and (ii) is by MS, such as LC-MS. In some embodiments, the method further comprises calculating a citrullination ratio. A “citrullination ratio,” as used herein, refers to a ratio of the first concentration (i) to the second concentration (ii). A citrullination ratio may be expressed in a variety of ways, for instance, as a percentage, or as any other fraction that is proportional to the aforementioned ratio of the first concentration to the second concentration.
In some cases, the method comprises assessing citrullination of more than one citrullination site. Accordingly, the method can comprise measuring the first concentration and the second concentration for each of a plurality of (meaning two or more) different proteins or peptides that each contain a citrullination site. In some such cases, the method comprises measuring the first concentration and the second concentration for each of the plurality of proteins or peptides. In some such cases, at least two different peptides of the plurality are nonoverlapping fragments of the same protein and contain different citrullination sites; in those cases, the second concentration is the same for the peptides that are fragments of the same protein. In some cases, the method comprises calculating a ratio of the first concentration to the second concentration for each of the plurality of different proteins or peptides. Exemplary citrullination sites, proteins, and peptide fragments thereof are discussed below and provided in Table A and Tables 3-18 herein. In some cases, the biological sample is obtained from a subject. In some cases, the biological sample is a whole blood, plasma, serum, blood supernatant, or synovial fluid sample. In other cases, the biological sample is a tissue sample. In some cases, the method further comprises comparing the first and second concentrations, or a ratio of the first and second concentrations to first and second concentrations or a ratio of first and second concentrations obtained from a control.
In some cases where citrullination of a biological sample from a subject is assessed herein, the method further comprises determining one or more of the following based on the first and second concentrations or a ratio of the first and second concentrations: probability of a clinical outcome, risk for developing a citrullination-related disease, diagnosis of a citrullinati on-related disease, and selecting the subject from which the biological sample was derived for a treatment of a citrullination-related disease. For instance, in some cases, the subject either has a citrullination-related disease or may be at risk of developing a citrullination-related disease, and an assessment of citrullination in a biological sample, according to methods herein, may be used to confirm a diagnosis of such a disease, or to determine whether or not the subject is at risk of developing such a disease. In some cases, the method may allow for monitoring of citrullination in a subject’s biological samples, in order to determine a probability of clinical outcome, for example, based on comparison to reference subjects whose clinical outcomes are known, or to determine whether a 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. Thus, for instance, in one example, the method further comprises determining, based on the first and second concentrations or a ratio of the first and second concentrations, whether the subject should receive treatment, for instance, in some cases, for a citrullination-related disease. In some cases, the method further comprises determining, based on the first and second concentrations or a ratio of the first and second concentrations, whether the subject should receive treatment with a therapeutic agent. Nonlimiting examples of such an agent include those that may be used for treatment of citrullination-related diseases, and those that target a biological pathway, such as, for example, NETosis or METosis, which may also involve PALM, such as, for example, an anti-histone antibody (e.g., CIT-013, for example), as well as PALM modulators, such as PALM inhibitors. In other cases, the method comprises determining whether a subject already receiving treatment with a therapeutic agent or other therapy should have the treatment adjusted, such as to increase or decrease a dose of a therapeutic agent being administered, or to start treatment with an agent such as a PAD4 modulator or an agent that targets a molecule involved in a biological pathway that involves PAD4. In some cases, 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. In some cases, the PAD4 modulator is a PAD4 inhibitor. Exemplary PAD4 modulators are provided in this disclosure, and include, for example, therapeutic agents that can 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. Such therapeutic agents may, in some cases, comprise large molecules such as antibodies or fusion proteins, or may comprise small molecules. In some embodiments, a method of assessing the activity of a therapeutic agent, such as a PAD4 modulator, comprises assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample from a subject. In such methods, the biological sample has been exposed to a 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. In some cases, the therapeutic agent being assessed is a PAD4 modulator. In some cases, the PAD4 modulator is a PAD4 inhibitor.
In any of the methods described herein, in some embodiments, at least two different citrullination sites are assessed. In some cases, at least three different sites are assessed. In some cases, at least four different sites are assessed. In some cases, citrullination of at least two different proteins is assessed. In some cases, citrullination of at least three different proteins is assessed. In some cases, citrullination of at least four different proteins is assessed. In some cases, citrullination of 2-4 citrullination sites and/or 2-4 different proteins is assessed.
In any of the methods herein, in some cases the protein comprising the citrullination site for assessment is selected from one or more of: 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 heavy variable 1-69, immunoglobulin heavy variable 1-69D, inter-alpha-trypsin inhibitor heavy chain H4, immunoglobulin lambdalike polypeptide 1, immunoglobulin heavy constant mu, inter-alpha-trypsin inhibitor heavy chain H2, isoform 2 of complement C4-A, plasminogen, plasminogen-like protein A, alpha- 1-microglobulin/bikunin precursor, prothrombin, proteoglycan 4, serotransferrin, serum amyloid A-l protein, serum amyloid A-2 protein, serum amyloid A-4 protein, stromelysin- 1, vitamin D-binding protein, and vitronectin. Thus, in some embodiments, a citrullination site from a protein above, or a peptide fragment thereof, is assessed. For example, in some embodiments, the method herein comprises a method of assessing the activity of a 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.
In some cases, the citrullination site to be assessed is selected from one or more citrullination sites shown in Table A below. In the table below, the amino acid residue corresponding to a citrullination site is noted in the left column, with a reference accession number that may be used to locate the citrullination site in the publicly available UniProt database is listed in the right column. The UniProt accession number for each protein listed is provided in the table. The public database provides an amino acid and gene sequence corresponding to the accession number (e.g., P01023 for alpha-2-macroglobulin). It is understood that each of the proteins listed in the table below may have more than one naturally occurring amino acid sequence. Different isoforms of a protein can arise, for instance, due to the natural occurrence of different alleles reflecting substitutions, insertions, and/or deletions in the amino acid sequence, as well as due to alternative promoters, splicing and/or translation initiation sites. Accordingly, the accession number and its associated sequence listed for a given protein in Table A, and also in further tables herein that also provide citrullination sites and reference protein accession numbers or sequences, is merely a reference, which may be used to identify the citrullination site in a protein having the same sequence as the reference sequence as well as to identify the citrullination site in a naturally occurring variant protein, for example, by alignment of the variant sequence against the reference sequence. In some embodiments only one of the citrullination sites of Table 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. In some cases, citrullination sites of only one protein are assessed, while in other cases, citrullination sites of two or more proteins, such as at least 2, 3, 5, 6, 7, or 8 proteins are assessed. In some cases, citrullination sites of 2-5, 2-10, 5-10, 2-20, 5-10, or 10-20 different proteins are assessed.
Table A, provided below, for example, 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).
Table A - Exemplary Citrullination Sites
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
In some cases, the citrullination site is a site designated by underlining on one or more peptides shown in Tables 3-18. In some cases, the method comprises assessing citrullination of a peptide fragment of a protein, wherein the sequence of the peptide fragment comprises a peptide sequence shown in any one of Tables 3-18. In some cases, the method comprises assessing citrullination of a peptide fragment of a protein, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 3. In some cases at least two peptide sequences as shown in Table 3 are assessed, such as 2, 3, 4, or all shown in Table 3. 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 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. In some cases at least two peptide fragment sequences as shown in Table 6 are assessed, such as 2, 3, 4, or all shown in Table 6. 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 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. 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 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. 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 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. 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 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. In some cases at least two peptide fragment sequences as shown in Table 16 are assessed, such as 2, 3, 4, or all shown in Table 16. In some cases, the method comprises assessing citrullination of a peptide fragment of a protein, wherein the sequence of the peptide fragment comprises a peptide sequence shown in Table 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.
In some cases, 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 P02787), wherein all of the code names beginning with Q or P followed by a number are UniProt accession numbers for the proteins. In some cases, these citrullination sites may be assessed from one or more peptide fragments of a protein, wherein the sequences of the peptide fragments comprise (where the citrullination site is bold and underlined): 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), or DLLFRDDTVCLAK (SEQ ID NO: 257). In some cases, 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:
249), SGQSEDRQPVPGQQMTLK (SEQ ID NO: 250), QLGLPGPPDVPDHAAYHPFRR (SEQ ID NO: 252), or GPCRAFIQLWAFDAVK (SEQ ID NO: 253). In some cases, 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 serotransferrin (corresponding to R651 of P02787). In any of the above cases, in some embodiments 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. In some cases, 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. In some such cases, the biological sample is a whole blood, serum, plasma, blood supernatant, or synovial fluid sample. In some such cases, the biological sample is a whole blood, serum, plasma, or blood supernatant sample. In some cases in which the biological sample comprises synovial fluid, 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. In some such cases, the citrullination site selected from one or more citrullination sites shown in Table 11, or is a citrullination site located on a peptide fragment comprising 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), or GPCRAFIQLWAFDAVK (SEQ ID NO: 253). 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 protein of interest comprises fibrinogen alpha chain and/or gelsolin. In such cases, 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. In some cases, the citrullination site for assessment may comprise the gelsolin citrullination site located at R32 of P06396, which in some cases may be assessed via a peptide of sequence ATASRGASQAGAPQGR (SEQ ID NO: 255), wherein the underlined R is the citrullination site. In some cases in which the citrullination site is from fibrinogen and/or gelsolin, 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. In some embodiments, the assessing comprises quantitatively determining citrullination at a particular citrullination site (e.g., a citrullination site disclosed herein). In some embodiments, quantitatively determining citrullination at a particular citrullination site comprises determining a “citrullination ratio” as defined above.
In some cases, mass spectrometry (MS) is used to assess citrullination at a citrullination site. MS can detect differences between the molecular weights of a protein or peptide that is citrullinated and the corresponding protein or peptide that is not citrullinated. As described herein in the examples, intensity measurements obtained with MS can be used to measure the concentration of a citrullinated peptide. MS can also be used to measure the total concentration of corresponding protein from which a citrullinated peptide is derived. For instance, such total concentration of protein can be measured by measuring the concentration of a signature peptide. As used herein, such a “signature peptide” is a peptide that is not modified and is present in both modified and unmodified forms of the protein; accordingly, the concentration of the signature peptide represents the concentration of the corresponding total protein, including modified and unmodified forms of the protein. For example, in the case of FGA, 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. Bioanalysis (11 April 2023) 10.4155/bio-2022- 0241 C In some cases, assessing citrullination of the citrullination site comprises performing liquid chromatography to assist in separating protein or peptide species, followed by mass spectrometry (LC-MS). For example, liquid chromatography (e.g., HPLC, RPLC, nLC, pLC, or the like) may be used to separate peptides or proteins for analysis by MS.
In methods herein, a sample may be assessed directly after being obtained, or alternatively, a sample may be first prepared in some fashion prior to assessment of citrullination, for example, in order to improve the detection of the proteins or peptides of interest in the sample. For instance, a sample may be diluted with a buffer, a sample comprising cells may be treated to lyse the cells or to separate the cells from the sample fluid such as by centrifugation or filtration. For example, a protein of interest or peptide fragments thereof may first be enriched by separating them from other components of the starting sample prior to assessment of citrullination. For instance, one method of enriching a protein or peptide of interest is to isolate it from the sample. Thus, such a protein or peptide could be exposed to an affinity reagent such as an antibody and thus targeted for purification or removal from the sample. In some cases, such an affinity reagent may be immobilized on a solid surface such as a bead, chip, plate, or well of a plate, in order to facilitate such enrichment. In some cases, the proteins of the sample may also be enzymatically digested to form peptide fragments, may be denatured to remove tertiary structure, and/or a sample may be diluted or treated to remove or separate certain components prior to assessment. In some cases, other components could be degraded or removed from the sample, such as nucleic acids for instance, by enzymatic degradation or by alcohol precipitation.
In some cases, a sample may also be treated so as to promote release of endogenous 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. In some cases, for instance in order to boost the overall PAD4 activity in the biological sample, the sample may be incubated with exogenous PAD4 prior to assessing citrullination. For example, in some cases, 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. In some cases, exogenous calcium ion (Ca2+) is also added to the sample. In some cases, where citrullination from additional PAD proteins is also assessed, those may also be added exogenously, such as PAD2, which could be added, for example, at concentrations of 2-4 nM, such as at 3 nM or about 3 nM. In other cases, the sample is not exposed to exogenous PAD4 or is not exposed to any exogenous PAD proteins. Thus, in some cases, all PAD4 activity in a biological sample is from endogenous PAD4 present in the sample. In some instances, 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. In some cases, such incubation is conducted at 34-40 °C, such as at 36-39 °C, 36-38 °C, or 37 °C. In some cases, the incubation is conducted in a TruCulture® null tube (Rules Based Medicine, Austin, TX), or a similar container comprising culture media for preservation or analysis of biological samples. In some cases where the method involves assessing an exogenous PAD4 modulator, the PAD4 modulator may be added before the incubation of the sample. In other cases, a PAD4 modulator is added after the incubation. Where 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. In some cases, such incubation is conducted at 34-40 °C, such as at 36-39 °C, 36-38 °C, or 37 °C. In some cases, both exogenous PAD4 and an exogenous PAD4 modulator may be added together. In other cases, a PAD4 modulator may be added after the incubation with exogenous PAD4. In some cases an incubation is conducted in a CO2 incubator. In some cases, the activity of exogenous PAD4 added to a sample is quenched by adding EDTA to the sample. Thus, for example, in some cases the method comprises adding EDTA to the biological sample after incubation with the exogenous PAD4 in order to quench the activity of the PAD4.
In some cases, prior to analysis, a sample could be treated to expose one or more proteins or peptide fragments thereof comprising citrullination sites for later assessment. For example, proteins in a sample may be denatured or digested with enzymes. For instance, proteins may be denatured to remove tertiary structure, for example, by incubation at above physiological temperatures, such as at 60-90°C, such as 60-80°C, 70-85°C, or 80°C, for a period of 5-30 minutes, such as 10-30, 10-20, 15-25, or 20 minutes. In other cases, a chemical denaturant such as urea could be applied. A proteolytic enzyme such as trypsin, LysC, rLysC, LysN, GluC, AspN, rAspN, or a combination of proteolytic enzymes, such as more than one of trypsin, LysC, rLysC, LysN, GluC, AspN, and rAspN, could be used for enzymatic digestion, for example, to form peptide fragments of proteins of interest for later assessment.
Accordingly, overall, there are a variety of ways in which a sample may be prepared for assessment of citrullination herein.
In embodiments herein in which assessment of citrullination of a biological sample is used to assess a PAD4 modulator or other therapeutic agent, there are also several ways in which the biological sample may be exposed to the PAD4 modulator or other therapeutic agent herein. In some cases, 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. In other cases, 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. For example, 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. In other cases, a biological sample may comprise tissue, such as from a joint, a neoplasm, or a tumor. In some methods herein, the biological sample comprises synovial fluid. In some methods, the biological sample comprises blood or is derived from blood; in some cases the biological sample comprises serum, plasma, or blood supernatant (e.g. a supernatant that forms after centrifugation or filtration of blood). In some cases, the biological sample is fresh, meaning that it has not been frozen and thawed prior to use. In other cases, the sample has been frozen and thawed before use in the method. Thus, in some cases the method comprises freezing and thawing the biological sample before assessing citrullination of the citrullination site. For example, the sample may be frozen at a temperature of -70 °C or below, or -80 °C or below. In some embodiments, the biological sample is prepared for assessment prior to the assessing, such as according to methods provided above. For instance, in some cases the sample is treated by enzymatically digesting proteins, such as with one or more protease enzymes. In some cases, proteins comprising citrullination sites are enriched, in some cases by being removed from the sample, such as with an antibody or affinity reagent, and/or in some cases other proteins or contaminants are removed from the sample. For example, in some cases an antibody that specifically binds to the protein to be assessed may be used to enrich that protein for later assessment of citrullination. In some cases, the antibody or affinity reagent may be immobilized, such as placed on a matrix such as a bead or chip or well of a plate, other solid surface. In some cases, the biological sample may be diluted before assessment of citrullination. In some cases, proteins in the biological sample may be denatured to remove tertiary structure prior to assessment of citrullination. In some cases, any combination of the above treatments may be conducted on the sample.
In some cases, assessing citrullination of a citrullination site comprises measuring a citrullination ratio, as described above. In some cases, the method comprises comparing the citrullination assessment, such as a citrullination ratio or the concentration of a citrullinated protein or peptide fragment thereof, to that of a reference or control. For example, in some cases, the comparison is to a reference citrullination ratio, which may be obtained, for example, from a reference sample, or which may be a value or range of values that represents those obtained from reference samples. Accordingly, in some cases, the method comprises assessing comprises determining a difference between the citrullination ratio for the biological sample and a reference citrullination ratio. The terms “reference” and “control,” when referring to a biological sample, are used interchangeably to refer to a biological sample against which a measurement from a sample at interest is compared. In some cases, such a control is a sample that has not been exposed to PAD4 but is otherwise comparable to the sample of interest. In some cases, the control is a “baseline” sample, which is a sample taken from a subject prior to some event of interest, such as prior to treatment with a therapeutic agent, e.g., 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. In some cases, the control is a sample from the subject taken prior to treatment with a PAD4 modulator. In some cases, the 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. In other cases, 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. Thus, in some embodiments, citrullination assessments are compared to those of one or more control samples (e.g., baseline samples or samples from other points of time from the same subject, or samples from other subjects), or to values associated with one or more control samples (e.g., from a pool of healthy subjects). For example, in some cases, a citrullination assessment from a biological sample herein may be controlled to a group of control samples representing typical values found in a particular type of subject, or representing values found across a range of subjects.
In some embodiments, the present disclosure includes methods of assessing citrullination in a biological sample of a subject comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the biological sample. In some cases, the subject has not received treatment with any PALM modulator. In other cases, the subject has received treatment with a PALM modulator. In some cases, the subject has not received treatment with any therapeutic agent. In other cases, the subject has received treatment with a therapeutic agent. The present disclosure also encompasses methods of assessing the activity of a 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. In some cases, these methods further comprises comparing the assessment of the citrullination to that of a control biological sample, such as described above. In some cases, where the subject has been treated with a PAD4 modulator or other therapeutic agent, the control biological sample is a baseline sample obtained from the subject prior to treatment with the PAD4 modulator or other therapeutic agent. In some cases, the method 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. In some cases, 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.
In some cases, 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. In some cases, the sample is a biological fluid sample, such as plasma or serum, blood supernatant, whole blood, or synovial fluid. In some cases, it is plasma or serum. In some cases, the biological sample is prepared as described above prior to the assessing. In some cases, the concentration of exogenous 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). In some cases, another PAD protein (e.g., PAD2) is also added. In some cases, calcium ion (Ca2+) is also added to the sample. For instance, in some cases, the sample is treated to enrich a protein of interest, and/or to denature and/or enzymatically digest proteins of interest for later assessment, and/or to deplete proteins such as serum albumin. In some cases, the protein of interest is enriched by immunoenrichment techniques such as described below, for example, which may comprise using affinity reagents to isolate or remove the protein of interest. In some embodiments, the methods comprise in vitro methods of assessing the activity of a 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. In some cases, such methods further comprise assessing citrullination of the citrullination site on the protein or a peptide fragment in a control biological sample, such as described above. Methods herein also comprise, for example, an in vitro method of assessing the activity of a 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. In some cases, 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. In some cases, the incubating is in a TruCulture® null tube. Such incubation may be for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60- 72 hours. In some cases, the incubation period is 48 to 96 hours. In some cases, the incubating is at a temperature of 35 to 40°C, at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C. In some cases, the sample is prepared for assessment of citrullination, such as to enrich the protein or peptide to be assessed. In some cases, the method comprises, for example, separating the plasma or supernatant from the whole blood sample, such as by centrifuging or filtering the whole blood sample. These methods may also comprise (i) incubating the plasma or supernatant with a protein depletion resin, and (ii) recovering depleted plasma or supernatant that has flowed through the resin to obtain depleted flowthrough, optionally wherein the recovering comprises centrifugation. They may also comprise enzymatically digesting polypeptides in the depleted flowthrough before the assessing, and optionally cleaning up enzymatically digested peptides, such as with an iST-BCT kit (PreOmics, Planegg/Martinsried, Germany) or a similar kit, for instance. Such kits may be used in some embodiments to purify a sample prior to MS analysis and/or to minimize artificial protein modifications induced by reagents added to a sample, such as deamidation and oxidation modifications. In some cases, the whole blood sample has not been frozen and thawed. In other cases, the whole blood sample has been frozen and thawed. In some cases, the method comprises freezing the plasma or supernatant after it has been separated from the whole blood and subsequently thawing the plasma or supernatant before the assessing.
The disclosure herein also encompasses an in vitro method of assessing changes in citrullination in a subject, comprising (a) incubating a whole blood sample from a subject at 34-40°C for an incubation period (such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60-72 hours), wherein the sample is obtained from the subject following administration of a 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. In some cases, the method further comprises (d) incubating a second whole blood sample from the subject at 34-40°C for an incubation period (such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60-72 hours), wherein the second whole blood sample is obtained from the subject before administration of the 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. In some cases, an outcome of the assessing in step (f) is compared with an outcome of the assessing in step (c). Methods herein also include, for example, a method of assessing effects of a PAD4 modulator, the method comprising (a) exposing a whole blood sample from a subject to a PAD4 modulator in vitro, (b) incubating the whole blood sample at 34-40°C for an incubation period (such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60-72 hours), (c) after the incubation period, separating plasma or supernatant from the whole blood sample, and (d) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant. In some embodiments, the method further comprises performing a parallel set of steps on at least one control whole blood sample. In some embodiments, the method further comprises (e) incubating a control whole blood sample at 34-40°C for an incubation period (such as at a temperature of 36 to 39°C, at a temperature of 36 to 38°C, or at a temperature of 37°C for at least 12 hours, at least 18 hours, 12-96 hours, 24-96 hours, 24-72 hours, 24-36 hours, 36-96 hours, 36-72 hours, 48-96 hours, 48-72 hours, 60-96 hours, 48-60 hours or 60-72 hours), (f) after the incubation period, separating control plasma or supernatant from the control whole blood sample, and (g) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the control plasma or supernatant. In some cases, an outcome of the assessing in step (g) is compared with an outcome of the assessing in step (d). The disclosure herein also encompasses, for example, an in vitro method of assessing 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). In any of these methods herein, the sample can be a plasma or supernatant from a whole blood sample. In some cases the plasma or supernatant sample has not been frozen before assessment, while in other cases the sample has been frozen and thawed, and in some instances the method further comprises freezing and thawing the plasma or supernatant after separation from whole blood but prior to the assessing. In some cases, a sample may be contacted with a reagent, such as an affinity reagent, to enrich or purify a protein or a peptide fragment thereof harboring a citrullination site to be analyzed. For example, in some cases the method comprises “immunoenriching” a sample for a protein or a peptide fragment thereof that is of interest, the protein or peptide harboring a citrullination site for assessment. Such “immunoenriching” may comprise contacting the sample with an antibody reagent that specifically binds to the protein and/or to a peptide fragment thereof (e.g., comprising an epitope of the protein of interest) that contains a citrullination site. In some cases, the antibody reagent binds to the protein or peptide in both the non-citrullinated and citrullinated forms. Accordingly, the protein or peptide fragment thereof may be enriched prior to assessment of citrullination. In some cases, an antibody or other affinity reagent may be immobilized, such as by attachment to a solid surface, such as a bead, chip, plate or well of a plate. Such immobilization, for instance, may assist in separating a protein of interest for assessment of citrullination from other proteins in the sample. In some cases, the reagent used for immunoenriching is an antibody attached to a solid surface, such as a bead, such as a magnetic bead, or a chip, plate, or well of a plate. In some such cases, the sample may be a sample that comprises or is whole blood, serum, plasma, synovial fluid, pleural fluid, interstitial fluid, sputum, urine, or another biological sample.
Accordingly, methods of the disclosure herein also include, for example, a method comprising: (a) immunoenriching a sample for a protein of interest or a peptide fragment thereof to form an immunoenriched sample, wherein the immunoenriching comprises contacting the serum or plasma sample with an antibody that specifically binds to both citrullinated and noncitrullinated forms of the protein of interest or protein fragment thereof, the protein of interest or peptide comprising a citrullination site, and (b) assessing citrullination in the immunoenriched sample at the citrullination site. In some cases, a method disclosed herein comprises: (a) immunoenriching a sample for a protein of interest to form an immunoenriched sample, wherein the immunoenriching comprises contacting the serum or plasma sample with an antibody that binds to both citrullinated and noncitrullinated forms of the protein of interest or peptide fragment thereof, the protein of interest or peptide fragment thereof comprising a citrullination site, (b) enzymatically digesting the immunoenriched sample to form digested peptides from the protein of interest, wherein steps (a) and (b) can be in any order, and (c) assessing citrullination of the digested peptides at the citrullination site. In other cases, the method comprises (a) immunoenriching a sample by contacting the sample with an immobilized antibody that binds to both citrullinated and noncitrullinated forms of a protein of interest, the protein of interest comprising a citrullination site, (b) eluting protein bound to the immobilized antibody, (d) enzymatically digesting the eluted protein to form digested peptides, and (e) assessing citrullination of the digested peptides at the citrullination site. In any of the above methods, the sample may be diluted prior to the immunoenriching step, such as by 2-1000 fold. In any of the above methods, the sample may be a whole blood, plasma, serum, or synovial fluid sample. In some embodiments, it is a plasma or serum sample. In some cases, the immunoenrichment is conducted prior to enzymatic digestion of the protein of interest. In other cases, the protein of interest is first enzymatically digested, and then one or more peptide fragments thereof are immunoenriched. In some cases, the sample may be diluted to a volume of at least 5 pl, e.g., to a volume of 5 pl- 1 OOpl, 5 pl-50pl, 5 pl-25 pl, 5 pl- 15 pl, e.g., to a volume of lOpl. In any of the above methods, the immunoenriching may comprise incubating the sample with the immobilized antibody for at least 30 minutes, such as for a period of 30-90 minutes. In some cases, the incubation is for 30-60 minutes, 40-80 minutes, 50 to 70 minutes, 55 to 65 minutes, or for 60 minutes. The incubating may be at a temperature of 22-28°C, or may be at room temperature. In some cases, the immunoenriching comprises shaking during the incubating (e.g., at 800-1200 rpm or at 1000 rpm). In some cases, the immunoenriching comprises removing the immobilized antibody from the sample and washing the immobilized antibody prior to the eluting, for example with a wash buffer, e.g., a wash buffer comprising PBS (phosphate buffered saline). In some cases, the eluting comprises washing the immobilized antibody with an elution composition. A variety of elution compositions may be used, including, for example elution compositions that elute a protein of interest based on a change of pH, such as elution compositions that are acidic, and/or that comprise a detergent such as a zwitterionic detergent. In some cases, the elution buffer may comprise an acidic solution, optionally further comprising a buffer or ion, such as a zwitterion. In some cases, the buffer is exchanged prior to assessing citrullination, and optionally prior to intermediate steps such as enzymatic digestion, if such steps are performed. For example, if an acidic elution composition is used, then the buffer may be altered to a neutral pH range for subsequent steps, for example. In some cases, proteins that have been immunoenriched according to methods herein are denatured and/or enzymatically digested prior to assessment of citrullination. For example, proteins may be denatured to remove tertiary structure, for example, by incubation at above physiological temperatures, such as at 60-90°C, such as 60- 80°C, 70-85°C, or 80°C, for a period of 5-30 minutes, such as 10-30, 10-20, 15-25, or 20 minutes. In other cases, a chemical denaturant such as urea could be applied. A proteolytic enzyme such as trypsin, LysC, rLysC, LysN, GluC, AspN, rAspN, or a combination of proteolytic enzymes, such as more than one of trypsin, LysC, rLysC, LysN, GluC, AspN, and rAspN, could be used for enzymatic digestion.
In these above methods, the protein of interest may comprise any of the proteins listed herein, such as those listed in Table A above, and the citrullination site to be assessed may comprise any one or more of those listed in Table A or in Tables 3-18, herein, for example. In some cases, the protein of interest comprises fibrinogen alpha chain and/or gelsolin. In such cases, 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. In such cases, the citrullination site for assessment may comprise the gelsolin citrullination site located at R32 of P06396, which in some cases may be assessed via a peptide of sequence ATASRGASQAGAPQGR (SEQ ID NO: 255), wherein the underlined R is the citrullination site. An exemplary antibody that could be used to immunoenrich fibrinogen, for example, 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. In some such embodiments, immunoenrichment of the sample is conducted prior to enzymatic digestion.
In any of the methods above comprising immunoenrichment and enzymatic digestion, assessment of citrullination may be conducted by mass spectroscopy (MS), for example, by liquid chromatography followed by mass spectroscopy such as tandem mass spectroscopy (LC-MS or LC-MS/MS).
The above methods may be used in a wide variety of contexts, and with a wide range of PAD4 modulators, such as PAD4 inhibitors and PAD4 agonists, and with 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. For example, they may be used for testing new PAD4 modulators or other agents to determine their effect on citrullination of certain sites. Or they may be used to check the activity of known PAD4 modulators or other agents. Or they may be used to monitor the effect of PAD4 modulators or other therapeutic agents, for example, on citrullination levels, after administration to a patient. Thus, in some cases, a biological sample may be used for testing the effect of a 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. In other cases, 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. In some cases, the PAD4 modulator or other therapeutic agent is an anti -histone antibody, such as CIT-013. In some such cases, the PAD4 modulator or other therapeutic agent is a PAD4 modulator. In some cases, it is a PAD4 inhibitor, such as an anti-PAD4 antibody or small molecule PAD4 inhibitor. In some cases, a PAD4 modulator does not significantly affect the activity of any protein species other than PAD4. In some such cases, the PAD4 modulator does not significantly affect the activity of PAD2, for example. In other cases, the PAD4 modulator modulates the activity of PAD4 as well as at least one other protein, such as PAD2. Certain exemplary PAD4 modulators compatible with methods herein are described below.
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. Such diseases and related subjects are discussed in more detail in following sections herein. In some cases, the subject has been diagnosed with an autoimmune disorder or is at risk for developing an autoimmune disorder. In some cases, the citrullination-related disease is the citrullination-related disease is rheumatoid arthritis, lupus (e.g., systemic lupus erythematosus (SLE)), lupus nephritis, vasculitis (e.g., ANCA-associated vasculitis), thrombosis (e.g, venous thrombosis), or inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn’s disease). In some cases the subject has been diagnosed with rheumatoid arthritis (RA) or is at risk for developing rheumatoid arthritis. In other cases, however, the subject has not been diagnosed with an autoimmune disease such as RA, or the subject is not at considered to be at risk of developing such a disease. Thus, in some cases, is a normal, healthy subject. In some cases, the subject is positive for anti-citrullinated protein antibodies (ACPA positive). In some cases, the subject is positive for anti-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.
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. Thus, in some cases, is a normal, healthy subject. In some cases, the subject is positive for anti- citrullinated protein antibodies (ACPA positive). In some cases, the subject is considered at risk for developing an autoimmune disorder or citrullination-related disease.
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.
III. Exemplary PAD4 Modulators
Exemplary Small Molecule PAD4 Modulators
Examples of PAD4 modulators include various small molecule compounds that may increase or reduce PAD4 activity, such as citrullination of amino acid residues. Examples of small molecule PAD4 modulators include benzimidazole-derived compounds and heteroaryl compounds, as well as peptides, such as peptide macrocycles. Exemplary small molecule PAD4 modulators, for example, include compounds disclosed in International Patent Publication No. W02014/015905, which describes benzimidazole-derived PAD4 modulators such as PAD4 inhibitors, for instance 2-(azaindole-2-yl)benzimidazoles. Additional small molecule PAD4 modulators such as PAD4 inhibitors, are also described in International Patent Publication Nos. WO2016/185279, 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. Others include compounds disclosed in United States Patent application or publication numbers US11198681, US20220402950, US11524959, US20230203039, US20220348562, US11878965, US20220227787, US11976083. Others include, for example, GSK121, GSK199, AFM-30a, and GSK 484, described in Lewis et al. (2015) Nat Chem Biol 11(3): 189-191 and Chen et al., doi 10.1136/annrheumdis-2023- eular.2894 (2023; BMS-P5, described in Li et al. (2020) Mol Cancer Ther 19(7): 1530-1538; JBI-589, described in Deng et al. (2022) Cancer Res 82(19): 3561-3572; JBI-1044, described in US20200237771, and US2020276206; YW3-56, described in Wang et al. (2012) J Biol Chem 287(31): 25941-25953; ZD-E-1M, described in Zhu et al. (2022) Acta Pharm Sin B 12(5): 2592-2608; modified and unmodified Streptonigrin described in Dreyton et al. (2012) Probe Reports from the NIH Molecular Libraries Program and Dreyton et al. (2014) Bioorg and Med Chem 22(4): 1362-9; Cl-amidine, described in Luo et al. (2006) Biochemistry 45(39): 11727-11736, and its modifications including BB-Cl-amidine (Knight et al (2015) Ann Rhuem Dis , 74(12): 2199-2206), and o-Cl-amidine (Causey et al. (2011) J. Med Chem 54(19): 6919-6935); F-amidine, Luo et al. (2006) J Am Chem Soc 128(4): 1092-1093, and its modifications including BB-F-amidine (Muth et al. (2017) J Med Chem 60(7): 3198-3211), and o-F-amidine (Causey et al. (2011) J. Med Chem 54(19): 6919-6935); TDFA, described in Jones et al. (2012) ACS Chem Biol 7(1): 160-165; SC97362 and others, described in Aliko et al. (2019) Int J Mol Sci 20(9): 2174; NSC95397, ruthenium red, and sanguinarine, described in Lewallen et al. (2014) ACS Chem Biol 9(4): 913-921; chlorotetracycline, minocycline, and streptomycin, described in Knuckley et al. (2007) Bioorg Med Chem 16(2): 739-745; and Lucid-21-302, described in WO-2017 /027967. Other examples include the inhibitors described in WO2017/027967; WO2014/019092; Zhu et al. (2023) Eur J Med Chem 258: 115619; Zhu et al. (2024) J Med Chem 67(10):7973-7994; Jia et al. (2023) Biomed Pharmacother 168:115826; Muth et al. (2017) J Med Chem 60(7): 3198-3211; Sarswat et al. (2017) Bioorg Med Chem 25(9):2643-2656; Wei et al. (2014) J Med Chem 56(4) 1715-1722; Teo et al. (2017) Chem Biol Drug Des 90(6): 1134-1146; Nadzirin (2021) Comput Biol Chem 92: 107487; and Mondal et al. (2019) Acc Chem Res 52(3): 818-832.
In other cases, 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. Additionally, Sims et al, doi 10.1136/annrheumdis-2024-eular.3510 (2024) and Chen et al., doi 10.1136/annrheumdis- 2023-eular.2894 (2023) describe PAD2/PAD4 bispecific antibodies, and WO2016/155745 describes a cross-reactive antibody capable of binding both PAD2 and PAD4. Further examples of anti-PAD4 antibodies are described in this section and the sections that follow, and further in W02024/020579, and the amino acid sequences of the antibodies described below are also provided herein. Each of the above-referenced publications is incorporated herein by reference in its entirety.
Clone 13 and Related Anti-PAD4 Antibodies
One group of exemplary 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 cryo EM study of a clone 13 Fab binding to PAD4 and paratope mapping to identify the portions of the clone 13 variable regions and its humanized variants that directly contact PAD4 provided further structural information as to the parts of clone 13 variable regions and those of its related humanized antibodies that dictate PAD4 binding. Antibody hzl3-5 was also further modified to identify antibodies with pH dependent binding to PAD4. The clone 13 antibody and its humanized derivatives are 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.
For example, in some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. In particular, paratope mapping and structural analysis of antibodies comprising the above sets of heavy and light chain CDRs revealed that it is the HCDR1, HCDR3, and LCDR1 that make contact with PAD4. (See Figs. 8A-8B.) Hence, in some embodiments, antibodies herein comprise the above set of these three CDRs. In some embodiments, 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. In other embodiments, 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.
In some embodiments, 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. Each of these paratope regions was identified as contacting PAD4, as described in priority application No. 63/528,323 and international Patent Publication No. W02024/020579. In some embodiments, the antibody further comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 224.
In some embodiments, the antibody comprises a VH comprising a glycine at Kabat position 94 (Gly94). Thus, for example, the originally identified mouse anti-human clone 13 antibody comprises a glycine at Kabat position 94, but several human framework regions do not. Thus, in certain humanized antibodies, a back mutation is necessary to obtain a glycine at Kabat position 94. Gly94 is an amino acid residue in the loop adjacent to the VH CDR3 (i.e., the VH CDR3 loop). In some embodiments, Gly94 is implicated in the flexibility and/or the geometry of the VH CDR3 loop. In some embodiments, Gly94 may interact with VH CDR3. In some embodiments, Gly94 is implicated in the activity of the anti-PAD4 antibody. In some embodiments, 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. Accordingly, in some embodiments, 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).
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 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. 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 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some such cases, 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. Thus, in such cases, the variation in the VH and VL compared to the above listed sequence identification numbers is located in regions outside of these specific CDR or paratope sequences. In some embodiments, 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. In some embodiments, 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.
In yet further embodiments, 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. Thus, for example, 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: 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 (PAD4), 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;
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: 68 and a VL comprising the amino acid sequence of SEQ ID NO: 70.
In any of the above antibodies, in some cases the antibody binds to an epitope on PAD4 comprising SEQ ID NO: 217 and SEQ ID NO: 218.
In some embodiments herein, the anti-PAD4 antibody is an IgA, IgG, or IgM antibody. In some cases, the antibody is an IgG antibody, such as a human IgGl, IgG2, IgG3, or IgG4 antibody or a murine IgGl or IgG2 antibody. In some cases, the antibody comprises a wild-type, human IgGl, IgG2, or IgG4 heavy chain constant region. In some embodiments, 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.
In some embodiments, the anti-PAD4 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.
In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some embodiments, 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. In some embodiments, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 194. In some embodiments, the antibody comprises both a 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.
Clone 20 and Related Antibodies
The methods herein may also be conducted with 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. Accordingly, in some embodiments, 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.
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 any one of SEQ ID NOs: 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, or 134. 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: 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, or 136. In some embodiments, 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. In some such cases, 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. In some cases, 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. In some embodiments, 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. In some embodiments, 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.
In yet further embodiments, 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. Thus, for example, 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: 82 and a VL comprising the amino acid sequence of SEQ ID NO: 84;
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: 86 and a VL comprising the amino acid sequence of SEQ ID NO: 88;
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: 94 and a VL comprising the amino acid sequence of SEQ ID NO: 96;
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;
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: 102 and a VL comprising the amino acid sequence of SEQ ID NO: 104;
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: 106 and a VL comprising the amino acid sequence of SEQ ID NO: 108;
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: 110 and a VL comprising the amino acid sequence of SEQ ID NO: 112;
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: 114 and a VL comprising the amino acid sequence of SEQ ID NO: 116;
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 (PAD4), 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;
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: 134 and a VL comprising the amino acid sequence of SEQ ID NO: 136.
In any of the above antibodies, in some cases, the antibody binds to an epitope on PAD4 comprising SEQ ID NO: 219 and SEQ ID NO: 220. In some embodiments herein, the antibody is an IgA, IgG, or IgM antibody. In some cases, the antibody is an IgG antibody, such as a human IgGl, IgG2, IgG3, or IgG4 antibody or a murine IgGl or IgG2 antibody. In some cases, the antibody comprises a wild-type, human IgGl, IgG2, or IgG4 heavy chain constant region. In certain aspects, the antibody is of the human IgGl isotype. In certain aspects, the antibody is of the human IgGl isotype with a P329G, L234A and L235A (LALAPG; EU numbering) mutation to reduce Fc-region effector function. In other aspects, the antibody is of the human IgG2 isotype. In certain aspects, the antibody is of the IgG4 isotype with an S228P mutation (EU numbering) in the hinge region to improve stability of IgG4 antibody. In some aspects, the antibody (e.g., a non-humanized antibody) may have a non-human IgG constant region, and may be, for example, a murine IgG2a antibody such as a murine IgG2a LALAPG antibody. In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some embodiments, 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. In some embodiments, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 194. In some embodiments, the antibody comprises both a 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. pH Dependent Clone 13 -Related Antibodies
Further examples of anti-PAD4 antibodies to which methods herein apply include variants of parental antibody clone 13 or hzl3-5, such as to impact the pH dependence of PAD4 binding. These are 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
In some such embodiments, 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. In some embodiments above, 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. In other embodiments above, 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.
In some embodiments, the antibody comprises:
- a 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;
- a 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;
- a 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;
- a 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;
- a 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;
- a 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;
- a 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;
- a 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;
- a 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;
- a 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;
- 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: 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: 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 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: 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
- a 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.
In some embodiments, the antibody comprises:
- a 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;
- a 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;
- a 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;
- a 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;
- a 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;
- a 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;
- a 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;
- a 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;
- a 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;
- a 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 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: 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;
- 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 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: 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
- a 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.
In some embodiments above, 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. In some embodiments above, 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.
In some embodiments 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. In some embodiments above, the antibody comprises a VL comprising 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: 172. In some embodiments 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, and also a VL comprising the amino acid sequence of SEQ ID NO: 170. In some embodiments 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, and also a VL comprising the amino acid sequence of SEQ ID NO: 172. In some embodiments, 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. 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. 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. In some embodiments, 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. In some embodiments, 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. 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: 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: 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 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: 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: 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. 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. 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. 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. 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. 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: 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. In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. 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. 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. In some embodiments, 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. In some embodiments, 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. 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: 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: 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 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: 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: 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. 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. 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. In some embodiments, 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. In some embodiments, 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. 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: 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: 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 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: 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: 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. 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. 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. In some embodiments, 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. In some embodiments, 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. 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: 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: 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 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: 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: 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. 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. 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. In some embodiments, 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. In some embodiments, 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. 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: 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: 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 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: 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: 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. 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. 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. 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. 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. 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: 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. In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. 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. 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. In some embodiments, 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. In some embodiments, 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. 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: 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: 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 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: 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: 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. In some embodiments, the antibody comprises a VH that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 154. In some embodiments, the antibody comprises 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. In some embodiments, 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. In some embodiments, 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. 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: 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: 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 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: 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: 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. In some embodiments, 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: 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: 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.
In some embodiments, 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.
In some embodiments, 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. 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. 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. In some embodiments, 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. In some embodiments, 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. 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: 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: 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 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: 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: 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. 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. 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. In some embodiments, 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. In some embodiments, 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. 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: 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: 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 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: 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: 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.
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: 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. 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: 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. 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: 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.
In some embodiments, 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. 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. 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. 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. 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. 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: 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. In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. 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. 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. In some embodiments, 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. In some embodiments, 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. 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: 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: 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 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: 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: 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.
In some embodiments, 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.
In some embodiments, 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. 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: 172. In some embodiments, 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 comprising the amino acid sequence of residues 93-101 of SEQ ID NO: 170. In some embodiments, the antibody comprises a VH that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 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. In some embodiments, 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. In some embodiments, 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. 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: 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: 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 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: 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: 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. 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. 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. In some embodiments, 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. In some embodiments, 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. 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: 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: 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 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: 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: 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. 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. 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. In some embodiments, 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. In some embodiments, 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. 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: 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: 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 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: 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: 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.
In some embodiments, 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.
In some embodiments, 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.
In some cases, 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. In some cases, the antibody comprises a wild-type, human IgGl, IgG2, or IgG4 heavy chain constant region. In some embodiments, 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, single-chain Fv (scFv), Fab, Fab’, or (Fab’)2.
In some embodiments, 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.
In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some embodiments, 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. In some embodiments, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 194. In some embodiments, the antibody comprises both a 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.
IV. Exemplary Antibody Variants, Fragments, and Constant Regions
In many embodiments, an antibody PAD4 modulator may further incorporate any of the features, singly or in combination, as described in the sections that follow. A. Antibody Fragments
In certain embodiments, 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. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthiin, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5, ’87, 458. For discussion of Fab and F(a”)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.
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. In certain embodiments, 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.
B. Bispecific or Multispecific Antibodies
In certain embodiments, 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. In certain embodiments, one of the binding specificities is TREM2 and the other is for any other antigen. In certain embodiments, 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. some embodiments, 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. Techniques for making 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. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Imm“nol., 1”8(5): 1547-1553 (1992)); using“"diabod”" technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).
Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies,” are also included herein (see, e.g. US 2006/0025576).
C. Chimeric and Humanized Antibodies
In certain embodiments, 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)). In one example, 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. In a further example, 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.
In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, 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. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, 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.
[001] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Set. USA 86: 10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).
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. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271 :22611-22618 (1996)).
In some embodiments, the humanized antibodies may comprise a human IgGl, IgG2, IgG3, or IgG4 heavy chain constant region.
D. Glycosylation and Pegylation Variants
In certain embodiments, the glycosylation of an antibody is modified. For example, 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. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, 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. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.
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.
Additionally or alternatively, 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. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such 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. For example, EP 1,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. 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. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases {e.g., beta(l,4)-N- acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17: 176-180).
Another modification of the antibodies described herein is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an 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. In some embodiments, 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). As used herein, the term “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. In certain embodiments, 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.
E. Constant Regions
In some embodiments, an antibody described herein comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from K and A. In some embodiments, an antibody described herein comprises a human IgG constant region, such as an IgGl, IgG2, IgG3, or IgG4. In some embodiments, an antibody described herein comprises a human IgG4 heavy chain constant region. In some such embodiments, an antibody described herein comprises an S241P mutation in the human IgG4 constant region. In some embodiments, 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. Such effector function, in some embodiments, 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. In some methods of treatment, including methods of treating some cancers, 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. In some embodiments, when effector function is desirable, an antibody comprising a human IgGl heavy chain or a human IgG3 heavy chain is selected.
In certain embodiments, 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.
Where the antibody comprises an Fc region, 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. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibodies with certain improved properties. For example, in some embodiments 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. In some embodiments, 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. For example, 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.
The in vivo or serum half-life of 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).
In some embodiments of the invention, an afucosylated antibody mediates ADCC in the presence of human effector cells more effectively than a parent antibody that comprises fucose, Generally, 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.
In certain embodiments, 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. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, 322, 330, and/or 331 (EU numbering) 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.
In some examples, 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). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et al.
In some examples, 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. In some examples, 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,
283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312,
313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340,
360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438 or
439 (EU numbering). 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. Other modifications for reducing 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. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691. For example, 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. Other 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.)
Other modifications for enhancing FcyR and complement interactions include but are not limited to substitutions 298 A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691. 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.
Optionally, 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). These and other methods can utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.
In certain embodiments, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, this can be done by increasing the binding affinity of the Fc region for FcRn, For example, 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. Alternatively, to increase the biological half life, 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. 5,869,046 and 6,121,022 by Presta et al. Other 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. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 31 1A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields etal., Journal of Biological Chemistry, 2001, 276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 31 1 S, 433R, 433S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/311 S (Dall’Acqua et al. Journal of Immunology, 2002, 169:5171-5180, Dal’Acqua et al., 2006, Journal of Biological Chemistry 281 :23514-23524). Other modifications for modulating FcRn binding are described in Yeung et al, 2010, J Immunol, 182:7663-7671.
In certain embodiments, hybrid IgG isotypes with particular biological characteristics can be used. For example, 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. Thus 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. In some embodiments described herein, 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. Thus 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.
Moreover, the binding sites on human IgGl for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R.L. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to FcyRIII. Additionally, the following combination mutants were shown to improve FcyRIII binding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A, which has been shown to exhibit enhanced FcyRIIIa binding and ADCC activity (Shields et al., 2001). Other 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). Introduction of the triple mutations into antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2/neu-specific), rituximab (CD20- specific), and cetuximab (EGFR- specific) translated into greatly enhanced ADCC activity in vitro, and the S239D/I332E variant showed an enhanced capacity to deplete B cells in monkeys (Lazar et al., 2006). In addition, 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.
In certain embodiments, 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.
In certain embodiments, 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.
In certain embodiments, 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.
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.
In certain embodiments, the glycosylation of an antibody is modified. For example, 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. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, 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. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.
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.
Additionally or alternatively, 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. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such 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. For example, EP 1,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. 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. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases {e.g., beta(l,4)-N- acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17: 176-180).
Another modification of the antibodies described herein is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an 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. In some embodiments, 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). As used herein, the term “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. In certain embodiments, 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.
V. Therapeutic Compositions and Methods
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. For example, in some embodiments, 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. In some such cases, 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.
In other cases, 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. For instance, a sample might be assessed for a purpose such as determining a likely clinical outcome, determining risk for developing a citrullination-related disease, diagnosing a citrullination-related disease, and selecting the subject from which the biological sample was derived for a treatment of a citrullination-related disease. As used herein, a “citrullination-related disease” refers to a disease characterized by presence of increased citrullination of polypeptides in biological samples from disease subjects, and/or by one or more of presence of NETosis, presence of METosis, presence of anti-citrullinated protein antibodies (ACPA), and increased PAD4 expression.
A. Exemplary Citrullination-Related Diseases
In some embodiments, the subject may have a citrullination-related disease or be at risk of developing a citrullinati on-associated disease, or a subject’s sample may be tested to determine if they have a citrullination-related disease or are at risk of developing a citrullination-related disease. For example, in some embodiments a citrullination-related disease is associated with one or more of NETosis, METosis, presence of anti-citrullinated protein antibodies (ACPA), increased PALM expression, and increased citrullination of polypeptides in a biological sample. In some embodiments, the citrullination-related disease is an autoimmune disorder or an infectious disease or cancer.
For example, citrullination by PALM is a stress response and may serve as a signal for removal of stressed cells. (Brentville et al., Oncoimmunology 8: el576490 (2019).) Proteins citrullinated by 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. (See, e.g., Curran et al. Nat. Rev. Rheumatol. 16: 301-15 (2020); Brentville et al.) Thus, PALM activity may lead to generation of anti-citrullinated protein antibodies (ACPA). In neutrophils, PALM also plays a role in a process called NETosis, by which neutrophils extrude a complex of decondensed chromatin structures containing a DNA scaffold, citrullinated histones, and antibacterial neutrophilic granules. (Li et al. J. Exp. Med. 207: 1853-62 (2010).) These extruded complexes are called neutrophil extracellular traps (NET) and, during NETosis, these NETs trap and kill invading microbes as part of the innate immune response. (Chamardani et al., Mol. Cell. Biochem. 477: 673-88 (2022).) A similar process involving monocytes and macrophages is called METosis and involves formation of monocyte extracellular traps (MET).
In some embodiments, the disorder is an autoimmune disorder. In some embodiments, the autoimmune disorder comprises or is rheumatoid arthritis (RA). In some embodiments, the RA is juvenile-onset RA, juvenile idiopathic arthritis (JIA), or juvenile rheumatoid arthritis (JRA). In some embodiments, the subject has rheumatoid synovitis or significant systemic involvement secondary to RA (including but not limited to vasculitis, pulmonary fibrosis or Felty's syndrome). In some embodiments, the subject is positive for ACPA. In some embodiments, the subject is positive for anti -PALM autoantibodies. Typically, such anti-PAD4 autoantibodies activate PALM.
A variety of data suggest that 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. (See, e.g., Curran et al.; Yadav et al., J. Cyst. Fibres. 18: 636-45 (2018); Wang et al., Front. Immunol. 13: 895216 (2022); Fresneda Alarcon et al. Frong. Immunol. 12: 649693 (2021); Weeding et al., Clin. Immunol. 196: 110-116 (2018); Xu et al., Chinese J. Microbiology and Immunology 12: 115-121 (2020); Yoshida et al., Clin. Kidney J. 6: 308-12 (2013); O’Sullivan et al., Rheumatology, 58(Suppl. 2): kez061.024 (2019); Pan et al., Authorea Preprints, 2021, DOI: 10.22541/au.l61590650.07168461/vl.)
In some embodiments, the autoimmune disorder comprises or is rheumatoid arthritis (RA). In some embodiments, the disorder is RA, or the subject to be treated has been diagnosed with RA. In some embodiments, the subject is considered at risk of developing RA. In some embodiments, the RA is juvenile-onset RA, juvenile idiopathic arthritis (JIA), or juvenile rheumatoid arthritis (JRA). In some embodiments, the subject has rheumatoid synovitis or significant systemic involvement secondary to RA (including but not limited to vasculitis, pulmonary fibrosis or Felty's syndrome). In some embodiments, the subject is positive for anti-citrullinated protein antibodies (ACPA). In some embodiments, the subject is positive for anti-PAD4 autoantibodies. Typically, such anti-PAD4 autoantibodies activate PAD4.
Rheumatoid arthritis (RA) is a major autoimmune disease the pathobiology of which commonly includes the presence of auto-antibodies including anti-citrullinated protein antibodies (ACPA). 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. 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. 2020 Jun;16(6):301-315. The importance of PAD4 in RA has been further supported by several lines of evidence. For example, 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. (Susuki et al., Nat. Genet. 34: 395-402 (2003).) Epigenetic changes at the promoter region of PADI4 were also found to correlate with RA disease activity and to the level of ACPA in RA subjects. (Kolarz et al., J. Clin. Med. 9: 2049 (2020); Reyes-Castillo et al., Clin. Exp. Immunol. 182: 119-31 (2015).) On a mechanistic level, PAD4 citrullinates various proteins known to be targets of ACPA, which antibodies are used as part of the classification and diagnosis of RA in subjects via the anti-CCP test. In addition to ACPA, anti-PAD4 antibodies are also present in a subset of RA patients and are significantly correlated with increased swollen joint count and RA disease severity. Among these anti-PAD4 antibodies found in RA subjects are antibodies that activate PAD4 in RA subjects. (Halvorsen et al., Ann. Rheum. Dis. 68: 249-52 (2009); Zhao et al., J. Rheumatol. 35: 969-74 (2008); Darrah et al., Sci. Transl. Med. 5: 186ra65 (2013).) 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. 2015 Aug 21;5: 13041.; Suzuki A et al., BMC Musculoskelet Disord. 2016 May 5;17:205; Fukui S. et al., Arthritis Rheumatol. 2022 Feb 15. doi: 10.1002/art.42093).
In some embodiments, a subject whose biological sample is assessed for citrullination herein is at risk for developing RA or is found to be at risk for developing RA at least in part on the basis of the assessment results herein. In some embodiments, the subject at risk for developing RA has a first-degree relative with RA (i.e., a parent or sibling) and/or presence of anti-citrullinated protein antibodies (ACPA) in serum and/or presence of rheumatoid factor (RF) in serum. For example, presence of anti-citrullinated protein antibodies may be determined in some cases using an anti-CCP test, such as an ELISA test. For example, anti- CCP antibody test positivity (i.e., presence of ACPA) was found in 46% of 340 individuals who did not meet the classification criteria for RA but nonetheless had anti-CCP testing performed, such as due to joint pain or lung disease. Those 46% went on to meet the classification for RA within the subsequent 5 years. (Ford et al., Rheum. Dis. Clin North Am 45: 101-112 (2019). In some cases, such anti-CCP test results may be positive up to 10 years prior to onset of RA symptoms. (See, e.g, Jones et al., Curr. Op. Drug Discov. Dev., 12(5): 616-627 (2009).) 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. 7: R268 (2005); Smolen et al., Nat. Rev. Dis. Priers 4: 18001 (2018).) Accordingly, in some cases, a subject at risk for developing RA has a history of smoking (e.g., cigarettes, cigars) or of using tobacco products (e.g., chewing tobacco), and/or has periodontal disease.
In some such cases, a subject at risk for developing RA does not have clinical symptoms of arthritis. In some embodiments, however, the subject shows subclinical symptoms of arthritis, such as joint inflammation visible by imaging, such as ultrasound or magnetic resonance imaging (MRI), presence of ACPA via an anti-CCP test, presence of rheumatoid factor (RF), or an SNP or other genetic alteration in the PADI4 gene or its promoter region characteristic of subjects with RA. In some cases, the subject has such subclinical symptoms along with one or both of a first-degree relative with RA and serum ACPA or serum rheumatoid factor (RF). In other cases, the subject has been diagnosed with arthralgia or undifferentiated arthritis. For example, “arthralgia” herein refers to symptoms of pain or aching in at least one joint, such as an ankle, toe, shoulder, elbow, wrist, knee, hip, or one or more joints of the hand, fingers, or spine. A subject with arthralgia may also have tenderness, redness, warmth, loss of mobility, stiffness, weakness, numbness and/or tingling in one or more joints. “Undifferentiated arthritis” refers to diagnosis of arthritis in a subject for which the type of arthritis, such as RA or osteoarthritis, is not specified or cannot be determined. In the case of a subject with arthralgia or undifferentiated arthritis, the subject may also have one or more of a first-degree relative with RA, ACPA in serum, RF in serum, and subclinical joint inflammation (e.g., by ultrasound or MRI). For example, in such subjects at risk of developing RA, the treating may comprise, for example, lessening effects of one or more present clinical symptoms and/or one or more present sub-clinical symptoms.
In some embodiments, an RA subject or a subject at risk of developing RA has a comorbidity. In some embodiments, the comorbidity is a lung disorder such as interstitial lung disease (ILD), pleural effusion, cricoarytenoiditis, constrictive or follicular bronchiolitis bronchiectasis, pulmonary vasculitis, or pulmonary hypertension. (See, e.g., S. Kadura & G. Raghu, Eur. Respiratory Rev. 30: 210011 (2021).) In some cases, the lung disorder is a parenchymal lung disease (e.g., pneumonia), an airway disease (e.g., cricoarytenoiditis), or a pleural disease (e.g., pleural effusion). (S. Kadura & G. Raghu.) In some embodiments, the comorbidity is a lung disorder characterized by inflammation and/or scarring (fibrosis) of the lung, such as interstitial lung disease (ILD), also known as pulmonary fibrosis.
In other embodiments, the subject has not been diagnosed with RA, but has a lung disorder, such as a disorder characterized by inflammation and/or scarring of the lung, such as interstitial lung disease (ILD), also known as pulmonary fibrosis, or has a parenchymal lung disease (e.g., pneumonia), an airway disease (e.g., cricoarytenoiditis), or a pleural disease (e.g., pleural effusion), or has interstitial lung disease (ILD), pleural effusion, cricoarytenoiditis, constrictive or follicular bronchiolitis bronchiectasis, pulmonary vasculitis, or pulmonary hypertension. For example, ILD can also be a comorbidity with other autoimmune diseases such as scleroderma, dermatomyositis and polymyositis, mixed connective tissue disease, Sjogren’s syndrome, and sarcoidosis, as well as result from certain infectious diseases such as pneumonia, or exposure to certain drugs or harmful substances such as asbestos, or can result from uncontrolled gastroesophageal reflux.
In some embodiments, the autoimmune disorder comprises or is a rheumatic autoimmune disease other than RA. 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. For example, 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. In the case of lupus nephritis, for example, 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. (See, for example, Massarenti et al., Scand. J. Rheumatol. 48(2): 133-140 (2019); Y. Liu et al., JCI Insight 3(23): el24729; N. Hanata et al., Front. Immunol. 11 : 1095 (2020).)
NETosis is associated with the pathophysiology of lupus and other autoimmune and renal diseases, including, for instance systemic lupus erythematosus, vasculitis (e.g., ANCA- associated vasculitis), antiphospholipid antibody syndrome, type 1 diabetes mellitus, and renal inflammatory diseases (gomerulophritides, e.g., proliferative glomerulonephritis and non-proliferative gromerulonephritis), and is also associated with the pathophysiology of cancer. (See, e.g., Li et al., Molecular Cancer Therapeutics, 19: 1530-38 (2020), Teijeira et al. Immunity: 56, 856-871 (2020), Gupta, S. and Kaplan, M.J. Nat Rev Nephrol 12(7):402- 413 (2016).) NETs are extracellular web-like structures composed of chromatin backbone and various peptides and proteins that are formed by neutrophils in response to various stimuli in a process called NETosis. NETosis has been found to involve the citrullination of histones, such as histone H3, which requires PALM activity. In vasculitis, for instance, NETosis is a key driver of disease. (See, for example, B. Ameth et al., Int. J. Med. Sci. 18: 1532-40 (2021); JM Berthelot et al., Joint Bone Spine 84(3): 255-262 (2017); ZL Wang et al., Beijing Da Xue Xue Bao Yi Xue Ban 46(2): 200-6 (2014).)
In some embodiments, the disease is cancer (e.g., a cancer disclosed herein), or an autoimmune disease, such as, e.g., lupus (e.g., systemic lupus erythematosus), vasculitis (e.g., ANCA-associated vasculitis), antiphospholipid antibody syndrome, type 1 diabetes mellitus, inflammatory bowel disease (IBD) (e.g., ulcerative colitis and Crohn’s disease), cystic fibrosis, or a renal disease such as renal inflammatory disease (e.g., proliferative glomerulonephritis and non-proliferative gromerulonephritis).
Furthermore, in thrombosis, either absence of PAD4 or inhibition of PAD4 has been shown to abrogate thrombus formation induced by heparin, for example. In addition, injection of recombinant human PAD4 in vivo induces the formation of von Willebrand factor platelet strings in mesenteric venules, which is dependent on PAD4 enzymatic activity. There is also a reduction of endogenous ADAMTS13 activity in the plasma of wild-type mice injected with recombinant human PAD4. Administration of recombinant human PAD4 also decreased time to vessel occlusion and significantly reduced thrombus embolization. (See, for example, J. Perdomo et al., Nature Commun. 10(1): 1322 (2019); N. Sorvillo et al., Circulation Res. 125(5): 507-519 (2019).)
In addition, 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. For example, elevated levels of PAD enzymes and/or citrullinated proteins have been found in all of those conditions. (See, e.g., Chumanevich et al., Am. J. Physiol. Gastrointest. Liver Physiol. 300(6): G929-G938 (2011); Jones et al., Curr. Op. Drug Discov. Dev., 12(5): 616-627 (2009).) For example, in MS, both in patients and in animal models, myelin basic protein was found to be abnormally deiminated and elevated levels of PAD4 were observed; while in Marburg MS, a particularly severe form of MS, levels of citrullinated myelin basic protein were found to be very high. (See, e.g., Jones et al., Curr. Op. Drug Discov. Dev., 12(5): 616-627 (2009).) Moreover, deamination of PAD substrates has been suggested to occur in response to TNF-alpha signaling, while anti-TNF- alpha antibodies have been used as treatments for various autoimmune conditions such as RA and IBD, suggesting that the elevated PAD activity may result from uncontrolled TNF-alpha signaling. (See Chumanevich et al., supra.) Chumanevich and colleagues, for example, found elevated levels of PAD4 in a colitis model and showed that a small molecule PAD inhibitor could be used to treat colitis in a dextran sulfate sodium (DSS)-induced murine colitis model. (Id.) Thus, in some embodiments, the autoimmune disorder comprises IBD. In some embodiments, the autoimmune disorder comprises colitis, such as ulcerative colitis, Crohn's disease, gluten-sensitive enteropathy, or Whipple’s disease.
In some embodiments, the autoimmune disorder comprises lupus, such as systemic lupus erythematosus, cutaneous lupus erythematosus, or lupus nephritis. In some embodiments, the autoimmune disorder comprises vasculitis. Exemplary types of vasculitis include Bechet’s Disease, Buerger’s Disease (Thromboangiitis Obliterans), eosinophilic granulomatosis with polyangiitis (EGPA; formerly known as Churg Strauss), cryoglobulinemia, giant cell arteritis (temporal arteritis), Henoch-Schbnlein purpura (HSP; IgA vasculitis), microscopic polyangiitis, polyarteritis nodosa, polymyalgia rheumatica, rheumatoid vasculitis, Takayasu’s arteritis, granulomatosis with polyangiitis (GPA; formerly known as Wegener’s), ANCA-associated vasculitis (such as PR3-ANCA associated vasculitis or MPO-ANCA associated vasculitis), hypersensitivity vasculitis, isolated aortitis, central nervous system vasculitis, primary angiitis of the central nervous system (PACNS), Kawasaki Disease, urticarial vasculitis, drug-induced vasculitis, relapsing polychondritis (RP). In some embodiments, the autoimmune disorder comprises thrombosis. In some cases, the autoimmune disorder comprises arthritis, such as, e.g., acute arthritis, chronic arthritis, gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, septic arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, menopausal arthritis, estrogen-depletion arthritis, ankylosing spondylitis, or rheumatoid spondylitis. In some cases, the autoimmune disorder comprises multiple sclerosis (MS), such may include: primary progressive multiple sclerosis (PPMS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), and progressive relapsing multiple sclerosis (PRMS). In some cases, the autoimmune disorder comprises systemic sclerosis (scleroderma), idiopathic inflammatory myopathy (such as, e.g., dermatomyositis, polymyositis, necrotizing autoimmune myopathy, or sporadic inclusion body myositis), Sjogren’s syndrome, sarcoidosis, autoimmune hemolytic anemia, immune pancytopenia, paroxysmal nocturnal hemoglobinuria, autoimmune thrombocytopenia (such as, e.g., idiopathic thrombocytopenic purpura, immune- mediated thrombocytopenia, acute thrombocytopenic purpura, chronic thrombocytopenic purpura), thyroiditis (such as, e.g., Grave's disease, Hashimoto’s thyroiditisjuvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephritis, tubulointerstitial nephritis), a demyelinating disease of the central and/or peripheral nervous system (such as, e.g., multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, or chronic inflammatory demyelinating polyneuropathy), a hepatobiliary disease (such as, e.g., infectious hepatitis (e.g., hepatitis A, B, C, D, E or other non-hepatotropic virus), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, or sclerosing cholangitis), inflammatory bowel disease (IBD)(such as, e.g., ulcerative colitis, Crohn's disease, glutensensitive enteropathy, or Whipple’s disease), an autoimmune or immune-mediated skin disease (such as, e.g., a bullous skin disease, erythema multiforme, contact dermatitis, or psoriasis), an allergic disease (such as, e.g., asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity, or urticaria), an immunologic disease of the lung (such as, e.g., eosinophilic pneumonia, idiopathic pulmonary fibrosis or hypersensitivity pneumonitis), a transplantation associated disease (such as, e.g., graft rejection or graft-versus-host-disease), fibrosis (such as, e.g., kidney fibrosis or hepatic fibrosis), cardiovascular disease, including atherosclerosis and coronary artery disease, cardiovascular events associated with chronic kidney disease, myocardial infarction, and congestive heart failure, diabetes, including type II diabetes, Bronchiolitis obliterans with organizing pneumonia (BOOP), or hemophagocytic syndrome, macrophage activation syndrome, sarcoidosis, or periodontitis). In some cases, the autoimmune disorder comprises a methotrexate-resistant autoimmune disorder, such as methotrexate-resistant RA, lupus, vasculitis, thrombosis, MS, or the like. In some cases, the autoimmune disorder comprises a renal disease, such as a renal inflammatory disease such as, e.g., kidney fibrosis, chronic kidney disease, proliferative glomerulonephritis, or nonproliferative gromerulonephritis.
In some cases, the subject has a disorder comprising: acid-induced lung injury, acne (PAPA), acute lymphocytic leukemia, acute respiratory distress syndrome, Addison’s disease, adrenal hyperplasia, adrenocortical insufficiency, ageing, AIDS, alcoholic hepatitis, alcoholic liver disease, allergen induced asthma, allergic bronchopulmonary, aspergillosis, allergic conjunctivitis, alopecia, Alzheimer’s disease, amyloidosis, amyotrophic lateral sclerosis, weight loss, angina pectoris, angioedema, anhidrotic ecodermal dysplasia-ID, ankylosing spondylitis, anterior segment, inflammation, antiphospholipid syndrome, aphthous stomatitis, appendicitis, arthritis, asthma, atherosclerosis, atopic dermatitis, autoimmune diseases, autoimmune hepatitis, bee sting-induced inflammation, Bechet’s disease, Bechet’s syndrome, Bells Palsy, berylliosis, Blau syndrome, bone pain, bronchitis, bronchiolitis, bums, bursitis, cardiac hypertrophy, carpal tunnel syndrome, catabolic disorders, cataracts, cerebral aneurysm, chemical irritant-induced inflammation, chorioretinitis, chronic heart failure, chronic lung disease of prematurity, chronic lymphocytic leukemia, chronic obstructive pulmonary disease, colitis, complex regional pain syndrome, connective tissue disease, COPD, corneal ulcer, Crohn’s disease, cryopyrin- associated periodic syndromes, cryptococcosis, cystic fibrosis, deficiency of the interleukin- 1-receptor antagonist (DIRA), dermatitis, dermatitis endotoxemia, dermatomyositis, diffuse intrinsic pontine glioma, dry eye disease, endometriosis, endotoxemia, epicondylitis, erythroblastopenia, familial amyloidotic polyneuropathy, familial cold urticarial, familial Mediterranean fever, fetal growth retardation, glaucoma, glomerular disease, glomerular nephritis, gout, gouty arthritis, graft-versus-host disease, gut diseases, head injury, headache, hearing loss, heart disease, hemolytic anemia, Henoch-Scholein purpura, hepatitis, hereditary periodic fever syndrome, herpes zoster and simplex, HIV-1, Hodgkin’s disease, Huntington’s disease, hyaline membrane disease, hyperammonemia, hypercalcemia, hypercholesterolemia, hyperimmunoglobulinemia D with recurrent fever (HIDS), hypoplastic and other anemias, hypoplastic anemia, idiopathic thrombocytopenic purpura, incontinentia pigmenti, infectious mononucleosis, inflammatory bowel disease, inflammatory lung disease, inflammatory neuropathy, inflammatory pain, insect bite-induced inflammation, iritis, irritant-induced inflammation, ischemia/reperfusion, juvenile rheumatoid arthritis, keratitis, kidney disease, kidney injury caused by parasitic infections, kidney injury caused by parasitic infections, kidney transplant rejection prophylaxis, leptospirosis, Lewy body dementia, Loeffler’s syndrome, lung injury, lupus, lupus nephritis, meningitis, mesothelioma, mixed connective tissue disease, Muckle-Wells syndrome (urticaria deafness amyloidosis), multiple sclerosis, multiple system atrophy, muscle wasting, muscular dystrophy, myasthenia gravis, myocarditis, mycosis fungoides, myelodysplastic syndrome, myositis, nasal sinusitis, necrotizing enterocolitis, neonatal onset multisystem inflammatory disease (NOMID), nephrotic syndrome, neuritis, neuropathological diseases, non-allergen induced asthma, obesity, ocular allergy, optic neuritis, organ transplant, osteoarthritis, otitis media, Paget’s disease, pain, pancreatitis, Parkinson’s disease, pemphigus, pericarditis, periodic fever, periodontitis, peritoneal endometriosis, pertussis, pharyngitis and adenitis (PFAPA syndrome), plant irritant-induced inflammation, pneumonia, pneumonitis, pneumocystis infection, poison ivy or urushiol oil-induced inflammation, polyarteritis nodosa, polychondritis, polycystic kidney disease, polymyositis, psoriasis, psychosocial stress diseases, pulmonary disease, pulmonary hypertension, pulmonary fibrosis, pyoderma gangrenosum, pyogenic sterile arthritis, renal disease, retinal disease, rheumatic carditis, rheumatic disease, rheumatoid arthritis, sarcoidosis, seborrhea, sepsis, severe pain, sickle cell, sickle cell anemia, silica-induced disease, Sjogren’s syndrome, skin diseases, sleep apnea, spinal cord injury, spondylitis, spondyloarthropathy, Stevens- Johnson syndrome, stroke, subarachnoid hemorrhage, sunburn, temporal arteritis, tenosynovitis, thrombocytopenia, thyroiditis, tissue transplant, TNF receptor associated periodic syndrome (TRAPS), toxoplasmosis, transplant, traumatic brain injury, tuberculosis, type 1 diabetes, type 2 diabetes, ulcerative colitis, urticarial, uveitis (including nongranulamotous uveitis and granulomatous uveitis), wound healing, Wegener’s granulomatosis, interstitial lung disease, psoriatic arthritis, juvenile idiopathic arthritis, antineutrophil cytoplasmic antibody (ANCA)- associated vasculitis, antiphospholipid antibody syndrome, deep vein thrombosis, fibrosis, Alzheimer’s, scleroderma or CREST syndrome.
In some embodiments, the disorder is cancer. For example, neutrophil inflammation, neutrophil extracellular traps (NET), and/or monocyte extracellular traps (MET) have been identified in cancers, and are associated with poorer prognosis. (See, e.g., Li et al., Molecular Cancer Therapeutics, 19: 1530-38 (2020).) For example, studies have shown that PAD4- catalyzed NET formation is upregulated in multiple tumors, and that PALM is overexpressed in a variety of cancers. (H. Chen et al., Cell Mol. Biol. Lett 26:9 (2021).) A small molecule PALM inhibitor was also shown to inhibit tumor growth and to inhibit histone H3 citrullination in a cancer model. (See Id.) In addition, PAD4 has been reported to be highly expressed in certain tumor tissues and in blood samples of cancer patients. (See, e.g., Wang et al., Biomedicine & Pharmacotherapy 153: 113289 (2022).) PALM has also been reported to promote radioresistance, survival, migration and invasion of cancer cells. (Chen et al Cell Mol Biol Lett (2021) 26:9.) In some embodiments, the cancer is a cancer that is typically responsive to immunotherapy. In some embodiments, the cancer is a cancer that is not typically responsive to immunotherapy. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a blood malignancy (liquid tumor).
In some embodiments, the cancer is carcinoma, lymphoma, blastoma, sarcoma, or leukemia. In some embodiments, the cancer is squamous cell cancer, small-cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma squamous cell carcinoma, small-cell lung cancer (SCLC), non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell renal carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer (including triple-negative breast cancer, ER positive breast cancer, ER negative breast cancer, node positive breast cancer, and node negative breast cancer), colon carcinoma, and head and neck cancer (or carcinoma), gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer/T-cell lymphoma, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain cancer, brain stem glioma, pituitary adenoma, Kaposi’s sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, an environmentally-induced cancer (e.g., a cancer induced by asbestos, a virus-related cancer or a cancer of viral origin (e.g., human papilloma virus (HPV-related or -originating tumors)), a hematologic malignancy derived from either of the two major blood cell lineages (i.e., the myeloid cell line (which produces granulocytes, erythrocytes, thrombocytes, macrophages and mast cells) or lymphoid cell line (which produces B, T, NK and plasma cells), such as, e.g., a leukemia, lymphoma, or myeloma (of any type), e.g., acute, chronic, lymphocytic and/or myelogenous leukemia, such as acute leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), undifferentiated AML (MO), myeloblastic leukemia (ML), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3 V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma; a lymphoma, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B cell hematologic malignancy, e.g., B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large-cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angio-immunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma/leukemia (T-Lbly/T- ALL), peripheral T- cell lymphoma, lymphoblastic lymphoma, post-transplantation lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, B cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B- cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmocytoma, and multiple myelomas, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; a hematopoietic tumor of myeloid lineage, a tumor of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; seminoma, teratocarcinoma, a tumor of the central or peripheral nervous system, such as astrocytoma, schwannoma; a tumors of mesenchymal origin, such as fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; or another tumor, such as melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer, teratocarcinoma, a hematopoietic tumor of lymphoid lineage, for example a T-cell or B-cell tumor, such as a T-cell disorder such as T-prolymphocytic leukemia (T-PLL), such as of the small cell or cerebriform cell type; a large granular lymphocyte leukemia (LGL) of the T-cell type; a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma (pleomorphic or immunoblastic subtype); angiocentric (nasal) T-cell lymphoma; cancer of the head or neck, renal cancer, rectal cancer, cancer of the thyroid gland; acute myeloid lymphoma, or any combinations of said cancers. The methods described herein can also be used for treatment of metastatic cancers, unresectable cancers, refractory cancers (e.g., cancers refractory to previous immunotherapy, e.g., with an anti-CTLA-4 or anti-PD-1 antibody), and/or recurrent cancers.
In some embodiments, the disease is an infectious disease. For example, occurrence of NETosis has also been found in various infections. (See, e.g., Li et al., Molecular Cancer Therapeutics, 19: 1530-38 (2020).) Infectious diseases may include, for example, viral diseases (including AIDS (HIV infection), hepatitis (A, B, C, D, and E), and herpes), bacterial infections, fungal infections, protozoal infections and parasitic infections. Examples of pathogenic infections include, but are not limited to, HIV, Hepatitis (A, B, & C), Influenza, Herpes (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, Epstein Barr virus), Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa, adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, Lyme disease bacteria, Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absihlorambzopus), Sporothrix schenkii. Blastomyces dermalilidis. Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum, Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis. In some cases, the infectious disease is caused by a viral pathogen. In other cases, it is caused by a bacterial pathogen.
B. Combination Therapy
A 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.
For treatment of rheumatoid arthritis, for example, a subject may be treated with therapeutic agents, for example, such as a disease-modifying anti-rheumatic drug (DMARD) such as methotrexate (Trexall® or Otrexup®), adalimumab (Humira®), etanercept (Enbrel®), infliximab (Remicade®), hydroxychloroquine (Plaquenil®), sulfasalazine (Azulfidine®), leflunomide (Arava®), abatacept (Orencia®), anakinra (Kineret®), Certolizumab (Cimzia®), golimumab (Simponi®), rituximab (Rituxan®), sarilumab (Kevzara®), tocilizumab (Actemra®), baricitinib (Olumiant®), tofacitinib (Xeljanz®), upadacitinib (Rinvoq®), and Orencia® (abatacept); an non-steroidal anti-inflammatory drug (NS AID) such as ibuprofen (Advil, Motrin, and diclofenac) and naproxen sodium; a COX-2 inhibitor (celecoxib or etoricoxib); a steroid such as prednisolone or prednisone. In some cases, 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. For treatment of autoimmune conditions, 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® (Leustatin). In some cases, the anti-PAD4 antibody may be administered with methotrexate.
For the treatment of lupus, for example, a subject may be treated with one or more therapeutic agents such as cyclosporine, tacrolimus, cyclophosphamide, azathioprine (Imuran®), mycophenolate (CellCept®), rituximab (Rituxan®), and Belimumab (Benlysta®), steroids (e.g., prednisone or prednisolone), blood pressure medication (e.g., antiotensin- convertin enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs)).
For the treatment of vasculitis, for example, includes administration of therapeutic agents such as steroids (e.g., prednisone, prednisolone, methylprednisolone, or dexamethasone), methotrexate (Trexall®), azathioprine (Imuran®, Azasan®), mycophenolate (CellCept®), cyclophosphamide, tocilizumab (Actemra®), rituximab (Rituxan®), Avacopan, plasma exchange, mycophenolate mofetil (MMF), azathioprine (AZA), leflunomide (LEF), belimumab, meprolizumab, and omalizumab. For treatment of cancer, a subject may be treated with anti-cancer agents, such as an immune checkpoint inhibitor, a chemotherapeutic agent, growth inhibitory agent, radiotoxic agent, immunosuppressive agent, anti-cancer vaccine such as a gene therapy vaccine, anti-angiogenesis agent and/or anti-neoplastic composition.
Examples of immune checkpoint inhibitors include molecules that inhibit particular signaling pathways that regulate the immune system. See e.g., Weber (2010) Semin. Oncol. 37:430; Pardoll (2012) Nat. Rev. Cancer 12:252. Immune checkpoint inhibitors, in some embodiments, comprise an antagonist of PD-1, PD-L1, CTLA4, LAG-3, Galectin 1, Galectin 9, CEACAM-1, BTLA, CD25, CD69, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM1, TIM3, TIM4, ILT4, IL-6, IL-10, TGFp, VEGF, KIR, LAG-3, adenosine A2A receptor, PI3Kdelta, or IDO. In some embodiments, an immune checkpoint inhibitor comprises an agonist of B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, 0X40, OX40L, GITR, GITRL, CD27, CD40, CD40L, DR3, CD28H, IL-2, IL-7, IL-12, IL-15, IL-21, IFNa, STING, or a Toll-like receptor agonist such as a TLR2/4 agonist. In some embodiments, an immune checkpoint inhibitor comprises an agent that binds to a member of the B7 family of membrane-bound proteins such as B7-1, B7-2, B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. In some embodiments, an immune checkpoint inhibitor binds to a member of the TNF receptor family or a co-stimulatory or co- inhibitory molecule binding to a member of the TNF receptor family such as CD40, CD40L, 0X40, OX40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL/ Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fnl4, TWEAK, BAFFR, ED AR, XEDAR, EDAI, EDA2, TACI, APRIL, BCMA, LTPR, LIGHT, DeR3, HVEM, VEGL/TL1A, TRAMP/DR3, TNFR1, TNFP, TNFR2, TNFa, ip2, FAS, FASL, RELT, DR6, TROY, or NGFp. In some embodiments, an immune checkpoint inhibitor antagonizes or inhibits a cytokine that inhibits T cell activation such as IL-6, IL- 10, TGFP, VEGF. In some embodiments, an immune checkpoint inhibitor comprises an agonist of a cytokine that stimulates T cell activation such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNa. In some embodiments, the at least one immune stimulating agent comprises an antagonist of a chemokine, such as CXCR2, CXCR4, CCR2, or CCR4. In some embodiments, an immune checkpoint inhibitor comprises an antibody. In some embodiments, an immune checkpoint inhibitor comprises a vaccine, such as a mesothelin-targeting vaccine or attenuated listeria cancer vaccine such as CRS-207.
Exemplary non-limiting example targets of immune checkpoint inhibitors are CTLA- 4, PD-1, and PD-L1. Non-limiting examples of such immune checkpoint inhibitors include anti-CTLA4, anti-PD-1, and anti-PD-Ll antibodies, such as, e.g., pembrolizumab (Keytruda®), ipilimumab (Yervoy®), nivolumab (Opdivo®), atezolizumab (Tecentriq®), avelumab (Bavencio®), dostarlimab (Jemperli®), cemiplimab (Libtayo®), and durvalumab (Imfinzi®).
Non-limiting examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and Cytoxan®/Neosar® cyclosphosphamide; lenalidomide (Revlimid®); bortezomib (Velcade®); bendamustine (Treanda®); rituximab (Rituxan®); alemtuzumab (Campath®); ofatumumab (Kesimpta®); everolimus (Afinitor®, Zortress®); carfilzomib (Kyprolis™); ifosamade; dexamethasone; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophy cin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil (Leukeran®), chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Inti. Ed. EngL, 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine (Fludara®), 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as gemcitabine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; chlorambucaxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2- ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone’ 2”2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxanes, e.g., Taxol® paclitaxel (Bristol- Myers Squibb Oncology, Princeton, N. J.), Abraxane® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and Taxotere® doxetaxel (Rhone- Poulenc Rorer, Antony, Franl56hlorambucilbucil; Gemzar® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine (Oncovin®); thalidomide (Thalomid®); Navelbine® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluorometlhyl ornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
Further nonlimiting exemplary chemotherapeutic agents include anti-hormonal agents that act to regulate or inhibit hormone action on cancers such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and Fareston® toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, Megase® megestrol acetate, Aromasin® exemestane, formestanie, fadrozole, Rivisor® vorozole, Femara® letrozole, and Arimidex® anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGF expression inhibitor (e.g., Angiozyme® ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapy vaccines, for example, Allovectin® vaccine, Leuvectin® vaccine, and Vaxid® vaccine; Proleukin® rIL-2; Lurtotecan® topoisomerase 1 inhibitor; Abarelix® rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
In some embodiments, an anti-angiogenesis agent may be administered. Non-limiting examples of an anti-angiogenesis agent can include an antibody or other antagonist to an angiogenic agent, e.g., antibodies to VEGF-A (e.g., bevacizumab (Avastin®)) or to the VEGF-A receptor (e.g., KDR receptor or Fit- 1 receptor), anti-PDGFR inhibitors such as Gleevec® (Imatinib Mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668, Sutent®/SUl 1248 (sunitinib malate), AMG706, or those described in, e.g., international patent application WO 2004/113304). Anti-angiogensis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D’Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 (e.g., Table 3 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).
In some embodiments, a tumor growth inhibitory agent may be administered. Nonlimiting examples of growth inhibitory agents include, but are not limited to, agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S- phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (W.B. Saunders, Philadelphia, 1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (Taxotere®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (Taxol®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
For treatment of inflammatory or autoimmune or infectious disease conditions or cancer, in some embodiments, an anti-inflammatory drug may be administered. The antiinflammatory drug can be, e.g., a steroid or a non-steroidal anti-inflammatory drug (NSAID). In cases where it is desirable to render aberrantly proliferative cells quiescent in conjunction with or prior to treatment with anti-PAD4 antibodies described herein, 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, Toremifene, or goserelin (ZOLADEX®), can also be administered to the patient.
In some cases, a subject with a disease may receive a vaccination protocol. Many experimental strategies for vaccination against infectious diseases and tumors have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). Examples are cell-based therapies such as dendritic cells, or vaccine-like particles (VLPs).
In other cases, a subject may be administered other therapies, such as radiation therapy in the case of a tumor, surgical interventions, or the like.
Any of the above agents may, in some cases, be administered with a 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. EXAMPLES
Example 1: Anti-PAD4 Antibodies hz!3-5 and hz!3-5 D31E Inhibit PAD4 Activity In Vitro
Antibodies hzl3-5 and hzl3-5D31E, among the anti-PAD4 antibodies described herein, were tested for their ability to inhibit the activity of PAD4 on a peptide substrate in vitro.
A. Methods for ELISA
Microtiter plates (96 well Nunc MaxiSorp™ 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). In a buffer containing 50 mM NaCl, 2 mM CaCh 1 mM DTT (Invitrogen, Cat# P/N 46-2250) and 25 mM HEPES (Gibco Cat#l 5630-080), recombinant human PAD4 (rhPAD4) (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). Wells were then further washed with washing buffer and incubated with 100 pl of horse radish peroxidase (HRP) conjugated anti-citrulline monoclonal antibody Clone 1D9 (Cayman chemicals, Cat# 30773, 1 :2000 in PBS-0.05% Tween®-20) for 1.5 hrs at room temperature. Subsequently, the plates were washed three times in washing buffer and incubated with a peroxidase substrate (TMB). After 30 minutes, the color reaction was stopped by adding 2N sulfuric acid (VWR Cat# VW3500-1). Optical density (OD) was measured at 450 nm using the SpectraMax™ 190. Data were acquired using Soft Max Pro™ 7.1.
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.
All assays were performed in triplicate. All data are shown as means and ranges of triplicate measurements. Percentage of inhibition was calculated by percentage reduction of OD values from indicated concentration of PAD4 alone, after subtracting the background OD. The results are presented as IC50 values, which 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.
B. hz!3-5 D31E Inhibited PAD4 In Vitro
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.
Similar experiments were also performed with the hzl3-5 antibody (IC50 of 0.70 +/- 0.34 at 13.5 nM rhPAD4 to 5.02 +/- 0.88 at 108 nM rhPAD4).
Figure imgf000162_0001
Example 2: Anti-PAD4 Antibodies Reduce Citrullinated Histone H3 Levels
Anti-PAD4 antibodies were analyzed for ability to reduce extracellular citrullinated H3 and secretion of cytokines in human blood monocytes stimulated by lipopolysaccharide (LPS). A. Methods for the LPS-Stimulated Monocyte Assay
Several antibodies were formatted with an IgG1.3f constant region (SEQ ID NO: 178). 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 EasySep™ 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).
7xl04 of human CD 14+ monocytes added to each well of a 96-well u-bottom polystyrene plate. The monocytes were incubated with different concentrations of clone 13- based IgG1.3f antibodies or the isotype control antibody, and 10 pg/mL LPS. The incubation volume in each well was 200 pL. The plate was incubated at 37°C, 5% CO2 for 24 hrs.
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.
B. Functional Results
The antibodies reduced the amount of extracellular Cit-H3 in a dose-dependent fashion (FIG. 2A-FIG. 2F).
Example 3: Activity of Anti-Human PAD4 Antibodies in an LPS-Induced Acute Lung Inflammation Model
An LPS ALI PD model was used to determine the in vivo activity of anti-human PAD4 antibodies (Ab) in human PAD4 knock-in (HU-PAD4KI) mice.
A. Methods Related to the Murine ALI PD Model
The methods related to the murine ALI PD model in this Example were as described in Example 13 of each of priority application No. 63/528,323 and international Patent Publication No. W02024/020579, with the following modifications. C57B1/6 human-PAD4 knock-in (Hu-PAD4 KI) 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. B. Results
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.
Specifically, 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. The humanized derivatives of mAb20, hz20-2 (FIG. 3B) and hz20-7 (FIG. 3C), showed 12% and 16% reduction of extracellular Cit-H3, respectively, at 30 mg/kg. Reduction of extracellular Cit- H3 was greater at 100 mg/kg for both these antibodies (FIG. 3B and FIG. 3C). 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.
In addition, 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). A first humanized derivative of mAbl3, hzl3-12, was just as effective in reducing extracellular Cit-H3, demonstrating a 54% reduction at 10 mg/kg and a 58% reduction at 30 mg/kg (FIG. 4B). A second humanized derivative of mAbl3, hzl3-5, had an even greater impact on extracellular Cit-H3 reduction by demonstrating 86% reduction at 6 mg/kg and 95% at 30 mg/kg (FIG. 4C). Similarly, both hzl3-12 and hzl3-5 reduced extracellular Cit-ITIH4 by more than 80% at a dose of 6 mg/kg dose and above (FIG. 4D-FIG. 4E).
The results show that anti human PAD4 mAbs modulated PAD4 function in a preclinical LPS ALI model of inflammation. Specifically, the antibodies reduced the level of extracellular Cit-H3.
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.
Figure imgf000165_0001
Example 4: Activity of Anti-Human PAD4 Antibodies in an LPS-Induced Acute Joint Inflammation Model
An LPS-induced AJI model was used to determine in vivo activity of anti-human PAD4 antibodies (Ab) in the joints of human PAD4 knock-in (HU-PAD4KI) mice.
A. Methods Related to the LPS AJI Model
The methods related to the LPS-induced AJI model in this Example (FIG. 5A-FIG. 5B) were as described in Example 14 of each of priority application No. 63/528,323 and international Patent Publication No. W02024/020579, with the following modifications: C57B1/6 human-PAD4 knock-in (Hu-PAD4 KI) mice were used as well as WT mice. At multiple time points post ia injection, mice patella were removed and explanted to extract extra-cellular proteins (FIG. 5B), and Cit-PRG4, Cit-ITIH4, and hPAD4 expression was determined by LC/MS (FIG. 5C- FIG. 5E).
In a first experiment, the following antibodies were tested: humanized antibodies hzl3-5, hzl3-12, and hz20-2. 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. In a second experiment, 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.
B. Results
First, 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.
Next, in order to test the ability of the antibodies to inhibit PALM function in the LPS AJI PD model, the antibodies were administered as described in the method section above and as shown in FIG. 5A-FIG. 5B. Clone 13 -based hzl3-12 antibody (FIG. 6 A), clone 13- based hzl3-5 antibody (FIG. 6B) and clone 20-based hz20-2 antibody (FIG. 6C) reduced relative levels of Cit-ITIH4 in a dose-dependent manner. Clone 13-based hzl3-5 had the strongest effects, providing 81% reduction at 1 mg/kg and 95% reduction at 30 mg/kg.
In the first experiment, 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) also reduced relative levels of Cit-ITIH4 in a dose-dependent manner. Clone 13-based hzl3-5 had the strongest effects, providing 83% reduction at 1 mg/kg and 100% reduction at 30 mg/kg.
In the second experiment, both hzl3-5 D31E and hzl3-5 inhibited citrullination of ITIH4 (FIG. 8A) and PRG4 (FIG. 8B). The antibody hzl3-5 D31E showed dose dependent inhibition of citrullination of ITIH4 and PRG4.
The results as a whole indicate that anti human PALM mAbs inhibited PALM function in this LPS-induced AJI in vivo model.
Example 5: Citrullination in human serum induced in vitro by exogenous PAD4 and inhibited by the anti-PAD4 antibody hz!3-5 D31E
Global proteomics experiments were carried out to identify sites on human proteins that are citrullinated by PALM and where citrullination is inhibited by PAD4 inhibitors. The anti-PAD4 antibody hzl3-5 D31E was used as an exemplary PALM inhibitor.
A. Methods
Induction of citrullination in vitro by exogenous PAD4 in human serum. In this series of experiments, 20 pl of enzyme reaction mix was added to 80 pl of serum in plates (100 nM PALM, 100 mM Tris, 2 mM DDT, and 1 mM CaCh final concentrations). Plates were incubated at 37°C in CO2 incubator for 2 hours. Reactions were stopped with 10 pl of 0.5 M EDTA. Plates were sealed and stored at -80°C.
Inhibition curves. To produce inhibition curves, 7.5 pl of Ab hzl3-5 D31E serial dilutions in PBS (final concentrations ranging from 0.0038 nM to 100 nM) or isotype control (final concentration 100 nM) were added to 80 pl of serum in plates, mixed and stored overnight at -80°C. Serum plates were thawed and 12.2 pl of PAD4 enzyme reaction mix with or without 2 mM CaCh (7 nM or 13.5 nM PAD4, 100 mM Tris, 2 mM DDT final) was added and incubated at 37°C in CO2 incubator for 2 hours. Reactions were stopped with 10 pl of 0.5 M EDTA. Plates were sealed and stored at -80°C.
Global proteomics. The serum mixture (serum + enzyme + antibody, prepared as described above) was thawed and diluted (1 part serum mixture to 10 parts water). Then 5 pl of the resulting diluted serum mixture was combined with 45 pl iST-BCT LYSE buffer (PreOmics, cat# P.O.00116) and heated for 10 minutes at 95°C. Samples were enzymatically digested with addition of trypsin and LysC, and resulting peptides cleaned up according to manufacturer’s protocol using iST-BCT kit (PreOmics, cat# P.O.00116).
Peptides were analyzed by nanoLC-MS on Bruker timsTOF mass spectrometer using a 13 minute diaPASEF method. Data dependent acquisition (DDA) data was collected on samples with 100 nM PAD4 without compound to generate a spectra library. Data independent acquisition (DIA) runs were subsequently analyzed using the Fragpipe and DIANN workflow (Demichev, V., Szyrwiel, L., Yu, F. et al. dia-PASEF data analysis using FragPipe and DIA-NN for deep proteomics of low sample amounts. Nat Commun 13, 3944 (2022). doi . org/ 10.1038/s41467-022-31492-0).
To identify citrullination sites that showed statistically significant dose response to Ab hzl3-5 D3 IE, a four-parameter non-linear model was applied to a data matrix containing citrullination ratio (CitRatio) of each citrullinated peptide calculated by dividing the intensity of the citrullinated peptide by the total protein intensity (summarized based maxLFQ algorithm). For each citrullinated site, CitRatio was fitted against doses in a four-parameters (upper bound, lower bound, ec50 and hill) non-linear model. The non-linear model was then compared against a null linear regression model using chi-square statistics. The null model fits the mean (CitRatio) against doses. This process generates a p. value (DRC.p.value) that represents goodness of fit and effect size of the dose dependency for each individual citrullination site. B. Global Proteomics Results
The addition of 100 nM exogenous PAD4 induced more than 500 unique citrullinated peptide precursors which originated from more than 100 serum proteins, many of which were inhibited by 100 ug/ml Ab hzl3-5 D3 IE. When PAD4 enzyme concentrations at more physiologically relevant levels were used in the same assay, many citrullinated proteins were induced even at these lower concentrations and were inhibited by Ab hzl3-5 D3 IE in a dose dependent fashion. A list of citrullinated peptides detected in at least 2/3 of all the samples and with a p value (for goodness of fit) < 0.01 with addition of 13.5 nM PAD4 or 7 nM PAD4, and with or without added calcium in enzyme reaction mix are shown in Tables 3-6 (below). Tables 3-6 also show particular arginine (R) residues in each peptide or protein that were citrullinated, using bold face and underlining. The peptides in the tables below also in some cases were found to also include chemical modifications at other residues, such as oxidation of methionine (M), 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. 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.
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Figure imgf000172_0002
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000175_0001
Figure imgf000175_0002
Figure imgf000176_0001
Figure imgf000176_0002
Figure imgf000177_0001
Peptides that showed consistent induction by PAD4 and inhibition by Ab hzl3-5
D31E across all four conditions can be seen by comparison of Table 3, Table 4, Table 5 and Table 6. The IC50s and IC90s of seven such peptides are shown in Tables 7 and 8 below. As expected, they were on average higher when 13.5 nM of PALM was used vs. 7 nM of PALM.
Figure imgf000178_0001
Figure imgf000178_0002
Example 6: Baseline citrullination in human plasma
Citrullination in human plasma was assessed. A. Methods
Sample collection. Plasma was separated by centrifugation from blood drawn on heparin from healthy volunteers and the plasma was frozen for further use.
Global proteomics. 5 pl plasma was thawed and incubated with 100 pl High-Select™ Top 14 Abundant Protein depletion resin (Thermo Fisher, cat# A36372) at room temperature for 15 minutes. Depleted plasma samples were recovered in flowthrough by centrifugation, and subsequently enzymatically digested and cleaned up using iST-BCT kit (PreOmics, cat# P.O.00116) following the manufacturer’s protocol.
Peptides were analyzed by nanoLC-MS on Bruker timsTOF mass spectrometer using a 13 -minute diaPASEF (DIA) method. A pooled sample generated by combining peptides from all samples were fractionated into 24 fractions using high PH reversed phase HPLC. Data dependent acquisition (DDA) data were collected on the fractions to generate a spectra library. DIA runs were analyzed using the Fragpipe and DIANN workflow (Demichev, V., Szyrwiel, L., Yu, F. et al. dia-PASEF data analysis using FragPipe and DIA-NN for deep proteomics of low sample amounts. Nat Commun 13, 3944 (2022). doi.org/10.1038/s41467- 022-31492-0).
B. Global Proteomics Results
Citrullinated peptides were detected by unbiased proteomics in plasma from 10 healthy donors (Table 9). Table 9 shows particular arginine positions of each peptide that were found to be citrullinated. The citrullination of the peptides at the sites shown in Table 9 (in bold and underlining) was also found in Example 5 to be induced by exogenously added
PAD4 and inhibited by Ab hzl3-5 D3 IE.
Figure imgf000179_0001
Figure imgf000180_0001
Example 7: Induction of citrullination ex vivo in incubated blood and inhibition of citrullination by a PAD4 inhibitor
Samples obtained from normal healthy volunteers were analyzed for citrullination of specific peptides by endogenous PAD4. The blood samples were incubated at 37°C to induce release of endogenous PAD4 from dying cells. The samples were also treated with a PALM inhibitor (the anti-PAD4 antibody hzl3-5 D31E) and analyzed for inhibition of citrullination by Ab hzl3-5 D31E.
A. Methods
Sample generation. PAD4 inhibitor (hzl3-5 D3 IE), isotype control, or PBS were added to whole blood collected in Sodium Heparin tubes or TruCulture® null tubes. Blood was then incubated at 37°C and 5% CO2 for 72 hours. Plasma was collected from Sodium Heparin tubes; supernatant from the blood was collected from TruCulture® tubes, and the resulting samples (plasma or TruCulture® supernatants) were stored at -80°C until analysis for citrullinated peptides identification and quantification. Reproducibility between donors, and within donors, between 2 timepoints that were 1 week apart were evaluated (N=6).
Analysis method for identification and measure of citrullinated peptides: Global proteomics. 5 pl of thawed sample (plasma or supernatant) was incubated with 100 pl High- Select™ Top 14 Abundant Protein depletion resin (Thermo Fisher, cat# A36372) at room temperature for 15 minutes. Depleted samples were recovered in flowthrough with centrifugation, and subsequently enzymatically digested and cleaned up using iST-BCT kit (PreOmics, cat# P.O.00116) following manufacture protocol. The LC-MS method was as discussed above in Example 6, with one exception: spectra library was created using HPLC fractions from pooled samples of the 72-hour incubation group without PAD4 antibody treatment.
B. Results
After 48 hours or 72 hours of incubation at 37°C, induction of specific citrullinated peptides was observed. Citrullination was inhibited by Ab hzl3-5 D31E in a dose dependent manner (Table 10 and FIG. 9 A to FIG. 9D). The expectation was that PAD4 would be released following cell death during the incubation. Consistent with this expectation, MPO levels as well as PAD4 levels showed increases between 0 hours and 48 hours (results not shown). Table 10 also shows the particular arginine positions of each peptide that were citrullinated sites in bold text and underlining.
Figure imgf000181_0001
Targeted proteomics analysis confirmed induction and inhibition of citrullination of GSN and FGA peptides (see Example 10 and FIG. 11 A to FIG. 1 IB).
Example 8: Identification of citrullinated peptides in RA synovial fluid (SF)
A. Methods
In this example, 5 pl synovial fluid (SF) (instead of serum) was used as starting material. LC-MS sample preparation and analysis methods were as discussed in Example 6 above. Spectra libraries were made for SF from pooled samples generated by combining a portion of each sample.
B. Results
Spearman correlation analysis was performed to assess the correlation between percent citrullination (calculated as citrullinated peptide intensity divided by total protein intensity) and MPO LC-MS intensity. MPO was used as a surrogate for NETosis. MPO and PAD4 levels measured by unbiased proteomics in this SF sample set are correlated (Spearman p=0.56 p=2.9xl0'3). The correlation of MPO with PAD4 was confirmed by measuring PAD4 concentrations using an affinity capture LC-MS assay on the same SF samples (Spearman p=0.69, p=2.8xl0'7).
The expectation is that citrullinated peptides that are positively correlated with MPO levels are relevant to NETosis and PAD4 activity in the joint. Citrullinated peptide precursors that were detected in at least 35% of patients with Spearman p >0.3, and p-value <0.05 are listed in Table 11 below. The charged form of the peptide that correlates the best with MPO was selected to provide correlation coefficient and p-value. Table 11 also shows particular arginine (R) amino acid positions of each peptide that were citrullinated sites, as depicted in bold and underlining. As in Tables 3-6, peptides in the tables below also in some cases were found to also include modifications at other residues, such as oxidation of methionine and carbamidomethylation at certain cysteine residues, or modifications at the N-terminus of the peptide. These residues are also underlined, or in the case of an N-terminal modification, the first peptide residue of the sequence is underlined.
Figure imgf000183_0001
Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000186_0001
Based on the global proteomics analysis, a subset of the peptides from Table 11 were selected for targeted LC-MS assay of rheumatoid arthritis synovial fluid (RA SF) for several reasons: (1) they showed in vivo inhibition in mouse models with Ab hzl3-5 D3 IE (PRG4 peptide is also present in mice); (2) they showed a good prevalence across donors (especially PRG4, FGA, AMBP and C3 peptides); (3) they were citrullinated by exogenous PAD4 in serum and inhibited with Ab hzl3-5 D31E (AMBP, FGA and C3 peptides); and (4) they were also detected in plasma (ITIH4). These selected peptides are listed in Table 12 below.
Figure imgf000187_0001
The level of citrullinated ITH44 is lower than other citrullinated peptides listed in Table 12; this observation might be due to a natural variant in the amino acid sequence of ITIH4 (669Q to L), which may have resulted in a variant peptide was not detected by mass spectrometry and/or levels of citrullination below the limit of detection in some donor samples.
The levels of the citrullinated peptides listed in Table 12 above were compared between anti-citrullinated protein antibodies positive (ACPA+) and ACPA negative (ACPA-) patients and no differences were observed. Example 9: Analysis of serum or plasma for citrullination by exogenous PAD4 and inhibition of citrullination by a PAD4 inhibitor
This example relates to an ex vivo assay for the measurement of citrullination inhibition by a PAD4 inhibitor (such as Ab hzl3-5 D3 IE) in serum or plasma. The assay may be used as a pharmacodynamic assay.
A. Method
Plasma or serum samples from human subjects were collected and analyzed to assess citrullination of particular citrullination sites.
Sample collection. Serum and plasma samples from normal healthy volunteers were used in this example. To obtain serum, blood was collected in serum tubes (Vacutainer SST blood collection tubes) to separate serum. To obtain plasma, whole blood samples were collected in sodium heparin vacutainer tubes and plasma was isolated from the whole blood samples using standard methods.
Ex Vivo Treatment with PAD 4 Inhibitor for Generating Inhibition curves. Serum or plasma aliquots of 80 pl were pipetted into 96 well microplates (plate cat# 351190, Falcon). Various concentrations of PAD4 inhibitor (anti-PAD4 antibody hzl3-5 D31E) were titrated into the samples in the wells and the microplates were incubated for 6 h at 37degrees C. Then the samples were frozen.
In vitro citrullination by exogenous PAD4. Exogenous PAD4 was added to thawed serum or plasma samples to induce citrullination of proteins. 12.2 pl of PAD4 enzyme mix (13.5 nM PAD4, lOOmM Tris, 2 mM DDT) was added to each sample. The reactions were incubated at 37°C in a CO2 incubator for 2 hours. Each reaction was stopped with 10 pl of 0.5 M EDTA and frozen prior to subsequent treatment and proteomics analysis.
Preparations Prior to Proteomic Analysis. Ten (10) pl of serum or plasma was thawed and diluted 15-fold with PBS. Sixty (60) pl of 15-fold diluted serum or plasma was used for depletion of human serum albumin using CUSTOM PhyTip 200 pL CaptureSelect™ Human Albumin tips (Biotage, San Jose, CA) containing 20 pL CaptureSelect™ human albumin affinity matrix. Five (5) pl of albumin-depleted serum or plasma was combined with 45 pl ist-BCT LYSE buffer and heated for 20 minutes at 80°C. Samples were enzymatically digested with addition of trypsin and LysC according to manufacturer’s protocol. Peptides were cleaned up using iST-BCT kit (PreOmics, cat# P.O.00116).
Targeted proteomic analysis. Peptides listed in Table 13 below were analyzed by nanoLC-MS on Bruker timsTOF of mass spectrometer using a 30-minute prm-PASEF method. Data dependent acquisition (DDA) data was acquired for samples with high PAD4 concentrations with spiked internal standards (heavy peptides) for each peptide of interest. Skyline software was used to generate a list of cit-peptide and unmodified peptide sequences from cit-proteins previously identified by global proteomics experiments. Next, the library in Skyline was created using MaxQuant® generated msms.txt; mqpar.xml, and modifications. xml files. Ion mobility library was created after importing DDA data and using the imported data to generate a spectra library. Three parameters for each analyte were exported from Skyline to build a prm-PASEF method in TimsTOF Control 3.0 software: retention time (RT); Ion Mobility (IM), and precursor m/z. Data was analyzed using Skyline workflow.
Citrullination of specific peptides, listed in Table 13, was quantified on nanoLC- TimsTOF platform using Skyline data processing software. In the peptides of Table 13, the cysteine at position 3 of the AMBP peptide and the cysteine at position 10 of the TF peptide were also found to be modified by carb amidomethylation, as noted by underlining of those residues.
Figure imgf000189_0001
Analyses on TripleQuad® platform were also performed. TripleQuad® platform is much higher throughput and can be used to achieve faster turnaround times. Peptides that were analyzed on TripleQuad® are shown in Table 14.
Figure imgf000190_0001
B. Results
The results showed dose-dependent inhibition of citrullination by the PAD4 inhibitor hzl3-5 D3 IE. Inhibition curves were generated using the citrullination ratios (ratio of citrullinated peptide to total corresponding protein) for each peptide and IC50s were calculated. The IC50 results (reported as mean ± standard deviation) for representative peptides, as measured in plasma and serum samples, are shown in Table 15 and inhibition curves for three donors are shown in FIG. 10A to FIG. IOC. These results (shown in Table 15 and FIG. 10A to FIG. IOC) were generated from measurements on nanoLC-TimsTOF platform. Measurements from the TripleQuad® platform yielded similar results (results not shown).
Figure imgf000190_0002
These results show that the assay can be used as an in vitro pharmacodynamic assay to track the citrullination inhibition by a PAD4 inhibitor. In clinical applications, baselinesamples (prior to treatment with a PAD4 inhibitor) can be collected, processed and analyzed as described in this Example. PAD4 inhibition by a PAD4 inhibitor can be assessed using samples obtained from a subject at one or more timepoints after treatment with the PAD4 inhibitor. For instance, PAD4 inhibition (% decrease in each of one or more citrullinated peptides) in post-treatment samples may be calculated using inhibition curves generated with baseline (pre-treatment) samples to which titrated concentrations of PAD4 inhibitor have been added. Example 10: Analysis of whole blood for ex vivo citrullination by endogenous PAD4 and inhibition of citrullination by a PAD4 inhibitor
This example relates to an ex vivo assay for the measurement of citrullination by endogenous PAD4 and its inhibition by a PAD4 inhibitor using whole blood samples.
A. Method i. Sample Collection and Preparation
Sample collection. Whole blood samples were collected from healthy subjects in sodium heparin vacutainer tubes or TruCulture® tubes. Various concentrations of PAD4 inhibitor (anti-PAD4 antibody hzl3-5 D31E) were titrated whole blood aliquots from each donor to generate titration curves.
Ex vivo citrullination in whole blood by endogenous PAD4. Fresh whole blood samples that had been treated with PAD4 inhibitor or not treated with PAD4 inhibitor (samples not treated with PAD4 inhibitor were treated with PBS or isotype control) were incubated (in the vacutainer tubes or TruCulture® tubes) on heat blocks at 37°C for 72 hours to trigger release of endogenous PALM. Plasma was collected from sodium heparin tubes, supernatant was collected from TruCulture tubes. Shorter incubation times (<72 h) were also tested (data not shown). With the 72 hour incubation, the supernatant separated without centrifugation; if short incubation times (e.g., 18 hours or less) are used or if non-incubated samples are tested, centrifugation can be used to separate supernatant. Centrifugation was used to obtain supernatant for control samples that were treated with PBS and not incubated. The plasma or supernatant samples were frozen prior to further preparation and proteomic analysis.
Preparation for Proteomic analysis. Frozen samples were thawed and further prepared for analysis by LC-MS to quantify citrullination at the sites specified in Table 13 above. Ten (10) pl of sample was thawed and diluted 15-fold with PBS. Sixty (60) pl of 15- fold diluted supernatant was used for depletion of human serum albumin using CUSTOM PhyTip 200 pL CaptureSelect™ Human Albumin tips (Biotage, San Jose, CA) containing 20 pL CaptureSelect™ human albumin affinity matrix. Twenty five (25) pl of the albumin- depleted sample was combined with 25 pl ist-BCT LYSE 2-fold buffer and heated for 20 minutes at 80°C. Samples were enzymatically digested with addition of trypsin and LysC according to manufacturer’s protocol. Peptides were cleaned up using iST-BCT kit (PreOmics, cat# P.O.00116). ii . Targeted Proteomic Analysis
Samples were analyzed by LC-MS and citrullination of specific peptides was analyzed and quantified as described in Example 9 above. Peptides assessed included those listed in Table 13. Changes in citrullination at specific citrullination sites were measured as in Example 9.
B. Results
IC50 results (mean ± standard deviation) for two citrullinated peptides measured in ex vivo endogenous PAD4 assay on TimsTOF platform are listed in Table 16. These results illustrate detection of inhibition of citrullination by a PAD4 inhibitor.
Figure imgf000192_0001
Induction and inhibition of citrullination of GSN and FGA peptides is also shown in FIG. 11 A and FIG. 1 IB, respectively. All results (Table 16 and Fig. 11 A-l IB) were generated from measurements on nanoLC-TimsTOF platform.
The results show that this assay can be used to assess inhibition by a PAD4 inhibitor (e.g., Ab hzl3-5 D31E) using whole blood samples. The assay can be used, for instance, to assess citrullination in samples obtained from subjects before and/or after treatment with a PAD4 inhibitor. This and other assays disclosed herein can be used to assess citrullination in subjects that are treated with an agent or considered for a treatment (e.g., treatment with a PAD4 inhibitor or another agent that affects citrullination and/or related biological mechanisms such as NETosis or METosis).
Example 11: Targeted proteomic analysis of citrullinated fibrinogen and citrullinated gelsolin after immunocapture enrichment
This example describes a method for performing proteomic analysis of two particular citrullinated proteins, fibrinogen (FGA) and gelsolin (GSN).
Immunocapture enrichment and trypsin digestion
Samples were collected in TruCulture® null tubes and treated as described in the Methods subsections of Example 9 titled “ Sample collection” and “Ex vivo citrullination in whole blood by endogenous PAD 4” . Before quantifying citrullinated and total FGA and GSN, the samples were immunoenriched using anti-FGA and anti-GELS monoclonal antibodies (mAh) immobilized on tosylated magnetic beads. The antibodies used for the immunoenrichment have high affinity to both citrullinated and non-citrullinated FGA and GSN.
TruCulture® supernatants were thawed and diluted 200- fold for FGA and 10-fold for GSN in a 96-well KingFisher plate. 40 pL of beads containing 0.10 pg/pL of anti-FGA mAb (ab244636, Abeam) and 40 pL of beads containing 0.05 pg/pL anti-GSN mAb (ab247406, Abeam) were added to 200 pL of diluted sample. Samples were incubated for 60 min at 25 °C with shaking at 1000 rpm. After incubation, 96-well plate was placed on the KingFisher robotic system. Beads were washed twice with 500 pL of PBST buffer, followed by one wash with 500 pL of PBS buffer. Proteins bound to the beads were eluted using 110 pL of 12 mM HC1 with 0.05% Zwittergent. Samples were neutralized with 10 pL of 500 mM NH iHCO, buffer and transferred into a clean LoBind 96-well plate. Samples were incubated at 80 °C for 20 min to denature proteins. After cooling the plate down to room temperature, 2.0 pg of Promega trypsin (20 pL of 100 pg/mL trypsin solution in 100 mM NELHCOa buffer) was added to each sample. Samples were incubated at 50°C for 60 min with shaking at 500 rpms. After incubation, the digestion was stopped by the addition of 10 pL of the internal standard working solution in 10% formic acid in 80/20 water/acetonitrile.
HPLC-MS/MS conditions
The digested peptides were monitored with selective reaction monitoring on Sciex 6500± mass spectrometer using a 15 min LC gradient. Chromatographic separation was performed on an ACQUITY UPLC HSS T3 100A, 1.8 pm, 2.1 mm x 50 mm column (Waters Corporation, MA, USA) with the column temperature set to 60 °C. Mobile phase A contained 0.1% formic acid (FA) in water and mobile phase B contained 0.1% FA in acetonitrile. A gradient separation program was used as following: 0-0.5 min 2% B; 0.5-13.0 min 2-30% B; 13.0-13.1 min 30-95% B; 13.1-14.0 min hold at 95% B; 14.0-14.1 min 95-2% B; and the run was stopped at 15.0 min. The flow rate was set at 0.6 mL/min and the injection volume was 40 pL. The mass spectrometer was operated using positive ion electrospray ionization. The following optimized MS conditions were used: curtain gas and collision gas were set as 20 and 10; the turbo spray voltage was set at 5500 V and ion source gas 1 and gas 2 were both set at 50 psi. The probe temperature was set at 500°C. FGA and GSN peptides were quantified using SRM transitions of m/z 682.03 > 870.89 (CE: 29.7 eV), m/z 571.11 > 610.68 (CE: 29.4 eV), m/z 496.53 > 585.64 (CE 24.6 eV), and m/z 920.00 > 1135.21 (CE 42.0 eV) for QFTSSTSYNRGDSTFESK (SEQ ID NO: 249) (Cit-FGA signature), GSESGIFTNTK (SEQ ID NO: 258) (total FGA signature), ATASRGASQAGAPQGR (SEQ ID NO: 255) (Cit-GSN signature), and TPSAAYLWVGTGASEAEK (SEQ ID NO: 367) (total GSN signature), respectively.
Results
Representative results for FGA peptide QFTSSTSYNRGDSTFESK (SEQ ID NO: 249) measured in TruCulture® samples after the immunocapture enrichment described above are shown in FIG. 12. Percent (%) citrullination was calculated using the concentration ratio of citrullinated FGA to total FGA (total FGA includes modified and unmodified versions) that was multiplied by 100%. The results showed that the assay detects citrullination and its inhibition by hzl3-5 D31E.
Example 12: Targeted proteomic analysis of FGA and GSN
To quantify citrullinated and total FGA and GSN in plasma samples from patients (e.g., rheumatoid arthritis (RA) patients), samples are immunoenriched using anti-FGA and anti-GSN monoclonal antibodies (mAb) immobilized on tosylated magnetic beads. The selected antibodies have high affinity to both citrullinated and non-citrullinated FGA and GSN. The patient plasma samples are collected in sodium heparin tubes using standard blood collection procedures. Samples may be collected before and/or after patients are treated with a PAD4 inhibitor (e.g., hzl3-5D31E).
Baseline samples collected before treatment (e.g., with a PAD4 inhibitor) may be subjected to ex vivo treatment (e.g., with the PAD4 inhibitor or isotype control), or one or more control treatments (e.g., PBS) as disclosed herein in other Examples for the purpose of assessing citrullination and determining IC50s.
The patient plasma samples are diluted 500- fold for assessment of FGA and 20-fold for assessment of GSN in a 96-well KingFisher plate. Exemplary FGA and GSN peptides are disclosed herein, for instance, in Table 14 and Table 15. 40 pL of beads containing 0.10 pg/pL of anti-FGA mAb (ab244636, Abeam) and 40 pL of beads containing 0.05 pg/pL anti- GSN mAb (ab247406, Abeam) are added to 200 pL of diluted plasma sample. Samples are incubated for 120 min at 25 ° with shaking at 1000 rpm. After incubation samples are processed and analyzed as described in Example 11. Example 13: Targeted Analysis of Citrullination of EGA in Cultured Human Blood by IC-LC-MS/MS
A targeted analysis of citrullination of FGA in cultured human blood samples by IC- LC-MS/MS was developed and can be used, for instance, in evaluating samples from subjects participating in clinical testing of PALM inhibitors such as anti-PAD4 antibodies.
Summary and assay qualification experiments
Percent citrullination of FGA was measured in whole blood from normal, healthy volunteers in TruCulture® null tubes at 37 °C to trigger release of endogenous PALM. Two samples were collected from each donor, one was incubated at 37 °C for 72 hours before freezing (TruCulture-72h) and the other was frozen immediately (TruCulture-Oh), and which was expected to have low levels of endogenous PALM. Each sample was thawed and diluted 200-fold in assay buffer, incubated with magnetic beads conjugated to anti-FGA antibody (Abeam catalog no. ab244636, rabbit monoclonal antibody to fibrinogen), after which beads were washed, and immunocaptured (IC) proteins were eluted with a low pH buffer. Beads were Dynabeads M-280, tosylactivated, at 30 mg/mL (ThermoFisher catalog no. 14204). Trypsin was added to the samples to digest the FGA protein. Internal standards were spiked into all samples, and samples were analyzed by LC-MS/MS on an Acquity® UPL HCC T3 column (100 Angstrom, 1.8 pm, 2.1 mm x 50 mm) for liquid chromatography and a Sciex® TripleQuad™ 7500 system, operated in positive ion electrospray mode, with detection by MRM, for MS/MS analysis.
Citrullination was assessed at FGA peptide QFTSSTSYNRGDSTFESK (SEQ ID NO: 249), with the citrullination site arginine bold and underlined. The total FGA peptide GSESGIFTNT (SEQ ID NO: 368) was also assessed to determine total FGA. Internal standard peptides were QFTSSTSYNCitGDSTFESLys (SEQ ID NO: 369) (13C6; 15N2) for Cit-FGA internal standard, and GSESGIFTNTLys (SEQ ID NO: 370) (13C6,15N2) for total FGA internal standard.
For assay qualification and calibration, synthetic FGA peptides were added to samples over a concentration range of 4-1000 nM for Cit-FGA peptide and 40-10,000 nM for total FGA peptide to obtain a standard curve.
To address donor-to-donor variability, TruCulture® samples from 12 healthy donors at the 0 and 72 hour timepoints were analyzed. The % cit (percentage citrullination) varied from 0.12% to 0.62% in the TruCulture Oh samples from the donors and varied from 0.51% to 3.65% in the TruCulture 72h samples from the donors. Protocol for assessing pharmacodynamic effects of a PAD4 inhibitor
Peripheral blood samples are collected in TruCulture® Null tubes and incubated for 72 hours at 37 °C to boost citrullination. TruCulture® supernatant is separated from blood cells after incubation (TruCulture-72h). Representative samples with low citrullinated FGA concentrations are obtained by processing TruCulture® Null samples immediately after collection (TruCulture-Oh). Samples for method qualification are collected. Only Truculture- 72h samples are collected from clinical subjects for testing. Samples are collected pre- and post-dose of PALM inhibitor to measure inhibition of FGA citrullination after each dose. Citrullinated FGA (Cit-FGA) concentrations are expected to decrease with increasing doses of PALM inhibitor.
Cit-FGA and total FGA are quantified by IC-LC-MS/MS assay as described above. % citrullination is calculated as follows: [Cone. Cit-FGA (nM)/ Cone. Total FGA (nM)] X 100%.
Example 14: Therapeutic PAD4 antibody maintained potency in presence of endogenous PAD4 antibodies from rheumatoid arthritis patients
A. Introduction
PALM is released extracellularly in the inflamed joint through neutrophil activation, NETosis and cell death. PAD enzymes catalyze the modification of an arginine into a citrulline residue and drive the formation of citrullinated neoantigens that are recognized by anti-citrullinated peptide antibodies (ACPA), which are a hallmark of rheumatoid arthritis. Formation of immune complexes between ACPAs and citrullinated proteins are believed to drive tissue damage and perpetuate inflammation. Besides ACPA, about 25-35% of RA patients express anti-PAD4 IgG, of which about 20-40% cross-react between PAD3/PAD4 and have the potential to enhance PAD4 activity leading to more erosive disease. In this Example, the impact of the presence of endogenous anti-PAD4 antibodies on the potency of antibody hzl3-5 D3 IE was investigated in serum or with purified immunoglobin G (IgG).
B. Materials and methods a. Serum samples
The samples were serum samples from rheumatoid arthritis (RA) patients with established disease. Serum from healthy controls (NHV) was collected in BD Vacutainer SST Blood collection tubes (Cat#367988). All serum samples were frozen until aliquoting in plates. b. PAD4 antibody ELISA
Serum anti-PAD4 autoantibodies were measured using PAD4 Autoantibody ELISA kit (500930) from Cayman Chemicals following manufacturer's protocol. Serum samples were tested at 1 : 150 dilution in an assay buffer provided in the ELISA kit. c. IgG purification
Human IgG was purified using Melon™ gel IgG Spin Purification Kit (45206) from Thermo Scientific. Briefly, 500 pl purification gel was loaded to spin columns and washed twice with 300 pl of purification buffer. 80 pl of serum was diluted 5-fold with purification buffer and added to purification gel containing spin columns. Columns were mixed endover-end for 5 minutes and centrifuged to collect purified IgG. Concentration of purified IgG was determined using NanoDrop™ One instrument from Thermo Scientific. d. Citrullinated H3 enzymatic assay
All reagents were calculated for 100 pl final reaction. In 42.5 pl volume, recombinant PAD4 (Cayman #10500) was pre-incubated with purified IgG on ice for 45 minutes. To produce inhibition curves, 7.5 pl of hzl3-5 D31E serial dilutions in PBS or isotype control were added to the PAD4 / IgG mix and incubated on ice for an additional 45 minutes. Histone H3 in buffer containing calcium chloride was added, and the reaction (final concentrations: 13.5 nM PAD4, 2 mM CaC12, 10 pg/ml H3, 100 pg/ml purified IgG) was incubated at 37°C in an incubator for 2 hours. The H3 citrullination reaction was stopped with 10 pl of 0.5M EDTA. Citrullinated histone H3 was measured using Citrullinated Histone H3 (Clone 11D3) ELISA kit (501620) from Cayman Chemicals following manufacturer's protocol. Citrullinated samples were tested @ 1 : 100 dilution in an assay buffer provided in the ELISA kit. e. Citrullination induction in serum
To produce inhibition curves, 7.5 pl of hzl3-5 D31E serial dilutions in PBS or isotype control were added to 80 pl of thawed serum in plates, mixed, incubated at 37 °C in CO2 incubator for 6h then stored overnight at -80 °C. Plates containing serum mixed with antibody were thawed and 12.2 pl of PAD4 enzyme reaction mix (13.5 nM PAD4, 100 mM Tris, 2mM DTT final) was added and incubated at 37°C for 2h. Reactions were stopped with 10 pl of 0.5 M EDTA. Plates were sealed and stored at -80 °C. f. Measure of citrullinated peptides by LC-MS
Ten (10) pl of serum was thawed and diluted 15-fold with PBS. Sixty (60) pl of 15- fold diluted plasma was used for depletion of human serum albumin using CUSTOM PhyTip 200 pL CaptureSelect™ Human Albumin tips (Biotage, San Jose, CA) containing 20 pL CaptureSelect™ human albumin affinity matrix. Five (5) pl of albumin-depleted serum was combined with 45 ul LYSE BCT buffer and heated for 20 minutes at 80 °C. Samples were enzymatically digested with addition of trypsin and LysC according to manufacturer’s protocol. Peptides were cleaned up using preOmics BCT kit (cat# P.O.00116).
Peptides were analyzed by nanoLC-MS on Bruker’s timsTOF of mass spectrometer using a 30-minute prm-PASEF method. Chromatographic separation was done using Bruker’s NanoElute™ LC on AURORA ELITE (AUR-15075C18-CSI) column. Data dependent acquisition (DDA) data was acquired for samples with high PAD4 concentrations with spiked internal standards (heavy peptides) for each peptide of interest. Skyline® software was used to generate a list of cit-peptide (citrullinated peptide) and unmodified peptide sequences from cit-proteins previously identified by global proteomics experiments. Next, the library in Skyline® was created using MaxQuant® generated msms.txt; mqpar.xml, and modifications. xml files. Ion mobility library was created after importing DDA data and using the imported data to generate a spectra library. Three parameters for each analyte were exported from Skyline® to build a prm-PASEF method in TimsTOF Control 3.0 software: retention time (RT); Ion Mobility (IM), and precursor m/z. Data was analyzed using Skyline® workflow. g. Calculation of IC50
IC50 calculations and statistics were performed in GraphPad® Prism software. Citrullinated H3 or % citrullination were plotted against the log of the concentration of the antibody and a non linear fit (Hill Slope = -1) was performed.
C. Results a. PAD4 antibody status
The presence of endogenous PAD4 autoantibodies was evaluated in the serum samples from the 21 RA patients and 10 healthy controls (NHV) by ELISA. On average the OD was significantly higher in RA than in NHV (1.3 in RA vs 0.38 in NHV, p<0.0001, Mann Whitney T test). Using 1 as a cutoff for positivity, 13 out of 21 (62%) RA sera and none of the NHV sera could be considered anti-PAD4 IgG+. FIG. 13 A shows serum anti- PAD4 autoantibodies measured by OD450 by ELISA in 21 RA and 10 NHV purified IgG. b. Hzl3-5 D3 IE potency in the presence of purified IgG
Potency of hzl3-5 D31E on inhibition of H3 citrullination in the presence of 100 ug/ml purified IgG from the 21 RA and 6 NHV sera was evaluated (3 plates containing each 7 RA and 2 NHV donors). hzl3-5 D3 IE inhibited in vitro PAD4 driven H3 citrullination in the presence of purified IgG and there was no difference in IC50 whether purified IgG came from RA or NHV sera (FIG. 13B, Table 17), despite the fact that a large proportion of RA IgG had anti-PAD4 autoantibodies. The mean IC50 (nM) for the RA sera (n = 21) was 8.2 (standard deviation of 1.3), while the mean IC50 (nM) for the NHV sera (n = 6) was 9.0 (with standard deviation 2.5). c. Hzl3-5 D3 IE potency in serum from RA patients
Serum from 21 RA and 10 NHV donors was incubated with 13.5 nM PAD4 to induce citrullination, without or with different doses of hzl3-5 D3 IE. Citrullination of 5 selected peptides for proteoglycan 4 (PRG4), fibrinogen A (FGA), Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha- 1-microglobulin/bikunin precursor (AMBP) and gelsolin (GSN) was measured by LC-MS and reported as a percent of citrullinated over total peptide. Citrullination of these peptides was observed in all samples with inter-donor variability. hzl3-5 D3 IE inhibited citrullination of each of these peptides in a dose dependent manner and potency similar between NHV and RA (Table 17, no statistical differences between NHV and RA by Mann Whitney test). In addition, there was no correlation between any of the IC50s and the presence of anti-PAD4 autoantibodies (Spearman, not shown), as measured by ELISA (FIG. 13 A), suggesting that the presence of endogenous PAD4 antibodies do not affect potency of hzl3-5 D31E. Table 17 shows IC50 of hzl3-5 D31E on citrullination of 5 serum peptides from RA or NHV donors. SD refers to the standard deviation. The sequences shown in Table 17 are SEQ ID NOs: 236-240, respectively, from top to bottom.
Table 17
SEQ ID Peptide sequence IC50 IC50
Protein NO: (citrullination sites that were (nM) (nM) assessed are bold and underlined) mean mean
(SD) for (SD) for
RA NHV
(n=21) (n=10)
PRG4 247 AITTRSGQTLSK 4.9 (3 5) 4.4 (2 3)
FGA 249 QFTSSTSYNRGDSTFESK 4.5(1 6) 5.5(2 3)
ITIH4 252 QLGLPGPPDVPDHAAYHPFRR 5.2(1 5) 5.7(2 6) AMBP 253 GPCRAFIQLWAFDAVK 4.3(1.0) 5.1(2 1)
GSN 255 ATASRGASQAGAPQGR 7.1(3.6) 7.7(9.3)
With two independent assays, no shift in potency of hzl3-5 D3 IE in the presence of endogenous anti-PAD4 autoantibodies was observed in this set of 21 RA serum samples.
Example 15: Targeted proteomic analysis of citrullinated PRG4 after immunocapture enrichment of citrullinated signature peptide
This example describes a method for performing targeted analysis of citrullinated proteoglycan 4 (PRG4).
C. Materials and methods
Plasma samples from normal healthy volunteers and rheumatoid arthritis patients were analyzed in this example. To obtain plasma, whole blood samples were collected in sodium heparin vacutainer tubes and plasma was isolated from the whole blood samples using standard methods. All plasma samples were frozen until aliquoting into plates for sample preparation for analysis. Cit-PRG4 concentrations in plasma samples from 30 rheumatoid (RA) patients and 10 normal healthy volunteers (NHV) were analyzed.
1. Sample preparation for HPLC-MS/MS
Before quantifying Cit-PRG4, plasma proteins were denatured, trypsin-digested, and cit-peptide of interest (AITTRSGQTLSK (SEQ ID NO: 247)) was immunocaptured with rabbit anti-human PRG4 (1384-1397) polyclonal antibody (pAb). The antibody used for the immunoenrichment was custom generated by immunizing rabbits with (KLH)-C- TARAITTRSGQTLS (SEQ ID NO: 371), where KLH is a carrier protein keyhole limpet hemocyanin.
Twenty-five pL of each plasma sample was aliquoted into a 96-well KingFisher plate and 225 pL of 50 mM ammonium bicarbonate in water was added to dilute samples 10-fold. Proteins in the diluted samples were denatured for 20 min at 80 °C and digested with 5 pg of trypsin per sample. After digestion, trypsin was heat inactivated for 10 min at 95 °C. To immunocapture AITTRSGQTLSK (SEQ ID NO: 247), samples were cooled to room temperature and 40 pL of beads containing 0.20 pg/pL of anti-PRG4 pAb immobilized on tosylated magnetic beads were added to each well. After incubation for 60 min at 25 °C with shaking at 1000 rpm, 96-well plate was placed on the KingFisher robotic system. Beads were washed twice with 500 pL of PBST buffer, followed by one wash with 500 pL of PBS buffer. Peptides bound to the beads were eluted using 115 pL of 12 mM HC1 with 0.05% Zwittergent. Ten pL of the internal standard working solution in 80/20 water/acetonitrile with 10% formic acid was added to each well, samples were mixed, transferred to a clean LoBind 96-well plate, and analyzed by LC-MS.
2. HPLC-MS/MS conditions
The digested surrogate peptide AITTKSGQTLSK (SEQ ID NO: 372) was monitored with selective reaction monitoring (SRM) on Sciex 7500 mass spectrometer using a 7 min LC gradient. Chromatographic separation was performed on an AC QUIT Y UPLC HSS T3 100 A, 1.8 pm, 2.1 mm x 50 mm column (Waters Corporation, MA, USA) with the column temperature set to 60 °C. Mobile phase A contained 0.1% formic acid (FA) in water and mobile phase B contained 0.1% FA in acetonitrile. A gradient separation program was used as following: 0-1.0 min 2% B; 1.0-4.9 min 2-20% B; 4.9-5.0 min 20-95% B; 5.0-6.0 min hold at 95% B; 6.0-6.1 min 95-2% B; and the run was stopped at 7.0 min. The flow rate was set at 0.6 mL/min and the injection volume was 30 pL. The mass spectrometer was operated using positive ion electrospray ionization. The following optimized MS conditions were used: curtain gas and collision gas were set as 40 and 12; the turbo spray voltage was set at 2500 V and ion source gas 1 and gas 2 were both set at 55 psi. The probe temperature was set at 650°C. AITTKSGQTLSK (SEQ ID NO: 372) was quantified using SRM transition of m/z 422.14 > 540.59 (CE: 18.3 eV).
D. Results
Comparison of Cit-PRG4 (surrogate peptide AITTKSGQTLSK; SEQ ID NO: 372) concentrations measured in RA and NHV plasma samples is shown in FIG. 14. These results indicate that citrullination of PRG4 is higher in RA patients than in normal healthy volunteers.
Example 16: Citrullination in human serum induced in vitro by exogenous PAD4 and/or exogenous PAD2a
Global proteomics experiments were carried out using serum samples from normal healthy volunteers to identify sites on human proteins that are citrullinated by PAD4 and/or PAD2.
A. Materials and Methods
Induction of citrullination in vitro by exogenous PAD4 in human serum. A starting enzyme reaction mixture containing a concentration of 1 mM of the recombinant human (rh) enzyme (1 mM of rhPAD4 or ImM of rhPAD2), 1 M Tris, 20 mM DDT, and 10 mM CaCh was used. The starting enzyme reaction mixture was diluted as needed with a 10-fold reaction buffer that contained 1 M Tris, 20 mM DDT, and 10 mM CaCh to achieve a mixture with lOx the final desired enzyme concentration. Then, 4 pl of the relevant lOx enzyme reaction mixture was added to 36 pl of serum in plates (BD 353263) to provide a final concentration of lOnM PAD4 or 3nM of PAD2. The wells without added PAD enzyme served as noninduction controls. Plates were incubated at 37°C in CO2 incubator for 2 hours. Reactions were stopped with 2 pl of 0.5 M EDTA. Plates were sealed and stored at -80°C.
Global proteomics. The serum mixture (serum + enzyme + antibody, prepared as described above) was thawed and 5 pL was passed over lOOul Thermo Top 14 Depletion Igg agarose beads (Cat. No. A36372). This was performed on an Agilent microfilter plate (Cat. No. 200989-100) after shaking for 15min and eluting with 1200xG for 2min. The eluate was combined with 50 pl 2X iST-BCT LYSE buffer (PreOmics, cat# P.O.00120) and heated for 10 minutes at 95°C. Samples were enzymatically digested with addition of trypsin and LysC, and resulting peptides cleaned up according to manufacturer’s protocol using iST-BCT kit (PreOmics, cat# P.O.00116).
Peptides were analyzed by nanoLC-MS/MS on a Bruker timsTOF mass spectrometer using a 13min diaPASEF method with a 200ng load. diaPASEF data was searched against a previously generated Data dependent acquisition (DDA) spectral library generated from NHV serum. Data independent acquisition (DIA) runs were subsequently analyzed using the Fragpipe and DIANN workflow (Demichev, V., Szyrwiel, L., Yu, F. et al. dia-PASEF data analysis using FragPipe and DIA-NN for deep proteomics of low sample amounts. Nat Commun 13, 3944 (2022). doi. org/ 10.1038/s41467-022-31492-0).
For differential citrullinome analysis across different treatment groups, the precursor values were log2 -normalized and precursors carrying arginine residues with (citrullination) modification were annotated as citrullinated (see bold R residues in peptide sequences of table below). Precursors showing citrullination on the last arginine residue were removed from further analysis as they were deemed misassigned. Protein level intensity values were also log2 -transformed before downstream analysis. Both citrullinated precursor and protein data were modeled using linear regression (“limma” package in R) where donor-to-donor variation was accounted for. For assessing which citrullination sites were citrullinated by which PAD enzymes (PAD2, PAD4, or both), citrullinated precursors at 3nM PAD2 and lOnM PAD4 treatment groups were compared to no-treatment control group separately. Precursors that were significantly citrullinated by PAD2 at adjusted p-value cut-off of 0.01, but showed no statistically significant effect by PAD4 treatment, are designed in the results below as "PAD2-citrullinated". Similarly, precursors that were significantly citrullinated by PAD4, but showed no statistically significant effect by PAD2 treatment, are designated in the results below as PAD4-citrullinated. Precursors that were significantly citrullinated by both PAD2 and PAD4 are designated in the results below as “common” targets.
B. Global Proteomics Results
The addition of exogenous PAD proteins at various concentrations induced more than 452 unique citrullinated peptide precursors which originated from more than 350 serum proteins (only selected results are shown; see also Example 5 for results obtained with different experimental conditions). When PAD enzyme concentration levels considered physiologically relevant were used in the assay, as described in this Example, many citrullinated proteins were induced even at these lower concentrations. A list of selected, exemplary peptides that were citrullinated with the designated PAD enzymes, based on the analysis described above, are shown in Table 18 below. Table 18 also shows particular arginine (R) residues in each peptide or protein that were citrullinated, using bold face and underlining. The peptides in the table below also in some cases were found to also include chemical modifications at other residues, such as oxidation of methionine (M), carbamidomethylation at certain cysteine (C) residues, or modifications at the N-terminus of the peptide, which are noted by underlining those residues, or in the case of a modification at the N-terminus, underlining the first residue in the peptide sequence. Each row of the table 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.
Figure imgf000203_0001
Figure imgf000204_0001
Figure imgf000205_0001
Figure imgf000206_0001
Figure imgf000207_0001
References
R. Huang and G. Chen, Higher order structure characterization of protein therapeutics by hydrogen/deuterium exchange mass spectrometry. Analytical and Bioanalytical Chemistry, 2014, 406, 6541-6558. H. Wei, J. Mo, L. Tao, R. J. Russell, A. A. Tymiak, G. Chen, R. E. lacob, J. R. Engen, Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications. Drug Discovery Today, 2014, 19, 95-102.
L. M. Jones, J. B. Sperry, J. A. Carroll, M. L. Gross, Fast photochemical oxidation of proteins for epitope mapping. Analytical Chemistry, 2011, 83, 7657-7661.
H. Zhang, J. Wen, R. Huang, R. Blankenship, M. Gross, Mass spectrometry-based carboxyl footprinting of proteins: Method evaluation. International Journal of Mass Spectrometry, 2012, 312, 78-86.
C. Y. Tremblay, P. Limpikirati, R. W. Vachet. J. Am. Soc. Mass Spectrom. 2021, 32, 5, 1237-1248
Xu L et al. (2002) Chemistry & Biology 9: 933
Roberts RW and JW Szostak (1997) Proc. Natl. Acad. Sci. USA 94: 12297
Kurz et al. (2000) Nucleic Acids Res. 28(18):E83
Joubert, M.K., et al., Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody -Based Biotherapeutics. PLoS One, 2016. 11(8): p. e0159328.
Wang, P., et al., A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach. PLoS Comput Biol, 2008. 4(4): p. el000048.
De Groot, A.S. and W. Martin, Reducing risk, improving outcomes: bioengineering less immunogenic protein therapeutics. Clin Immunol, 2009. 131(2): p. 189-201.
Hua, F., et al., Anti-IL21 receptor monoclonal antibody (ATR-107): Safety, pharmacokinetics, and pharmacodynamic evaluation in healthy volunteers: a phase I, first-inhuman study. J Clin Pharmacol, 2014. 54(1): p. 14-22.
Prometheus, July 2012, Proleukin® (aldesleukin), accessdata.fda.gov/drugsatfda_docs/label/2012/103293s51301bl.pdf.
Li, M., Lin, C., Deng, H., Strnad, J., Bemabei, L., Vogl, D. T., Burke, J. J., & Nefedova, Y. (2020). A Novel Peptidylarginine Deiminase 4 (PAD4) Inhibitor BMS-P5 Blocks Formation of Neutrophil Extracellular Traps and Delays Progression of Multiple Myeloma. Molecular cancer therapeutics, 19(7), 1530-1538. https://doi.org/10.1158/1535- 7163.MCT-19-1020
Teijeira, A., Garasa, S., Gato, M., Alfaro, C., Migueliz, I., Cirella, A., de Andrea, C., Ochoa, M. C., Otano, I., Etxeberria, I., Andueza, M. P., Nieto, C. P., Resano, L., Azpilikueta, A., Allegretti, M., de Pizzol, M., Ponz-Sarvise, M., Rouzaut, A., Sanmamed, M. F., Schalper, K., . . . Melero, I. (2020). CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune
Cytotoxicity. Immunity, 52(5), 856-871. e8. https://doi.Org/10.1016/j.immuni.2020.03.001
Wang, Y., Liu, C., Zhang, N., Song, X., Song, Y., Cai, D., Fang, K., & Chang, X. (2022). Anti-PADI4 antibody suppresses breast cancer by repressing the citrullinated fibronectin in the tumor microenvironment. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 153, 113289. Advance online publication. https://doi.Org/10.1016/j.biopha.2022.113289
Yazdani, H. O., Roy, E., Comerci, A. J., van der Windt, D. J., Zhang, H., Huang, H., Loughran, P., Shiva, S., Geller, D. A., Bartlett, D. L., Tsung, A., Sheng, T., Simmons, R. L., & Tohme, S. (2019). Neutrophil Extracellular Traps Drive Mitochondrial Homeostasis in Tumors to Augment Growth. Cancer research, 79(21), 5626-5639. https://doi.org/10.1158/0008-5472.CAN-19-0800
DESCRIPTION OF ADDITIONAL SEQUENCES
The following table provides a listing of certain sequences referenced herein. In the antibody variable region sequences disclosed herein, for example, for certain anti-PAD4 antibodies, the heavy chain variable region (VH) CDR1, CDR2, and CDR3 sequences are located at Kabat positions comprising amino acids 26-35, 50-65, and 95-102, respectively, and which correspond to amino acid positions 26-35, 50-66, and 99-108 of SEQ ID NO: 10 and NO: 78 in the table below, and the light chain variable region (VL) CDR1, CDR2, and CDR3 sequences are located at Kabat positions comprising amino acids 24-34, 50-56, and 89-97, respectively, and which correspond to amino acid positions 24-38, 54-60, and 93-101 of SEQ ID NO: 12 and NO: 80 in the table below. In certain VH and VL sequences, the CDR sequences are in bold text, in some cases also with underlining. In certain humanized antibody VH and VL sequences, underlined amino acids in the framework regions indicate framework back mutations to mouse amino acids. In certain full-length heavy and light chain sequences, variable regions are in italics. In certain mutant constant region sequences, constant region residues that are altered compared to the corresponding wild-type sequence are shown in bold underlining. Additional sequences are denoted elsewhere in the description.
Figure imgf000209_0001
Figure imgf000210_0001
Figure imgf000211_0001
Figure imgf000212_0001
Figure imgf000213_0001
Figure imgf000214_0001
Figure imgf000215_0001
Figure imgf000216_0001
Figure imgf000217_0001
Figure imgf000218_0001
Figure imgf000219_0001
Figure imgf000220_0001
Figure imgf000221_0001
Figure imgf000222_0001
Figure imgf000223_0001
Figure imgf000224_0001
Figure imgf000225_0001
Figure imgf000226_0001
Figure imgf000227_0001
Figure imgf000228_0001
Figure imgf000229_0001
Figure imgf000230_0001
Figure imgf000231_0001
Figure imgf000232_0001
Figure imgf000233_0001
Figure imgf000234_0001
Figure imgf000235_0001
Figure imgf000236_0001

Claims

What is Claimed is:
1. A method of determining citrullination of a protein or peptide fragment thereof, comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof in a biological sample from a subject, wherein the biological sample has been exposed to a PAD4 inhibitor, wherein the 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 R748 of PO 1024),
R688 of inter-alpha-trypsin inhibitor heavy chain H4 (corresponding to R688 of QI 4624),
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).
2. A method of assessing the activity of a PAD4 inhibitor, the method comprising assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample from a subject, wherein the biological sample has been exposed to the PAD4 inhibitor, wherein the 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 R748 of PO 1024),
R688 of inter-alpha-trypsin inhibitor heavy chain H4 (corresponding to R688 of QI 4624),
R297 of alpha- 1-microglobulin/bikunin precursor (corresponding to R297 of P02760), R715 of alpha-2-macroglobulin (corresponding to R715 of P01023), 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).
3. The method of claim 2, wherein the PAD4 inhibitor reduces citrullination at the citrullination site in a dose-dependent manner.
4. The method of claim 1, 2, or 3, wherein the method comprises assessing citrullination of a peptide fragment of the protein, wherein the peptide fragment comprises the citrullination site.
5. The method of any one of claims 1-4, wherein the biological sample has been exposed to a PAD inhibitor in vivo in a subject as a result of the PAD inhibitor being administered to the subject.
6. The method of any one of claims 1-4, wherein the biological sample has been exposed to the PAD inhibitor by contacting the biological sample with the PAD inhibitor ex vivo.
7. The method of any one of claims 1-6, wherein the assessing citrullination of the citrullination site comprises mass spectrometry (MS).
8. The method of any one of claims 1-6, wherein the assessing citrullination of the citrullination site comprises liquid chromatography and mass spectrometry (LC-MS).
9. The method of any one of claims 1-6, wherein the assessing citrullination comprises selective reaction monitoring and chromatographic separation.
10. The method of any one of claims 1-9, wherein assessing citrullination of the citrullination site comprises measuring, in the biological sample, a first concentration of the citrullinated protein or peptide fragment thereof and a second concentration of corresponding total protein.
11. The method of claim 10, wherein assessing citrullination of the citrullination site comprises determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.
12. The method of claim 11, wherein the method comprises comparing the citrullination ratio to a reference citrullination ratio.
13. The method of claim 12, wherein the reference citrullination ratio is a citrullination ratio determined for a control biological sample.
14. The method of claim 13, wherein the control biological sample is a biological sample that: (a) has not been exposed to a PAD4 modulator or PAD4 inhibitor, (b) is from the same subject, and/or (c) is from the same subject prior to treatment with the PAD4 inhibitor.
15. The method of any one of claims 12-14, wherein 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 claims 1-15, wherein the method comprises contacting the biological sample with exogenous PALM.
17. A method comprising:
(i) contacting a biological sample from a subject with exogenous PALM, and
(ii) assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in a biological sample; wherein the citrullination site is selected from one or more of the following:
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 R748 of PO 1024),
R688 of inter-alpha-trypsin inhibitor heavy chain H4 (corresponding to R688 of
QI 4624),
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).
18. The method of claim 16 or 17, wherein the method comprises incubating the exogenous PALM with the biological sample.
19. The method of claim 18, wherein the method comprises incubating the exogenous PALM with the biological sample at a temperature of 36-38 °C.
20. The method of claim 18 or 19, wherein the incubating is for 1-3 hours.
21. The method of any one of claims 18-20, wherein the method comprises adding EDTA to the biological sample after the incubation with exogenous PAD4.
22. The method of any one of claims 1-21, wherein the method comprises incubating the biological sample for an incubation period before assessing citrullination.
23. The method of claim 22, wherein the incubating is performed at 35-40°C.
24. The method of claim 22 or 23, wherein the incubation period is 48 to 96 hours.
25. The method of any one of claims 1-24, comprising preparing the biological sample for assessment prior to the assessing.
26. The method of claim 25, wherein the preparing comprises enzymatically digesting proteins in the biological sample to form peptide fragments thereof.
27. The method of claim 25 or 26, wherein the preparing comprises depleting proteins or peptide fragments thereof from the biological sample that are not targeted by the assessing.
28. The method of any one of claims 25-27, wherein the preparing comprises diluting the biological sample.
29. The method of any one of claims 25-28, wherein the preparing comprises denaturing proteins or peptide fragments thereof in the biological sample.
30. The method of any one of claims 25-29, wherein the preparing comprises enriching the biological sample for a protein or peptide fragment thereof that comprises the citrullination site to be assessed.
31. The method of claim 30, wherein the preparing comprises contacting the biological sample with an antibody that specifically binds to citrullinated and noncitrullinated forms of the protein or peptide fragment thereof that comprises the citrullination site to be assessed.
32. The method of any one of claims 1-31, wherein the method comprises freezing and thawing the biological sample before assessing citrullination of the citrullination site.
33. The method of any one of claims 1-32, comprising:
(a) immunoenriching the sample for the protein or peptide fragment thereof to form an immunoenriched sample, wherein the immunoenriching comprises contacting the sample with an antibody that binds to both citrullinated and noncitrullinated forms of the protein and/or to both citrullinated and noncitrullinated forms of the peptide fragment thereof comprising the citrullination site, (b) enzymatically digesting proteins in the sample either before or after the immunoenriching to form the peptide fragment, and
(c) assessing citrullination of the peptide fragment at the citrullination site.
34. The method of claim 33, wherein the immunoenriching comprises contacting the sample with an immobilized antibody that binds to both citrullinated and noncitrullinated forms of the protein and/or to both citrullinated and noncitrullinated forms of the peptide fragment thereof comprising the citrullination site, and eluting protein and/or peptide bound to the immobilized antibody.
35. The method of claim 33 or 34, wherein the enzymatically digesting is conducted before the immunoenriching.
36. The method of claim 33 or 34, wherein the enzymatically digesting is conducted after the immunoenriching.
37. The method of any one of claims 33-36, wherein the immunoenriching comprises incubating the sample with the immobilized antibody for at least 30 minutes.
38. The method of claim 37, wherein the incubating is at a temperature of 22-28°C.
39. The method of claim 37 or 38, wherein the incubating is for a period of 30 to 90 minutes.
40. The method of any one of claims 33-38, wherein the immunoenriching comprises shaking during the incubating.
41. The method of any one of claims 34-40, wherein the immunoenriching comprises removing the immobilized antibody from the sample and washing the immobilized antibody prior to the eluting.
42. The method of any one of claims 34-41, wherein the eluting comprises washing the immobilized antibody with an elution composition under acidic conditions.
43. The method of claim 42, wherein the elution composition comprises a detergent.
44. The method of claim 43, wherein the detergent is a zwitterionic detergent.
45. The method of any one of claims 34-44, the method comprises neutralizing eluted protein in a buffer prior to assessing, and optionally prior to enzymatically digesting.
46. The method of any one of claims 33-45, wherein the antibody is immobilized by attachment to a solid surface.
47. The method of any one of claims 33-46, wherein the assessing citrullination comprises measuring a first concentration of citrullinated peptide in the digested peptides and a second concentration of signature peptide in the digested peptides, and optionally determining a citrullination ratio, wherein the citrullination ratio is a ratio of the first concentration to the second concentration.
48. The method of any one of claims 33-47, comprising denaturing proteins in the immunoenriched sample prior to the assessing.
49. The method of any one of claims 34-48, comprising denaturing the eluted protein prior to the assessing.
50. The method of claim 49, comprising denaturing the eluted protein prior to enzymatically digesting the eluted protein.
51. The method of any one of claims 33-50, comprising diluting the plasma or serum sample prior to the immunoenriching and performing the immunoenriching on the sample that has been diluted.
52. The method of claim 51, comprising diluting the plasma or serum sample by 2 to 1000 fold.
53. The method of claim 51 or 52, wherein the sample that has been diluted has a volume of at least 5 pl.
54. The method of any one of claims 1-53, wherein the 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).
55. The method of any one of claims 1-54, wherein the biological sample comprises whole blood, plasma, serum, or blood supernatant.
56. The method of claim 55, wherein the citrullination site is: R1391 of proteoglycan 4 (corresponding to R1391 of Q92954), R573 of fibrinogen alpha chain (corresponding to
Figure imgf000243_0001
R591 ofP02671 or R593 ofP02671.
57. The method of claim 56, wherein the 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.
58. The method of any one of claims 1-54, wherein the biological sample comprises synovial fluid.
59. The method of claim 58, wherein the 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).
60. The method of claim 59, wherein the 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).
61. The method of any one of claims 1-60, wherein the method comprises assessing citrullination of two or more proteins or peptide fragments thereof.
62. The method of any one of claims 1-60, wherein the method further comprises assessing citrullination of at least one additional citrullination site listed in Table A or in any one of Tables 3-18.
63. A method comprising:
(a) immunoenriching a biological 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 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
(b) assessing citrullination in the immunoenriched sample at the citrullination site.
64. A method comprising:
(a) immunoenriching a biological sample for a protein 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 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,
(b) enzymatically digesting the immunoenriched sample to form peptide fragments of the protein of interest, wherein parts (a) and (b) may be performed in any order, and
(c) assessing citrullination of the peptide fragments at the citrullination site.
65. 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,
(b) eluting protein bound to the immobilized antibody,
(d) enzymatically digesting the eluted protein to form peptide fragments thereof, and
(e) assessing citrullination of the peptide fragments at the citrullination site.
66. A method comprising
(a) enzymatically digesting proteins in a biological sample to form peptide fragments thereof,
(b) immunoenriching the enzymatically digested biological sample by contacting the sample with an immobilized antibody that specifically binds to both citrullinated and noncitrullinated forms of a peptide fragment of a protein of interest, the peptide fragment comprising a citrullination site,
(c) eluting peptides bound to the immobilized antibody, and
(d) assessing citrullination of the peptides at the citrullination site.
67. The method of any one of claims 63-66, wherein the sample comprises whole blood, plasma, serum, or blood supernatant.
68. A method of assessing the activity of a PAD4 inhibitor, the method comprising
(a) obtaining a biological sample from a subject following treatment of the subject with at least one dose of a PAD4 inhibitor, and
(b) assessing citrullination of a citrullination site on a protein or a peptide fragment thereof that is present in the biological sample.
69. An in vitro method of assessing the activity of a PAD4 inhibitor, the method comprising
(a) treating a biological sample from a subject with a PAD4 inhibitor to form a treated biological sample, and 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.
70. An in vitro method of assessing the activity of a PAD4 inhibitor, the method comprising
(i) dividing a biological sample obtained from a subject into a plurality of biological samples,
(ii) contacting each of the plurality of biological samples with a different dose of the PAD4 inhibitor, 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.
71. The method of claim 70, further comprising calculating an IC50 for the PAD4 inhibitor based on the outcome of the assessing.
72. The method of any one of claims 68 or 70-71, further comprising selecting or adjusting a dose of a PALM inhibitor to be administered to the subject based on the outcome of the assessing.
73. An in vitro method of assessing citrullination by endogenous PALM, the method comprising
(a) incubating a whole blood sample from a subject at 35-40°C for an incubation period,
(b) after the incubation period, separating plasma or supernatant from the whole blood sample, and
(c) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant.
74. An in vitro method of assessing changes in citrullination in a subject, the method comprising
(a) incubating a whole blood sample from a subject at 35-40°C for an incubation period, wherein the sample is obtained from the subject following administration of a PAD4 inhibitor to the subj ect,
(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.
75. The method of claim 74, further comprising
(d) incubating a second whole blood sample from the subject at 35-40°C for an incubation period, wherein the second whole blood sample is obtained from the subject before administration of the PAD4 inhibitor to the subject,
(e) after the incubation period, separating plasma or supernatant from the second whole blood sample, and
(f) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant from the second whole blood sample.
76. The method of claim 74, further comprising comparing an outcome of the assessing in step (f) with an outcome of the assessing in step (c).
77. A method of assessing effects of a PALM inhibitor, the method comprising
(a) exposing a whole blood sample from a subject to a PALM inhibitor in vitro,
(b) incubating the whole blood sample at 35-40°C for an incubation period,
(c) after the incubation period, separating plasma or supernatant from the whole blood sample, and
(d) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the plasma or supernatant.
78. The method of claim 75, further comprising
(e) incubating a control whole blood sample at 35-40°C for an incubation period,
(f) after the incubation period, separating control plasma or supernatant from the control whole blood sample, and
(g) assessing citrullination of a citrullination site on a protein or peptide fragment thereof that is present in the control plasma or supernatant.
79. The method of claim 78, further comprising comparing an outcome of the assessing in step (g) with an outcome of the assessing in step (d).
80. 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 35-40°C for an incubation period.
81. The method of claim 88, wherein the plasma or supernatant has been frozen and the method comprises thawing the plasma or supernatant prior to the assessing.
82. A method of assessing citrullination at a citrullination site, the method comprising
(i) measuring a first concentration of a citrullinated protein or a citrullinated peptide in a biological sample by mass spectrometry (MS), wherein the citrullinated protein or citrullinated peptide is citrullinated at a citrullination site, and
(ii) measuring a second concentration of corresponding total protein in the biological sample by MS.
83. The method of claim 82, wherein the method comprises (i) measuring a first concentration of a citrullinated peptide from a target protein in a biological sample by 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.
84. The method of claim 82 or 83, further comprising calculating a ratio of the first concentration to the second concentration
85. The method of any one of claims 82-84, wherein the method comprises measuring the first concentration and the second concentration for each of a plurality of different proteins or peptides.
86. The method of claim 85, wherein the method comprises measuring the first concentration for each of two different peptides, each of which are nonoverlapping fragments of the same protein and contain different citrullination sites.
87. The method of claim 85 or 86, wherein the method comprises calculating a ratio of the first concentration to the second concentration for each of the plurality of different proteins or peptides.
88. The method of any one of claims 63-87, wherein the citrullination site a site listed in Table A or in any one of Tables 3-18.
89. The method of any one of claims 63-88, wherein the 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 R748 of PO 1024), R688 of inter-alpha-trypsin inhibitor heavy chain H4 (corresponding to R688 of QI 4624),
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).
90. The method of claim 89, wherein 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).
91. The method of any one of claims 1-90, wherein the biological sample is obtained from a subject that is ACPA positive.
92 The method of any one of claims 1-91, wherein the biological sample is obtained from a subject that is positive for endogenous PAD4 antibodies.
93. The method of claim 92, wherein the subject is positive for endogenous PAD4 activating antibodies.
94. The method of any one of claims 1-93, wherein the biological sample is obtained from a subject that has been diagnosed with a citrullinati on-related disease or is at risk for developing a citrullination-related disease.
95. The method of any one of claims 1-94, wherein the method further comprises selecting the subject from which the biological sample was derived for treatment of a citrullinati on- related disease.
96. The method of claim 95, wherein the citrullination-related disease is an autoimmune disorder.
97. The method of claim 96, wherein the citrullination-related disease is rheumatoid arthritis, lupus (e.g., systemic lupus erythematosus (SLE)), lupus nephritis, vasculitis (e.g., ANCA- associated vasculitis), thrombosis (e.g, venous thrombosis), or inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn’s disease).
98. The method of any one of claims 1-97, wherein the biological sample is obtained from a subject following treatment of the subject with at least one dose of a PAD4 inhibitor.
99. The method of any one of claims 1-98, wherein the biological sample is obtained from a subject that is a normal, healthy subject.
100. The method of any one of claims 1-99, wherein the method further comprises administering a PAD4 inhibitor to the subject, and optionally assessing citrullinati on of a citrullinati on site on a protein or peptide fragment thereof in a sample from the subject collected following administering of the PAD4 inhibitor
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Citations (81)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0154316A2 (en) 1984-03-06 1985-09-11 Takeda Chemical Industries, Ltd. Chemically modified lymphokine and production thereof
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
EP0401384A1 (en) 1988-12-22 1990-12-12 Kirin-Amgen, Inc. Chemically modified granulocyte colony stimulating factor
EP0404097A2 (en) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Bispecific and oligospecific, mono- and oligovalent receptors, production and applications thereof
WO1993001161A1 (en) 1991-07-11 1993-01-21 Pfizer Limited Process for preparing sertraline intermediates
US5187458A (en) 1989-09-21 1993-02-16 Nihon Musen Kabushiki Kaisha Composite longitudinal vibration mechanical filter having central frequency deviation elimination means and method of manufacturing same
WO1993008829A1 (en) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions that mediate killing of hiv-infected cells
WO1993016185A2 (en) 1992-02-06 1993-08-19 Creative Biomolecules, Inc. Biosynthetic binding protein for cancer marker
WO1994029351A2 (en) 1993-06-16 1994-12-22 Celltech Limited Antibodies
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
WO1997030087A1 (en) 1996-02-16 1997-08-21 Glaxo Group Limited Preparation of glycosylated antibodies
US5714350A (en) 1992-03-09 1998-02-03 Protein Design Labs, Inc. Increasing antibody affinity by altering glycosylation in the immunoglobulin variable region
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO1998058964A1 (en) 1997-06-24 1998-12-30 Genentech, Inc. Methods and compositions for galactosylated glycoproteins
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
WO1999022764A1 (en) 1997-10-31 1999-05-14 Genentech, Inc. Methods and compositions comprising glycoprotein glycoforms
WO1999054342A1 (en) 1998-04-20 1999-10-28 Pablo Umana Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
WO2000042072A2 (en) 1999-01-15 2000-07-20 Genentech, Inc. Polypeptide variants with altered effector function
US6121022A (en) 1995-04-14 2000-09-19 Genentech, Inc. Altered polypeptides with increased half-life
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
US6248516B1 (en) 1988-11-11 2001-06-19 Medical Research Council Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors
WO2001058957A2 (en) 2000-02-11 2001-08-16 Lexigen Pharmaceuticals Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US6277375B1 (en) 1997-03-03 2001-08-21 Board Of Regents, The University Of Texas System Immunoglobulin-like domains with increased half-lives
WO2002006919A2 (en) 2000-07-18 2002-01-24 Aegis Analytical Corporation System, method and computer program product for mapping data of multi-database origins
EP1176195A1 (en) 1999-04-09 2002-01-30 Kyowa Hakko Kogyo Co., Ltd. Method for controlling the activity of immunologically functional molecule
WO2003011878A2 (en) 2001-08-03 2003-02-13 Glycart Biotechnology Ag Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity
WO2003035835A2 (en) 2001-10-25 2003-05-01 Genentech, Inc. Glycoprotein compositions
WO2004016750A2 (en) 2002-08-14 2004-02-26 Macrogenics, Inc. FcϜRIIB-SPECIFIC ANTIBODIES AND METHODS OF USE THEREOF
WO2004029207A2 (en) 2002-09-27 2004-04-08 Xencor Inc. Optimized fc variants and methods for their generation
WO2004035752A2 (en) 2002-10-15 2004-04-29 Protein Design Labs, Inc. ALTERATION OF FcRn BINDING AFFINITIES OR SERUM HALF-LIVES OF ANTIBODIES BY MUTAGENESIS
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
WO2004063351A2 (en) 2003-01-09 2004-07-29 Macrogenics, Inc. IDENTIFICATION AND ENGINEERING OF ANTIBODIES WITH VARIANT Fc REGIONS AND METHODS OF USING SAME
WO2004074455A2 (en) 2003-02-20 2004-09-02 Applied Molecular Evolution Fc REGION VARIANTS
WO2004099249A2 (en) 2003-05-02 2004-11-18 Xencor, Inc. Optimized fc variants and methods for their generation
WO2004113304A1 (en) 2003-05-22 2004-12-29 Abbott Laboratories Indazole, benzisoxazole, and benzisothiazole kinase inhibitors
WO2005040217A2 (en) 2003-10-17 2005-05-06 Cambridge University Technical Services Limited Antibodies having a mutated amino acid residue at position 268 (ch2 region) in constant regions
US20050123546A1 (en) 2003-11-05 2005-06-09 Glycart Biotechnology Ag Antigen binding molecules with increased Fc receptor binding affinity and effector function
WO2005070963A1 (en) 2004-01-12 2005-08-04 Applied Molecular Evolution, Inc Fc region variants
WO2005092925A2 (en) 2004-03-24 2005-10-06 Xencor, Inc. Immunoglobulin variants outside the fc region
US6982321B2 (en) 1986-03-27 2006-01-03 Medical Research Council Altered antibodies
US20060025576A1 (en) 2000-04-11 2006-02-02 Genentech, Inc. Multivalent antibodies and uses therefor
WO2006020114A2 (en) 2004-08-04 2006-02-23 Applied Molecular Evolution, Inc. Variant fc regions
US7087409B2 (en) 1997-12-05 2006-08-08 The Scripps Research Institute Humanization of murine antibody
US7317091B2 (en) 2002-03-01 2008-01-08 Xencor, Inc. Optimized Fc variants
US7527791B2 (en) 2004-03-31 2009-05-05 Genentech, Inc. Humanized anti-TGF-beta antibodies
WO2009089004A1 (en) 2008-01-07 2009-07-16 Amgen Inc. Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
US8101720B2 (en) 2004-10-21 2012-01-24 Xencor, Inc. Immunoglobulin insertions, deletions and substitutions
WO2014015905A1 (en) 2012-07-26 2014-01-30 Glaxo Group Limited 2 - (azaindol- 2 -yl) benz imidazoles as pad4 inhibitors
WO2014019092A1 (en) 2012-08-01 2014-02-06 The Hospital For Sick Children Inhibitors of peptidyl arginine deiminase (pad) enzymes and uses thereof
WO2016081746A2 (en) 2014-11-21 2016-05-26 Bristol-Myers Squibb Company Antibodies comprising modified heavy constant regions
WO2016143753A1 (en) 2015-03-06 2016-09-15 公立大学法人横浜市立大学 Novel anti-pad4 antibody
WO2016155745A1 (en) 2015-03-27 2016-10-06 Rigshospitalet Cross-reactive anti-pad antibodies
WO2016185279A1 (en) 2015-05-21 2016-11-24 Glaxosmithkline Intellectual Property Development Limited Benzoimidazole derivatives as pad4 inhibitors
WO2017007405A1 (en) 2015-07-03 2017-01-12 Catrina Anca Methods and compounds for the alleviation and/or prevention of bone loss
WO2017027967A1 (en) 2015-08-17 2017-02-23 University Health Network Inhibitors of peptidyl arginine deiminase (pad) enzymes and uses thereof
WO2017087678A2 (en) 2015-11-19 2017-05-26 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (gitr) and uses thereof
WO2017100594A1 (en) 2015-12-09 2017-06-15 Padlock Therapeutics, Inc. Bicyclic inhibitors of pad4
WO2017100601A1 (en) 2015-12-09 2017-06-15 Padlock Therapeutics, Inc. Aza-benzimidazole inhibitors of pad4
WO2017147102A1 (en) 2016-02-23 2017-08-31 Padlock Therapeutics, Inc. Heteroaryl inhibitors of pad4
WO2018022897A1 (en) 2016-07-27 2018-02-01 Padlock Therapeutics, Inc. Covalent inhibitors of pad4
WO2018049296A1 (en) 2016-09-12 2018-03-15 Padlock Therapeutics, Inc. Heteroaryl inhibitors of pad4
WO2019058393A1 (en) 2017-09-22 2019-03-28 Jubilant Biosys Limited Heterocyclic compounds as pad inhibitors
WO2019131769A1 (en) 2017-12-26 2019-07-04 公立大学法人名古屋市立大学 Novel anti-pad4 antibody
WO2020033490A1 (en) 2018-08-08 2020-02-13 Bristol-Myers Squibb Company Substituted thienopyrroles as pad4 inhibitors
WO2020033488A1 (en) 2018-08-08 2020-02-13 Bristol-Myers Squibb Company Substituted benzimidazoles as pad4 inhibitors
WO2020033514A1 (en) 2018-08-08 2020-02-13 Bristol-Myers Squibb Company Benzimidazole inhibitors of pad enzymes
WO2020033520A1 (en) 2018-08-08 2020-02-13 Bristol-Myers Squibb Company Indole and azaindole inhibitors of pad enzymes
US20200237771A1 (en) 2017-10-18 2020-07-30 Jubilant Epipad LLC Imidazo-pyridine compounds as pad inhibitors
WO2021158840A1 (en) 2020-02-06 2021-08-12 Bristol-Myers Squibb Company Macrocyclic pad4 inhibitors useful as immunosuppressant
WO2021163254A1 (en) 2020-02-12 2021-08-19 Bristol-Myers Squibb Company Heterocyclic pad4 inhibitors
WO2022140428A2 (en) 2020-12-22 2022-06-30 Gilead Sciences, Inc. Inhibitors of peptidylarginine deiminases
WO2022173722A1 (en) 2021-02-09 2022-08-18 Gilead Sciences, Inc. Thienopyrrole compounds
WO2022176970A1 (en) 2021-02-18 2022-08-25 田辺三菱製薬株式会社 Novel anti-pad4 antibody
WO2022221642A1 (en) 2021-04-16 2022-10-20 Gilead Sciences, Inc. Thienopyrrole compounds
WO2023083365A1 (en) 2021-11-15 2023-05-19 Qilu Regor Therapeutics Inc. Pad4 inhibitors and use thereof
US11878965B2 (en) 2020-12-22 2024-01-23 Gilead Sciences, Inc. Inhibitors of peptidylarginine deiminases
WO2024020579A1 (en) 2022-07-22 2024-01-25 Bristol-Myers Squibb Company Antibodies binding to human pad4 and uses thereof
US11976083B2 (en) 2020-04-30 2024-05-07 Gilead Sciences, Inc. Inhibitors of peptidylarginine deiminases

Patent Citations (92)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
EP0154316A2 (en) 1984-03-06 1985-09-11 Takeda Chemical Industries, Ltd. Chemically modified lymphokine and production thereof
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US6982321B2 (en) 1986-03-27 2006-01-03 Medical Research Council Altered antibodies
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5648260A (en) 1987-03-18 1997-07-15 Scotgen Biopharmaceuticals Incorporated DNA encoding antibodies with altered effector functions
US6248516B1 (en) 1988-11-11 2001-06-19 Medical Research Council Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors
EP0401384A1 (en) 1988-12-22 1990-12-12 Kirin-Amgen, Inc. Chemically modified granulocyte colony stimulating factor
EP0404097A2 (en) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Bispecific and oligospecific, mono- and oligovalent receptors, production and applications thereof
US5187458A (en) 1989-09-21 1993-02-16 Nihon Musen Kabushiki Kaisha Composite longitudinal vibration mechanical filter having central frequency deviation elimination means and method of manufacturing same
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO1993001161A1 (en) 1991-07-11 1993-01-21 Pfizer Limited Process for preparing sertraline intermediates
WO1993008829A1 (en) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions that mediate killing of hiv-infected cells
WO1993016185A2 (en) 1992-02-06 1993-08-19 Creative Biomolecules, Inc. Biosynthetic binding protein for cancer marker
US5714350A (en) 1992-03-09 1998-02-03 Protein Design Labs, Inc. Increasing antibody affinity by altering glycosylation in the immunoglobulin variable region
US6350861B1 (en) 1992-03-09 2002-02-26 Protein Design Labs, Inc. Antibodies with increased binding affinity
WO1994029351A2 (en) 1993-06-16 1994-12-22 Celltech Limited Antibodies
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US6121022A (en) 1995-04-14 2000-09-19 Genentech, Inc. Altered polypeptides with increased half-life
WO1997030087A1 (en) 1996-02-16 1997-08-21 Glaxo Group Limited Preparation of glycosylated antibodies
US6277375B1 (en) 1997-03-03 2001-08-21 Board Of Regents, The University Of Texas System Immunoglobulin-like domains with increased half-lives
WO1998058964A1 (en) 1997-06-24 1998-12-30 Genentech, Inc. Methods and compositions for galactosylated glycoproteins
WO1999022764A1 (en) 1997-10-31 1999-05-14 Genentech, Inc. Methods and compositions comprising glycoprotein glycoforms
US7087409B2 (en) 1997-12-05 2006-08-08 The Scripps Research Institute Humanization of murine antibody
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
WO1999054342A1 (en) 1998-04-20 1999-10-28 Pablo Umana Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US6602684B1 (en) 1998-04-20 2003-08-05 Glycart Biotechnology Ag Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
WO2000042072A2 (en) 1999-01-15 2000-07-20 Genentech, Inc. Polypeptide variants with altered effector function
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
EP1176195A1 (en) 1999-04-09 2002-01-30 Kyowa Hakko Kogyo Co., Ltd. Method for controlling the activity of immunologically functional molecule
WO2001058957A2 (en) 2000-02-11 2001-08-16 Lexigen Pharmaceuticals Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US20060025576A1 (en) 2000-04-11 2006-02-02 Genentech, Inc. Multivalent antibodies and uses therefor
WO2002006919A2 (en) 2000-07-18 2002-01-24 Aegis Analytical Corporation System, method and computer program product for mapping data of multi-database origins
WO2003011878A2 (en) 2001-08-03 2003-02-13 Glycart Biotechnology Ag Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity
WO2003035835A2 (en) 2001-10-25 2003-05-01 Genentech, Inc. Glycoprotein compositions
US7317091B2 (en) 2002-03-01 2008-01-08 Xencor, Inc. Optimized Fc variants
WO2004016750A2 (en) 2002-08-14 2004-02-26 Macrogenics, Inc. FcϜRIIB-SPECIFIC ANTIBODIES AND METHODS OF USE THEREOF
WO2004029207A2 (en) 2002-09-27 2004-04-08 Xencor Inc. Optimized fc variants and methods for their generation
WO2004035752A2 (en) 2002-10-15 2004-04-29 Protein Design Labs, Inc. ALTERATION OF FcRn BINDING AFFINITIES OR SERUM HALF-LIVES OF ANTIBODIES BY MUTAGENESIS
WO2004063351A2 (en) 2003-01-09 2004-07-29 Macrogenics, Inc. IDENTIFICATION AND ENGINEERING OF ANTIBODIES WITH VARIANT Fc REGIONS AND METHODS OF USING SAME
WO2004074455A2 (en) 2003-02-20 2004-09-02 Applied Molecular Evolution Fc REGION VARIANTS
WO2004099249A2 (en) 2003-05-02 2004-11-18 Xencor, Inc. Optimized fc variants and methods for their generation
WO2004113304A1 (en) 2003-05-22 2004-12-29 Abbott Laboratories Indazole, benzisoxazole, and benzisothiazole kinase inhibitors
WO2005040217A2 (en) 2003-10-17 2005-05-06 Cambridge University Technical Services Limited Antibodies having a mutated amino acid residue at position 268 (ch2 region) in constant regions
US20050123546A1 (en) 2003-11-05 2005-06-09 Glycart Biotechnology Ag Antigen binding molecules with increased Fc receptor binding affinity and effector function
WO2005070963A1 (en) 2004-01-12 2005-08-04 Applied Molecular Evolution, Inc Fc region variants
WO2005092925A2 (en) 2004-03-24 2005-10-06 Xencor, Inc. Immunoglobulin variants outside the fc region
US7527791B2 (en) 2004-03-31 2009-05-05 Genentech, Inc. Humanized anti-TGF-beta antibodies
WO2006020114A2 (en) 2004-08-04 2006-02-23 Applied Molecular Evolution, Inc. Variant fc regions
US8101720B2 (en) 2004-10-21 2012-01-24 Xencor, Inc. Immunoglobulin insertions, deletions and substitutions
WO2009089004A1 (en) 2008-01-07 2009-07-16 Amgen Inc. Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
WO2014015905A1 (en) 2012-07-26 2014-01-30 Glaxo Group Limited 2 - (azaindol- 2 -yl) benz imidazoles as pad4 inhibitors
WO2014019092A1 (en) 2012-08-01 2014-02-06 The Hospital For Sick Children Inhibitors of peptidyl arginine deiminase (pad) enzymes and uses thereof
WO2016081746A2 (en) 2014-11-21 2016-05-26 Bristol-Myers Squibb Company Antibodies comprising modified heavy constant regions
WO2016143753A1 (en) 2015-03-06 2016-09-15 公立大学法人横浜市立大学 Novel anti-pad4 antibody
US20180044434A1 (en) 2015-03-06 2018-02-15 Public University Corporation Yokohama City University Novel anti-pad4 antibody
WO2016155745A1 (en) 2015-03-27 2016-10-06 Rigshospitalet Cross-reactive anti-pad antibodies
WO2016185279A1 (en) 2015-05-21 2016-11-24 Glaxosmithkline Intellectual Property Development Limited Benzoimidazole derivatives as pad4 inhibitors
WO2017007405A1 (en) 2015-07-03 2017-01-12 Catrina Anca Methods and compounds for the alleviation and/or prevention of bone loss
WO2017027967A1 (en) 2015-08-17 2017-02-23 University Health Network Inhibitors of peptidyl arginine deiminase (pad) enzymes and uses thereof
WO2017087678A2 (en) 2015-11-19 2017-05-26 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (gitr) and uses thereof
WO2017100594A1 (en) 2015-12-09 2017-06-15 Padlock Therapeutics, Inc. Bicyclic inhibitors of pad4
WO2017100601A1 (en) 2015-12-09 2017-06-15 Padlock Therapeutics, Inc. Aza-benzimidazole inhibitors of pad4
US11198681B2 (en) 2016-02-23 2021-12-14 Padlock Therapeutics, Inc. Heteroaryl inhibitors of PAD4
WO2017147102A1 (en) 2016-02-23 2017-08-31 Padlock Therapeutics, Inc. Heteroaryl inhibitors of pad4
WO2018022897A1 (en) 2016-07-27 2018-02-01 Padlock Therapeutics, Inc. Covalent inhibitors of pad4
WO2018049296A1 (en) 2016-09-12 2018-03-15 Padlock Therapeutics, Inc. Heteroaryl inhibitors of pad4
WO2019058393A1 (en) 2017-09-22 2019-03-28 Jubilant Biosys Limited Heterocyclic compounds as pad inhibitors
US20200276206A1 (en) 2017-09-22 2020-09-03 Jubilant Epipad LLC Heterocyclic compounds as pad inhibitors
US20200237771A1 (en) 2017-10-18 2020-07-30 Jubilant Epipad LLC Imidazo-pyridine compounds as pad inhibitors
WO2019131769A1 (en) 2017-12-26 2019-07-04 公立大学法人名古屋市立大学 Novel anti-pad4 antibody
WO2020033488A1 (en) 2018-08-08 2020-02-13 Bristol-Myers Squibb Company Substituted benzimidazoles as pad4 inhibitors
US20220402950A1 (en) 2018-08-08 2022-12-22 Bristol-Myers Squibb Company Substituted benzimidazoles as pad4 inhibitors
WO2020033514A1 (en) 2018-08-08 2020-02-13 Bristol-Myers Squibb Company Benzimidazole inhibitors of pad enzymes
WO2020033520A1 (en) 2018-08-08 2020-02-13 Bristol-Myers Squibb Company Indole and azaindole inhibitors of pad enzymes
US11524959B1 (en) 2018-08-08 2022-12-13 Bristol-Myers Squibb Company Indole and azaindole inhibitors of pad enzymes
WO2020033490A1 (en) 2018-08-08 2020-02-13 Bristol-Myers Squibb Company Substituted thienopyrroles as pad4 inhibitors
US20220348562A1 (en) 2018-08-08 2022-11-03 Bristol-Myers Squibb Company Benzimidazole inhibitors of pad enzymes
WO2021158840A1 (en) 2020-02-06 2021-08-12 Bristol-Myers Squibb Company Macrocyclic pad4 inhibitors useful as immunosuppressant
US20230203039A1 (en) 2020-02-06 2023-06-29 Bristol-Myers Squibb Company Macrocyclic pad4 inhibitors useful as immunosuppressant
WO2021163254A1 (en) 2020-02-12 2021-08-19 Bristol-Myers Squibb Company Heterocyclic pad4 inhibitors
US11976083B2 (en) 2020-04-30 2024-05-07 Gilead Sciences, Inc. Inhibitors of peptidylarginine deiminases
US20220227787A1 (en) 2020-12-22 2022-07-21 Gilead Sciences, Inc. Inhibitors of peptidylarginine deiminases
WO2022140428A2 (en) 2020-12-22 2022-06-30 Gilead Sciences, Inc. Inhibitors of peptidylarginine deiminases
US11878965B2 (en) 2020-12-22 2024-01-23 Gilead Sciences, Inc. Inhibitors of peptidylarginine deiminases
WO2022173722A1 (en) 2021-02-09 2022-08-18 Gilead Sciences, Inc. Thienopyrrole compounds
WO2022176970A1 (en) 2021-02-18 2022-08-25 田辺三菱製薬株式会社 Novel anti-pad4 antibody
WO2022221642A1 (en) 2021-04-16 2022-10-20 Gilead Sciences, Inc. Thienopyrrole compounds
WO2023083365A1 (en) 2021-11-15 2023-05-19 Qilu Regor Therapeutics Inc. Pad4 inhibitors and use thereof
WO2024020579A1 (en) 2022-07-22 2024-01-25 Bristol-Myers Squibb Company Antibodies binding to human pad4 and uses thereof

Non-Patent Citations (131)

* Cited by examiner, † Cited by third party
Title
"Anti-PADI4 antibody suppresses breast cancer by repressing the citrullinated fibronectin in the tumor microenvironment", BIOMEDICINE & PHARMACOTHERAPY = BIOMEDECINE & PHARMACOTHERAPIE, vol. 153, 2022, pages 113289
"UniProt", Database accession no. P10909-3
AGNEW, CHEM INTL. ED. ENGL., vol. 33, 1994, pages 183 - 186
ALIKO ET AL., INT J MOL SCI, vol. 20, no. 9, 2019, pages 2174
ALMAGROFRANSSON, FRONT. BIOSCI, vol. 13, 2008, pages 1619 - 1633
B. AMETH ET AL., INT. J. MED. SCI, vol. 18, 2021, pages 1532 - 40
BACA ET AL., J. BIOL. CHEM, vol. 272, 1997, pages 10678 - 10684
BRENNAN ET AL., SCIENCE, vol. 229, 1985, pages 81
BRENTVILLE ET AL., ONCOIMMUNOLOGY, vol. 8, 2019, pages e1576490
C. Y. TREMBLAYP. LIMPIKIRATIR. W. VACHET, J. AM. SOC. MASS SPECTROM, vol. 32, no. 5, 2021, pages 1237 - 1248
CARTE R ET AL., PRO C. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 4285
CAUSEY ET AL., J. MED CHEM, vol. 54, no. 19, 2011, pages 6919 - 6935
CHAMARDANI ET AL., MOL. CELL. BIOCHEM, vol. 477, 2022, pages 673 - 88
CHANG ET AL., ARTHITIS RES. THER, vol. 7, 2005, pages R268
CHEN ET AL., CELL MOL BIOL LETT, vol. 26, 2021, pages 9
CHUMANEVICH ET AL., AM. J. PHYSIOL. GASTROINTEST. LIVER PHYSIOL, vol. 300, no. 6, 2011, pages 929 - 938
CLARKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628
CURRAN AMNAIK PGILES JTDARRAH E, NAT REV RHEUMATOL, vol. 16, no. 6, June 2020 (2020-06-01), pages 301 - 315
CURRAN ET AL., NAT. REV. RHEUMATOL, vol. 16, 2020, pages 301 - 15
DAL' ACQUA ET AL., JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 281, 2006, pages 23514 - 23524
DALL' ACQUA ET AL., JOURNAL OF IMMUNOLOGY, vol. 169, 2002, pages 5171 - 5180
DALL' ACQUA ET AL., METHODS, vol. 36, 2005, pages 61 - 68
DARRAH ET AL., SCI. TRANSL. MED, vol. 5, 2013, pages 186ra65
DE GROOT, A.SW. MARTIN: "Reducing risk, improving outcomes: bioengineering less immunogenic protein therapeutics", CLIN IMMUNOL, vol. 131, no. 2, 2009, pages 189 - 201, XP026035888, DOI: 10.1016/j.clim.2009.01.009
DEMICHEV, V.SZYRWIEL, L.YU, F ET AL.: "dia-PASEF data analysis using FragPipe and DIA-NN for deep proteomics of low sample amounts", NAT COMMUN, vol. 13, 2022, pages 3944
DENG ET AL., CANCER RES, vol. 82, no. 19, 2022, pages 3561 - 3572
DREYTON ET AL., BIOORG AND MED CHEM, vol. 22, no. 4, 2014, pages 1362 - 9
DREYTON, PROBE REPORTS FROM THE NIH MOLECULAR LIBRARIES PROGRAM, 2012
FERRARAALITALO, NATURE MEDICINE, vol. 5, no. 12, 1999, pages 1359 - 1364
FLATMAN ET AL., J. CHROMATOGR. B, vol. 848, 2007, pages 79 - 87
FORD ET AL., RHEUM. DIS. CLIN NORTH AM, vol. 45, 2019, pages 101 - 112
FRESNEDA ALARCON ET AL., FRONG. IMMUNOL, vol. 12, 2021, pages 649693
FUKUI S ET AL., ARTHRITIS RHEUMATOL, 15 February 2022 (2022-02-15)
GRUBER ET AL., J. IMMUNOL., vol. 152, 1994, pages 5368 - 315
GUPTA, SKAPLAN, M.J, NAT REV NEPHROL, vol. 12, no. 7, 2016, pages 402 - 413
H. CHEN ET AL., CELL MOL. BIOL. LETT, vol. 26, 2021, pages 9
H. ZHANGJ. WENR. HUANGR. BLANKENSHIPM. GROSS: "Mass spectrometry-based carboxyl footprinting of proteins: Method evaluation", INTERNATIONAL JOURNAL OF MASS SPECTROMETRY, vol. 312, 2012, pages 78 - 86
HALVORSEN ET AL., ANN. RHEUM. DIS, vol. 68, 2009, pages 249 - 52
HINTON ET AL., J. BIOL. CHEM., vol. 279, no. 8, 2004, pages 6213 - 6216
HINTON ET AL., JOURNAL OF IMMUNOLOGY, vol. 176, 2006, pages 346 - 356
HOLLIGERHUDSON, NATURE BIOTECHNOLOGY, vol. 23, 2005, pages 1126 - 1136
HOLLINGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
HUA, F. ET AL.: "Anti-IL21 receptor monoclonal antibody (ATR-107): Safety, pharmacokinetics, and pharmacodynamic evaluation in healthy volunteers: a phase I, first-inhuman study", J CLIN PHARMACOL, vol. 54, no. 1, 2014, pages 14 - 22
HUDSON ET AL., NAT. MED, vol. 9, 2003, pages 129 - 134
HUDSON ET AL., NAT. MED., vol. 9, 2003, pages 129 - 134
J. PERDOMO ET AL., NATURE COMMUN, vol. 10, no. 1, 2019, pages 1322
JIA ET AL., BIOMED PHARMACOTHER, vol. 168, 2023, pages 115826
JM BERTHELOT ET AL., JOINT BONE SPINE, vol. 84, no. 3, 2017, pages 255 - 262
JONES ET AL., ACS CHEM BIOL, vol. 7, no. 1, 2012, pages 160 - 165
JONES ET AL., CURR. OP. DRUG DISCOV. DEV., vol. 12, no. 5, 2009, pages 616 - 627
JOUBERT, M.K. ET AL.: "Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics", PLOS ONE, vol. 11, no. 8, 2016, pages e0159328, XP055514895, DOI: 10.1371/journal.pone.0159328
KLAGSBRUND' AMORE, ANNU. REV. PHYSIOL, vol. 53, 1991, pages 217 - 39
KLIMKA ET AL., BR. J. CANCER, vol. 83, 2000, pages 252 - 260
KNIGHT ET AL., ANN RHUEM DIS, vol. 74, no. 12, 2015, pages 2199 - 2206
KNUCKLEY ET AL., BIOORGMED CHEM, 2007, pages 739 - 745
KOLARZ ET AL., J. CLIN. MED, vol. 9, 2020, pages 2049
KOSTELNY ET AL., J. IMM ''NOL., no. 5, 1992, pages 1547 - 1553
KURZ ET AL., NUCLEIC ACIDS RES, vol. 28, no. 18, 2000, pages E83
L. M. JONESJ. B. SPERRYJ. A. CARROLLM. L. GROSS: "Fast photochemical oxidation of proteins for epitope mapping", ANALYTICAL CHEMISTRY, vol. 83, 2011, pages 7657 - 7661
LEWALLEN ET AL., ACS CHEM BIOL, vol. 9, no. 4, 2014, pages 913 - 921
LI ET AL., J. EXP. MED., vol. 207, 2010, pages 1853 - 62
LI ET AL., MOL CANCER THER, vol. 19, no. 7, 2020, pages 1530 - 1538
LI ET AL., MOLECULAR CANCER THERAPEUTICS, vol. 19, 2020, pages 1530 - 38
LI, M.LIN, C.DENG, H.STRNAD, J.BERNABEI, L.VOGL, D. T.BURKE, J. J.NEFEDOVA, Y: "A Novel Peptidylarginine Deiminase 4 (PAD4) Inhibitor BMS-P5 Blocks Formation of Neutrophil Extracellular Traps and Delays Progression of Multiple Myeloma", MOLECULAR CANCER THERAPEUTICS, vol. 19, no. 7, 2020, pages 1530 - 1538, Retrieved from the Internet <URL:https://doi.org/10.1158/1535-7163.MCT-19-1020>
LOGOTHETIS, C, ASCO EDUCATIONAL BOOK SPRING, vol. 730, 2000, pages 738
LUO ET AL., JAM CHEM SOC, vol. 128, no. 4, 2006, pages 1092 - 1093
MARTÍN MONREAL MARÍA ET AL: "Applicability of Small-Molecule Inhibitors in the Study of Peptidyl Arginine Deiminase 2 (PAD2) and PAD4", FRONTIERS IN IMMUNOLOGY, vol. 12, 19 October 2021 (2021-10-19), Lausanne, CH, XP093210269, ISSN: 1664-3224, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8560728/pdf/fimmu-12-716250.pdf> DOI: 10.3389/fimmu.2021.716250 *
MASSARENTISCAND ET AL., J. RHEUMATOL, vol. 48, no. 2, 2019, pages 133 - 140
MILSTEINCUELLO, NATURE, vol. 305, 1983, pages 537
MONDAL ET AL., ACC CHEM RES, vol. 52, no. 3, 2019, pages 818 - 832
MORRISON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851 - 6855
MURAKAMIW.B. SAUNDERS ET AL., PHILADELPHIA, 1995
MUTH ET AL., J MED CHEM, vol. 60, no. 7, 2017, pages 3198 - 3211
N. HANATA ET AL., FRONT. IMMUNOL, vol. 11, 2020, pages 1095
N. SORVILLO ET AL., CIRCULATION RES, vol. 125, no. 5, 2019, pages 507 - 519
NADZIRIN, COMPUT BIOL CHEM, vol. 92, 2021, pages 107487
O'SULLIVAN ET AL., RHEUMATOLOGY, vol. 58, 2019
PADLAN, MOL. IMMUNOL, vol. 28, 1991, pages 489 - 498
PAN ET AL., AUTHOREA PREPRINTS, 2021
PARDOLL, NAT. REV. CANCER, vol. 12, 2012, pages 252
PETKOVA ET AL., INTERNATIONAL IMMUNOLOGY, vol. 18, no. 12, 2006, pages 1759 - 1769
PORTOLANO ET AL., J. IMMUNOL., vol. 151, 1993, pages 2623 - 887
QIU, X.I ET AL.: "Signature peptide selection workflow for biomarker quantification using LC-MS based targeted proteomics", BIOANALYSIS, 11 April 2023 (2023-04-11)
QUEEN ET AL., PROC. NAT'L ACAD. SCI. USA, vol. 86, 1989, pages 10029 - 10033
R. HUANGG. CHEN: "Higher order structure characterization of protein therapeutics by hydrogen/deuterium exchange mass spectrometry", ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 406, 2014, pages 6541 - 6558, XP035401219, DOI: 10.1007/s00216-014-7924-3
RESTIFO, NSZNOL, M., CANCER VACCINES, CH, vol. 61, pages 3023 - 3043
REYES-CASTILLO ET AL., CLIN. EXP. IMMUNOL, vol. 182, 2015, pages 119 - 31
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329
ROBERTS RWJW SZOSTAK, PROC. NATL. ACAD. SCI. USA, vol. 94, 1997, pages 12297
ROSOK ET AL., J. BIOL. CHEM., vol. 271, 1996, pages 22611 - 22618
S. KADURAG. RAGHU, EUR. RESPIRATORY REV, vol. 30, 2021, pages 210011
SARSWAT, BIOORG MED CHEM, vol. 25, no. 9, 2017, pages 2643 - 2656
SATO, INT. J. CLIN. ONCOL, vol. 8, 2003, pages 200 - 206
SHIELDS ET AL., JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 9, 2001, pages 6591 - 6604
SHIELDS, R.L ET AL., J. BIOL. CHEM., vol. 276, 2001, pages 6591 - 6604
SHIELDS, R.L ET AL., J. BIOL. CHEM., vol. 277, 2002, pages 26733 - 26740
SIMS ET AL., J. IMMUNOL, vol. 151, 1993, pages 2296
SMOLEN ET AL., NAT. REV. DIS. PRIERS, vol. 4, 2018, pages 18001
STROHL, CURRENT OPINION IN BIOTECHNOLOGY, vol. 20, 2009, pages 685 - 691
SUSUKI ET AL., NAT. GENET, vol. 34, 2003, pages 395 - 402
SUZUKI A ET AL., BMC MUSCULOSKELET DISORD., vol. 17, 5 May 2016 (2016-05-05), pages 205
TEIJEIRA, A., GARASA, S., GATO, M., ALFARO, C., MIGUELIZ, I., CIRELLA, A., DE ANDREA, C., OCHOA, M. C., OTANO, I., ETXEBERRIA, I.,: "CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune Cytotoxicity", IMMUNITY, vol. 52, no. 5, 2020, pages 856 - 871, Retrieved from the Internet <URL:https://doi.org/10.1016/j.immuni.2020.03.001>
TEO ET AL., CHEM BIOL DRUG DES, vol. 90, no. 6, 2017, pages 1134 - 1146
TONINI ET AL., ONCOGENE, vol. 22, 2003, pages 6549 - 6556
TRAUNECKER ET AL., EMBO J., vol. 10, 1991, pages 3655
TUTT ET AL., J. IMMUNOL, vol. 147, 1991, pages 60
UMANA ET AL., NAT. BIOTECH, vol. 17, 1999, pages 176 - 180
WANG ET AL., BIOMEDICINE & PHARMACOTHERAPY, vol. 153, 2022, pages 113289
WANG ET AL., BIOMEDICINE AND PHARMACOTHERAPY, vol. 153, 2022, pages 113289
WANG ET AL., FRONT. IMMUNOL, vol. 13, 2022, pages 895216
WANG ET AL., J BIOL CHEM, vol. 287, no. 31, 2012, pages 25941 - 25953
WANG FEI ET AL: "Identification of citrullinated peptides in the synovial fluid of patients with rheumatoid arthritis using LC-MALDI-TOF/TOF", CLINICAL RHEUMATOLOGY, ACTA MEDICA BELGICA, BRUXELLES, BE, vol. 35, no. 9, 8 April 2016 (2016-04-08), pages 2185 - 2194, XP036034762, ISSN: 0770-3198, [retrieved on 20160408], DOI: 10.1007/S10067-016-3247-4 *
WANG, P. ET AL.: "A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach", PLOS COMPUT BIOL, vol. 4, no. 4, 2008, pages e1000048, XP002598739, DOI: 10.1371/journal.pcbi.1000048
WEBER, SEMIN. ONCOL, vol. 37, 2010, pages 430
WEEDING ET AL., CLIN. IMMUNOL, vol. 196, 2018, pages 110 - 116
WEI ET AL., J MED CHEM, vol. 56, no. 4, 2014, pages 1715 - 1722
WERI Y ET AL., SCI REP, vol. 5, 21 August 2015 (2015-08-21), pages 13041
WRIGHT ET AL., TIBTECH, vol. 15, 1997, pages 26 - 32
XU ET AL., CHINESE J. MICROBIOLOGY AND IMMUNOLOGY, vol. 12, 2020, pages 115 - 121
XU L ET AL., CHEMISTRY & BIOLOGY, vol. 9, 2002, pages 933
Y. LIU ET AL., JCI INSIGHT, vol. 3, no. 23, pages e124729
YADAV ET AL., J. CYST. FIBROS, vol. 18, 2018, pages 636 - 45
YAZDANI, H. O.ROY, E.COMERCI, A. J.VAN DER WINDT, D. J.ZHANG, H.HUANG, H.LOUGHRAN, P.SHIVA, S.GELLER, D. A.BARTLETT, D. L.: "Neutrophil Extracellular Traps Drive Mitochondrial Homeostasis in Tumors to Augment Growth", CANCER RESEARCH, vol. 79, no. 21, 2019, pages 5626 - 5639, Retrieved from the Internet <URL:https://doi.org/10.1158/0008-5472.CAN-19-0800>
YEUNG ET AL., J IMMUNOL, vol. 182, 2010, pages 7663 - 7671
YOSHIDA ET AL., CLIN. KIDNEY J, vol. 6, pages 308 - 12
ZHAO ET AL., J. RHEUMATOL., vol. 35, 2008, pages 969 - 74
ZHOU ET AL., NAT CHEM BIOL, vol. 20, 2024, pages 742 - 750
ZHU ET AL., ACTA PHARM SIN B, vol. 12, no. 5, 2022, pages 2592 - 2608
ZHU ET AL., EUR J MED CHEM, vol. 258, 2023, pages 115619
ZHU ET AL., J MEDCHEM, vol. 67, no. 10, 2024, pages 7973 - 7994
ZL WANG ET AL., BEIJING DA XUE XUE BAO YI XUE BAN, vol. 46, no. 2, 2014, pages 200 - 6

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