WO2025155877A2 - Antibodies binding to pad4 and uses thereof - Google Patents

Abstract

The disclosure provides antibodies that specifically bind human PAD4 and methods of use thereof. In some aspects, the disclosure is directed to methods of treating a disease in a subject, comprising administering to the subject an anti-PAD4 antibody.

Description

ANTIBODIES BINDING TO PAD4 AND USES THEREOF

STATEMENT OF GOVERNMENT FUNDING

[0001] This invention was made with government support under K99 EB030587 awarded by the National Institutes of Health. The government has certain rights in the invention.

CROSS-REFERENCE TO EARLIER FILED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63/622,494, filed January 18, 2024 and U.S. Provisional Application No. 63/624,121, filed January 23, 2024, the contents of which are incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

[0003] The content of the electronically submitted sequence listing (Name: 5542_002PC02_SequenceListing_ST26.xml, Size: 92,459 bytes; and Date of Creation: January 14, 2025) is incorporated herein by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

[0004] Protein arginine deiminase 4 (PAD4) is a calcium-dependent enzyme that catalyzes hydrolysis of peptidyl arginine sidechains to citrulline in proteins. ^1,2,3 In neutrophils, PAD4- catalyzed chromatin decondensation plays a crucial yet ambiguous role in inducing an inflammatory form of neutrophil cell death called NETosis. Multiple reports have shown a correlation between PAD4 expression and histone citrullination leading to NETosis, but the link between citrullination and NET formation is still largely complicated. ⁴’^5,6,7 During this process, PAD4 and other intracellular contents are released to the extracellular space where PAD4 can create citrullinated neoepitopes. These neoepitopes are recognized by the immune system and further trigger inflammatory diseases such as rheumatoid arthritis (RA). ^8,9 Small-molecule PAD4 inhibitors were developed and found to be effective in alleviating RA phenotypes in mouse models, indicating the relevance of PAD4 in RA pathology. ^10,11 While small-molecule PAD4 inhibitors show promise in preclinical studies of targeting RA, they often lack specificity and potency due to direct targeting of the enzyme's active site which is highly conserved across PAD isoforms. Therefore, there is a need for potent PAD4 inhibitors. ^12,13,14 SUMMARY OF THE DISCLOSURE

[0005] One aspect of the present disclosure is directed to an antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises a VH complementarity determining region (CDR) 1, a VH-CDR2, and a VH-CDR3 and the VL comprises a VL-CDR1, a VL-CDR2, and a VL-CDR3; wherein the VH-CDR3 comprises an amino acid sequence set forth in SEQ ID NOs: 3, 13, 23, 33, or 43; the VH-CDR2 comprises an amino acid sequence set forth in SEQ ID NOs: 2, 12, 22, 32, or 42; the VH-CDR1 comprises an amino acid sequence set forth in SEQ ID NOs: 1, 11, 21, 31, or 41; the VL-CDR1 comprises an amino acid sequence set forth in SEQ ID NOs: 6, 16, 26, 36, or 46; the VL-CDR2 comprises an amino acid sequence set forth in SEQ ID NOs: 7, 17, 27, 37, or 47; and the VL-CDR3 comprises an amino acid sequence set forth in SEQ ID NOs: 8, 18, 28, 38, or 48.

[0006] In some aspects, the antibody or antigen binding portion thereof of the present disclosure, comprises

(a) the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the VH- CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, the VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 6, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 7, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 8;

(b) the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the VH- CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, the VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17 or antigen binding portion thereof, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18;

(c) the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21, the VH- CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 22, the VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 26, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 27, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28; (d) the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 31, the VH- CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32, the VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 33, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 36, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 37, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 38; or

(e) the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 41, the VH- CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 42, the VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 43, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 46, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 48.

[0007] In some aspects, the antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof of the present disclosure, comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises a VH complementarity determining region (CDR) 1, a VH-CDR2, and a VH-CDR3 of the VH comprising an amino acid sequence set forth in SEQ ID NOs: 4, 14, 24, 34, or 44, and the VL comprises a VL-CDR1, a VL-CDR2, and a VL-CDR3 of the VL comprising an amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, or 49.

[0008] In some aspects, the antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof of the present disclosure, comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, the VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 6, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 7, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0009] In some aspects, the antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof of the present disclosure, comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 1 2, the VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17 or antigen binding portion thereof, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18.

[0010] In some aspects, the antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof of the present disclosure, comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 22, the VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 26, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 27, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0011] In some aspects, the antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof of the present disclosure, comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 31, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32, the VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 33, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 36, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 37, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 38.

[0012] In some aspects, the antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof of the present disclosure, comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 41, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 42, the VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 43, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 46, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 48.

[0013] In some aspects, the antibody of the present disclosure has one or more following properties:

(a) the antibody inhibits PAD4 activity;

(b) the antibody inhibits PAD dimerization; (c) the antibody promotes PAD monomerization;

(d) the antibody disrupts Ca²⁺ and substrate binding; or

(e) any combination thereof.

[0014] In some aspects, the VH comprises an amino acid sequence at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 4, 14, 24, 34, or 44.

[0015] In some aspects, the VH comprises an amino acid sequence set forth in SEQ ID NOs: 4, 14, 24, 34, or 44.

[0016] In some aspects, the VL comprises an amino acid sequence at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, or 49.

[0017] In some aspects, the VL comprises an amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, or 49.

[0018] In some aspects, (a) the VH comprises the amino acid sequence set forth in SEQ ID NO: 4 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 9; (b) the VH comprises the amino acid sequence set forth in SEQ ID NO: 14 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 19; (c) the VH comprises the amino acid sequence set forth in SEQ ID NO: 24 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 29; (d) the VH comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 39; or (e) the VH comprises the amino acid sequence set forth in SEQ ID NO: 44 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 49.

[0019] In some aspects, (a) the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 51, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 52; (b) the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 53, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 54; (c) the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 55, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 56; (d) the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 57, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 58; or (e) the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 59, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 60.

[0020] Some aspects of the present disclosure are directed to an antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the antibody or antigen binding portion thereof cross competes with the antibody or antigen binding portion thereof of the present dislcosure.

[0021] Some aspects of the present disclosure are directed to an antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the antibody or antigen binding portion thereof binds to the same epitope as the antibody or antigen binding portion thereof of the present dislcosure.

[0022] In some aspects, the antibody of the present disclosure specifically binds human PAD4 with a KD of about 1 x 10'⁶ M M or less, wherein KD is measured by surface plasmon resonance (Biacore) analysis.

[0023] In some aspects, the antibody of of the present disclosure is an IgGl, an IgG2, an IgG3, an IgG4 or a variant thereof.

[0024] In some aspects, the antibody is an IgGl antibody. In some aspects, the antibody is non- fucosylated.

[0025] In some aspects, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0026] Some aspects of the present disclosure are directed to polynucleotide or a set of polynucleotides encoding the antibody of of the present disclosure.

[0027] Some aspects of the present disclosure are directed to a vector or a set of vectors comprising the polynucleotide or the set of polynucleotides of the present disclosure.

[0028] Some aspects of the present disclosure are directed to a cell comprising the antibody, the polynucleotide or the set of polynucleotides, or the vector or the set of vectors of the present disclosure.

[0029] Some aspects of the present disclosure are directed to an immunoconjugate comprising the antibody of the present disclosure and a therapeutic agent. In some aspects, the therapeutic agent is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive agent, a second antibody, an enzyme, an anti-neoplastic agent, and any combination thereof.

[0030] Some aspects of the present disclosure are directed to a pharmaceutical composition comprising the antibody, the polynucleotide or the set of polynucleotides, or the vector or the set of vectors, the cell, or the immunoconjugate of the present disclosure, and a pharmaceutically acceptable excipient.

[0031] Some aspects of the present disclosure are directed to a pharmaceutical composition comprising the antibody of the present disclosure, and a second antibody. In some aspects, the pharmaceutical composition of the present disclosure is formulated for intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrastemal, topical, epidermal, or mucosal administration.

[0032] Some aspects of the present disclosure are directed to a method of treating a disease in a subject in need thereof, comprising administering to the subject the antibody, the polynucleotide or the set of polynucleotides, or the vector or the set of vectors, the cell, or the immunoconjugate, or the pharmaceutical composition of the present disclosure.

[0033] Some aspects of the present disclosure are directed to a method of reducing shed PAD4 in the serum in a subject in need thereof, comprising administering to the subject the antibody, the polynucleotide or the set of polynucleotides, or the vector or the set of vectors, the cell, or the immunoconjugate, or the pharmaceutical composition of the present disclosure.

[0034] Some aspects of the present disclosure are directed to a method of promoting PAD monomerization in a subject in need thereof, comprising administering the antibody, the polynucleotide or the set of polynucleotides, or the vector or the set of vectors, the cell, or the immunoconjugate, or the pharmaceutical composition of the present disclosure.

[0035] Some aspects of the present disclosure are directed to a method of disrupting Ca²⁺ and substrate binding in a subject in need thereof, comprising administering the antibody, the polynucleotide or the set of polynucleotides, or the vector or the set of vectors, the cell, or the immunoconjugate, or the pharmaceutical composition of the present disclosure.

[0036] In some aspects, the antibody, the polynucleotide or the set of polynucleotides, or the vector or the set of vectors, the cell, or the immunoconjugate, or the pharmaceutical composition of the present disclosure is administered for intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, topical, epidermal, or mucosal administration.

[0037] In some aspects, the subject is a human. In some aspects, the method of the present disclosure further comprises administering to the subject a second therapy.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0038] FIGs. 1A-1E show PAD4 antibody selection and hits characterization. FIG. 1A presents a schematic representation of a PAD4 phage. The Fab-phage library (1) was depleted of non-specific streptavidin (SA) binders (2); the remaining soluble Fab-phage were allowed to bind to PAD4 immobilized on SA in the presence of 1 mM Ca²⁺ or 1 mM EDTA with TCEP and washed (3); the bound Fab-phage were eluted (4), amplified (5) and subjected to additional selection rounds before final characterization of individual hits (6). FIG. IB presents a schematic representation of a fluorescent-substrate PAD4 activity assay (derived from Sabulski, et. al.). Higher fluorescent signal is indicative of lower PAD4 activity. FIG. 1C presents a characterization of Fab binding effect on PAD4 activity from hPAD4 selection 1 and 2 and mPAD4 selection 4 (see FIG. 3E) via fluorescent- substrate activity assay. Tested clones were hI281, hA288, hA362, hI364, hI365, and mA342 are described in Example 1 below. FIG. ID presents the activity of hPAD4 in the presence of control antibody, an inhibitory Fab to human PAD4 (hI281), or activating Fabs (hA288, and hA362) measured by citrullination of protein substrate H3. hI281 reduces the activity of PAD4 while hA288/hA362 increases PAD4 activity. FIG. IE presents the activity of mPAD4 in the presence of control antibody, an activating IgG to murine PAD4 (mA342) and its variant mA342- c4 measured by citrullination of protein substrate H3.

[0039] FIGs. 1F-1M show expression and biophysical characterization of hPAD4. FIG. IF presents a map of hPAD4 gene expressed in C43 or BL21 E. coli. N-terminal 6x His tag and Avi tag are separated by a protease cleavage site to hPAD4 to remove tags. FIG. 1G presents a gel shift assay of full-length hPAD4 (left, 74 kDa) and biotinylated hPAD4 bound to NeutrAvidin (right, 134 kDa). FIG. 1H presents a structural representation of PAD4 enzyme. PAD4 enzyme contains 19 free cysteines, requiring storage with reducing agent to prevent oxidation of cysteines. FIG. II presents an SDS-PAGE showing reduction of the inter-heavy and light chain disulfide of a 4G10^4D5 antibody upon addition of reducing agents. The 4G10^4D5 antibody adopts the antibody scaffold of the Fab-phage library used for PAD4 antibody selection. 50 kDa band represents disulfide-linked Fab while 25 kDa bands represent separated heavy and light chains following TCEP reduction. FIG. 1 J presents a graphical representation of results obtained from biolayer interferometry (BLI) showing binding of Fab 4G10^4D5 to its cognate antibody is not influenced by various reducing agents. FIG. IK presents a protein immunoblot showing detection of PAD4-mediated Histone 3 citrullination (Cit-H3) showing PAD4 activity is calcium dependent. FIG. IL presents a differential scanning fluorimetry (DSF) plot showing melting temperatures of hPAD4 in the presence and absence of Ca²⁺. FIG. IM presents a table listing PALM melting temperatures measured by DSF.

[0040] FIGs. 2A-2E show antibodies that modulate hPAD4 dimerization influence enzymatic activity. FIG. 2A presents a schematic representation of antibodies influencing PAD4 activity through modulating its dimerization. Monomeric PAD4 is less active than dimeric PAD4, and hI281 blocks dimerization while hA288 and hA362 promote dimerization. FIG. 2B presents negative stain-EM 2D micrographs showing monomeric state of PAD4 in complex with hI281 and dimeric state in complex with hA288 or hA362. FIG. 2C presents SEC traces of PAD4 alone, in complex with Fab-hI281, and in complex with Fab -h A362. PAD4 alone exists in both the monomer and dimer form, while addition of hI281 promotes the monomer form and hA362 promotes the dimer form as evidenced by elution time. FIG. 2D presents graphical results obtained from a binding assay. Binding is correlated with antibody function using a trypsin substrate assay. Biological duplicates are shown. Fabs binding to mutants with less affinity are less able to inhibit enzymatic activity (Right). FIG. 2E presents a table displaying measured binding affinities of activating Fab hA362 to PAD4 mutants (Left). Binding is drastically decreased upon mutation of several residues at the PAD4 dimer interface.

[0041] FIGs. 2F-2G show a phage ELISA for characterizing hPAD4/mPAD4 cross reactivity and multi-point biolayer interferometry (BLI) binding data of lead candidate IgGs to hPAD4. FIG. 2F presents the binding of Fab-phage to hPAD4/mPAD4 measured by ELISA A280 signal. Very few clones from hPAD4 or mPAD4 phage selections (left, middle) were cross-reactive. By taking the round 3 phage pool and performing selections with the other antigen for two additional rounds, nearly all phage clones from round 5 are cross reactive with hPAD4 and mPAD4 (right). FIG. 2G presents multi point BLI data showing that hl364 and hl365 are dependent on calcium to bind hPAD4. hl281 and hA362 are calcium-independent binders although binding to the calcium-bound form of the enzyme is stronger. Binding of all clones to PAD4 at 2 mM Ca²⁺ is comparable to their binding at 10 mM Ca²⁺. KDS reported as <1 nM are due to bottoming out of off-rate measurements on BLI. [0042] FIGs. 3A-3D shows hA362 directly contributes to the PAD4 dimerization interface and helps order the substrate binding site. FIG. 3A shows two views of the cryo-EM map of PAD4 in complex with Fab hA362 (PDB: 8SMK). The N-terminal domain and C-terminal domain are also shown. FIG. 3B shows a model of PAD4/hA362 derived from the cryo-EM map shown as ribbon. The boxed region delineates Fab CDR interaction with I- and S-loop on PAD4. FIG. 3C shows a zoomed-in view of the boxed region in FIG. 3B and shows hA362 reaching across the PAD4 monomer to interact with the I loop on the other PAD4 monomer. This helps order the S loop via the R441-D465 salt bridge. FIG. 3D shows a model of the detailed hA362-PAD4 interactions. Both chains of the Fab pack a large number of aromatics against both monomers of PAD4 dimer. Ion and hydrogen bonds marked with dashed lines.

[0043] FIGs. 3E-3H show functional characterization of mPAD4 and h/mPAD4 cross-reactive antibodies. FIG. 3E presents data showing that hml400 is a functional inhibitor of both hPAD4 and mPAD4. FIG. 3F presents BLI results showing that mA342 interacts with the calcium bound form of mPAD4 but binds minimally to the calcium-free form. FIG. 3G presents an SEC trace showing improved biophysical properties of mA342-c4 as compared to WT mA342. FIG. 3H presents a BLI comparison of WT mA342 and its improved variant mA342-c4 in 10 mM Ca²⁺.

[0044] FIGs. 4A-4D present a Cryo-EM structure that illustrates the mechanism of calcium dependency and inhibitory function of hI365. FIG. 4A presents a diagram showing that inhibitory Fab hI365 can bind both the monomeric and dimeric form of hPAD4. FIG. 4B shows a Cryo-EM map (left, PDB: 8SML) and the resulting model (right) of hPAD4 in complex with hI365. The Ab primarily binds an N-terminal Ca²⁺-coordinated region, but the H3 loop extends to the C-terminal pocket and induces conformational change. FIG. 4C shows a comparison of the Ca²⁺-bound hPAD4 (PDB: 1WD9), Ca²⁺/substrate bound hPAD4 (PDB: 1WDA), and the Ca²⁺/ hI365 bound hPAD4. The interaction of hI365 with residues 340-352 in hPAD4 alters the structure and orientation of this fragment disrupting the organization of several residues (D350, R374, W347) involved in calcium and substrate binding. FIG. 4D presents a model of the detailed hA365-PAD4 interactions. The Fab interacts predominantly with one chain against one monomer of PAD4, burying considerable number of aromatics with some hydrogen bonds.

[0045] FIGs. 4E-4J show the biophysical characterization of PAD4 monomerization mutants and effect of PAD4 monomerization on antibody binding. FIG. 4E shows a computational modeling of the PAD4 dimer interface to identify key residues that contribute to dimerization (bolded). FIG. 4F shows key residues: R8, Y435, and W548 highlighted at the dimer interface. The selected control residue N438 is also highlighted. FIG. 4G shows an SEC trace showing earlier elution of dimeric, WT PAD4 compared to monomerizati on -promoting mutants R8E and Y435A. Control mutants, N438A and N438R, elute with WT PAD4. FIG. 4H shows a DSF plot of PAD4 mutants in the presence of 10 mM Ca²⁺ or 1 mM EDTA. FIG. 41 shows a table of estimated T_ms of PAD4 mutants from DSF plots. FIG. 4J shows BLI measurements of hA362 binding to WT hPAD4 and monomerization mutants.

[0046] FIGs. 5A-5F show antibody engineering strategies employed to improve affinity and inhibition activity of the calcium-dependent binder, hI365. FIG. 5A shows hPAD4-hI365 binding interface with CDRs L3, H1-H4 highlighted. CDR loops H1-H4 are forming contacts with PAD4, but L3 is too short to facilitate any contact with the enzyme. FIG. 5B shows SEC traces of WT hI365 and affinity matured clones. Selected hits show improved SEC profiles as indicated by earlier elution times. FIG. 5C shows CDR sequences of WT hI365 aligned with residues to be mutagenized via soft randomization; L3 is randomized to contain 9 or 10 amino acids. FIG. 5D shows CDR sequences of the two top engineered binders with good affinity and solubility. FIG. 5E shows PAD4 activity as measured by the fluorescent substrate assay shows that several Ab clones identified from soft randomization inhibit PAD4 more potently than WT hI365. 2 mM Ca²⁺ was used in this assay to mimic physiological extracellular conditions. Error bars represent mean ± standard deviation of three biological replicates. FIG. 5F presents a plot showing IC50s of lead candidates E3 and E6 as measured by PAD4 citrullination H3. IC50s of E3 and E6 determined to be 95 nM and 13 nM, respectively and represent mean ± SEM of three biological replicates.

[0047] FIGs. 5G-5K show a detailed view of Ca²⁺ ions and Fab/hPAD4 interactions with cryo- EM map. FIG. 5G shows three Ca²⁺ ions in the N terminal domain of hPAD4-hA362 structure. Model in ribbon and atoms on top, overlayed with cryo-EM map on the bottom. FIG. 5H shows a Ca²⁺ ion in the C terminal domain of the hPAD4-hA362 structure. One Ca²⁺ was removed from analysis, as the electron density was very weak. FIG. 51 shows a zoomed in view of the hA362 interactions with the hPAD4 I-loop with corresponding cryo-EM map. Adapted from FIG. 3C. FIG. 5 J shows Ca²⁺ ions in the N terminal domain of the hPAD4-hA365 structure. FIG. 5K shows a zoomed in view of the hl365 interactions with the hPAD4 active site with corresponding cryo- EM map. Adapted from FIG. 4C, right panel.

[0048] FIGs. 6A-6H show a rosetta antibody design (rAbD) guided optimization of hl365. FIG. 6A shows several G58 mutants are predicted to reduce interface energy of hl365 and hPAD4. FIG. 6B shows BLI measurements showing that point mutant G58D binds PAD4 with similar affinity as WT hl365. FIG. 6C shows an SEC trace showing improved biophysical properties of mutant G58D as compared to WT hl365, as evidenced by the earlier elution time. FIG. 6D shows the position of CDR L3 at the PAD4 binding interface and rAbD workflow for designing L3 variants. Though the loop sits at the interface, it is not forming any favorable interactions with PAD4 due to its short length. High-resolution structure of PAD4/hl365 interface served as the input model for rAbD. CDR L3 of the relaxed model was allowed to "Graft" and "Sequence" design or only "Sequence" design with or without flexible backbone design. Neighboring CDRs L 1 and H3 were repacked to avoid clashes and optimize interface interactions. FIG. 6E shows "Graft" and "Sequence" design generated models with low totalscore and interface energies shown by dG separated (in REU). "Sequence" design alone did not improve the interface with all models having poor dG separated score. The dG separated score of the cryo EM structure of PAD4/hl365 interface is —54.4 REU. FIG. 6F shows a rosetta antibody design (rAbD) predicted CDR L3 at lengths of 9 and 10 aa to have the most optimal interface energies. Different CDR L3 lengths and corresponding dG separated scores are shown. FIG. 6G shows an SEC chromatagram showing poor performance of L3 mutants compared to parent (hl365) and control antibodies (hl356). FIG. 6H shows the results of a fluorescent substrate activity assay showing inhibition of PAD4 in the presence of WT hl365 and the predicted L3 variants by rAbD. Error bars represent mean ± standard deviation of three biological replicates.

[0049] FIGs. 7A-7E show soft randomization of hl365. FIG. 7A shows sequence logos of library 1 showing amino acid preference for each CDR. FIG. 7B shows sequence logos of library 2 showing amino acid preference for each CDR. FIG. 7C shows the binding of top Fab-phage clones to PAD4 as measured by direct ELISAs. Top clones exhibit similar or improved binding to WT hl365 at both 20 nM and 5 nM PAD4. Top clones are less likely to be competed off PAD4, indicating a lower relative k_Off. FIG. 7D shows the binding of top Fab-phage clones to PAD4 as measured by competition ELISAs. Top clones exhibit similar or improved binding to WT hl365 at both 20 nM and 5 nM PAD4. Top clones are less likely to be competed off PAD4, indicating a lower relative k_off. FIG. 7E presents plots showing multi-point octet trace of leading clones, E3 and E6. Both appear to bind PAD4 at nanomolar affinity.

[0050] FIGs. 8A-8B show anti-PAD4 antibody specificity and effect on PAD4 on whole cell lysate detect via anti-modified citrulline western blot. FIG. 8A shows the binding of hl365, E3, and E6 to PAD4, PAD2, PAD3, and Trastuzumab negative control measured by BLI. All PAD4 antibodies bind only to PAD4 and not PAD2 or PAD4. FIG. 8B shows whole cell lysate treated with PAD4 +/- inhibitors. Functional antibodies, hl365, E3, and E6 inhibit PAD4-catalyzed citrullination on a variety of cytosolic, nuclear, and membrane-bound proteins. Trastuzumab IgG and pan-PAD inhibitor Cl-amidine included as negative and positive controls. An anti-actin loading control is included.

[0051] FIGs. 9A-9E show cryo-EM map statistics for hPAD4/hA362. FIG. 9A shows a map shaded by local resolution. FIG. 9B shows global resolution FSC plots generated from half maps. The FSC=0.143 cutoff is indicated. FIG. 9C shows directional FSC plots. Left: Histogram showing the percentage of per angle FSC vs resolution, showing agreement with global FSC and sphericity of 0.966. Right: Directional FSC curves. FIG. 9D shows a model of hPAD4/hA362. Left: Model shaded by per residue Q score; Right: Fab CDR loop showing model/map fit. PDB: 8SMK. FIG. 9E shows a cryo-EM data processing workflow for PAD4-hA362.

[0052] FIG. 10A-10E show cryo-EM map statistics for hPAD4/hl365. FIG. 10A shows a map colored by local resolution. FIG. 10B shows global resolution FSC plots generated from half maps. The FSC=0.143 cutoff is indicated. FIG. 10C shows directional FSC plots. Left: Histogram showing the percentage of per angle FSC vs resolution, showing some degree of anisotropy, but overall agreement with the global FSC and sphericity of 0.928. Right: Directional FSC curves demonstrating better than 3 A resolution in two directions and -4.5A in third direction. FIG. 10D shows a model of hPAD4/hA362. Left: Model shaded by per residue Q score; Right: Fab CDR loop showing model/map fit. PDB: 8SML. FIG. 10E shows cryo-EM data processing workflow for PAD4-hl365.

DETAILED DESCRIPTION OF DISCLOSURE

[0053] The present disclosure relates to antibodies and antigen binding portions thereof that specifically bind Peptidyl arginine deiminase 4 (PAD4) ("anti-PAD4 antibodies"). In some aspects, the PAD4 is human PAD4. Some aspects of the present disclosure relate to methods of treating a subject in need thereof, comprising administering to the subject an antibody or antigen binding portion thereof that specifically binds PAD4.

I. Terms

[0054] In order that the present description can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0055] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence," is understood to represent one or more nucleotide sequences. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0056] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0057] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of' and/or "consisting essentially of' are also provided.

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0059] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0060] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower).

[0061] The term "Peptidyl arginine deiminase 4," or "protein arginine deiminase 4," or "protein-arginine deiminase type-4," or "PAD4" as used herein is known to catalyze the citrullination/deimination of arginine residues of proteins such as histones, thereby playing a key role in histone code and regulation of stem cell maintenance. PAD4 is also known to citrullinate histone Hl at 'Arg-54' (to form HlR54ci), histone H3 at 'Arg-2', 'Arg-8', 'Arg-17' and/or 'Arg-26' (to form H3R2ci, H3R8ci, H3R17ci, H3R26ci, respectively) and histone H4 at 'Arg-3' (to form H4R3ci). PAD4 is known to act as a key regulator of stem cell maintenance by mediating citrullination of histone Hl : citrullination of 'Arg-54' of histone Hl (HlR54ci) results in Hl displacement from chromatin and global chromatin decondensation, thereby promoting pluripotency and stem cell maintenance. PAD4 is also known to promote profound chromatin decondensation during the innate immune response to infection in neutrophils by mediating formation of HlR54ci. PAD4 is required for the formation of neutrophil extracellular traps (NETs); NETs are mainly composed of DNA fibers and are released by neutrophils to bind pathogens during inflammation (By similarity). PAD4 also plays a role in citrullination of histone H3 prevents their methylation by CARMI and HRMT1L2/PRMT1 and represses transcription. In addition PAD4 is known to citrullinates EP300/P300 at 'Arg-2142', which favors its interaction with NC0A2/GRIP1.

[0062] Below are the amino acid sequences of the three known human PAD4 isoforms.

Q9UM07

MAQGTLIRVTPEQPTHAVCVLGTLTQLDICSSAPEDCTSFSINASPGVWDIAHGPPAKKKSTGS STWPLDPGVEVTLTMKVASGSTGDQKVQISYYGPKTPPVKALLYLTGVEISLCADITRTGKVKPT RAVKDQRTWTWGPCGQGAILLVNCDRDNLESSAMDCEDDEVLDSEDLQDMSLMTLSTKTPKDFFT NHTLVLHVARSEMDKVRVFQATRGKLSSKCSWLGPKWPSHYLMVPGGKHNMDFYVEALAFPDTD FPGLITLTISLLDTSNLELPEAWFQDSWFRVAPWIMTPNTQPPQEVYACSIFENEDFLKSVTT LAMKAKCKLTICPEEENMDDQWMQDEMEIGYIQAPHKTLPWFDSPRNRGLKEFPIKRVMGPDFG YVTRGPQTGGISGLDSFGNLEVSPPVTVRGKEYPLGRILFGDSCYPSNDSRQMHQALQDFLSAQQ VQAPVKLYSDWLSVGHVDEFLSFVPAPDRKGFRLLLASPRSCYKLFQEQQNEGHGEALLFEGIKK KKQQKI KNI LSNKTLREHNS FVERCIDWNRELLKRELGLAESDI IDI PQLFKLKEFSKAEAFFPN MVNMLVLGKHLGIPKPFGPVINGRCCLEEKVCSLLEPLGLQCTFINDFFTYHIRHGEVHCGTNVR RKPFSFKWWNMVP ( SEQ ID NO : 61 )

B1AQ67

[0063 ] MAQGTLIRVTPEQPTHAVCVLGTLTQLDICSSAPEDCTSFSINASPGVWDIAHGPP AKKKSTGSSTWPLDPGVEVTLTMKVASGSTGDQKVQISYYGPKTPPVKALLYLTGVDGVSPCHPG WSAMA ( SEQ ID NO : 62 )

[0064] The term "antibody" refers, in some aspects, to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In some antibodies, e.g., naturally-occurring IgG antibodies, the heavy chain constant region is comprised of a hinge and three domains, CHI, CH2 and CH3. In some antibodies, e.g., naturally-occurring IgG antibodies, each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from aminoterminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. A heavy chain may have the C-terminal lysine or not. Unless specified otherwise herein, the amino acids in the variable regions are numbered using the Kabat numbering system and those in the constant regions are numbered using the EU system.

[0065] An "IgG antibody", e.g., a human IgGl, IgG2, IgG3 and IgG4 antibody, as used herein has, in some aspects, the structure of a naturally-occurring IgG antibody, z.e., it has the same number of heavy and light chains and disulfide bonds as a naturally-occurring IgG antibody of the same subclass. For example, an anti-PAD4 IgGl, IgG2, IgG3 or IgG4 antibody consists of two heavy chains (HCs) and two light chains (LCs), wherein the two HCs and LCs are linked by the same number and location of disulfide bridges that occur in naturally-occurring IgGl, IgG2, IgG3 and IgG4 antibodies, respectively (unless the antibody has been mutated to modify the disulfide bridges).

[0066] Antibodies typically bind specifically to their cognate antigen with high affinity, reflected by a dissociation constant (KD) of 10'⁵ to 10'¹¹ M or less. Any KD greater than about 10'⁴ M is generally considered to indicate nonspecific binding. As used herein, an antibody that "binds specifically" to an antigen refers to an antibody that binds to the antigen and substantially identical antigens with high affinity, which means having a KD of 10'⁷M or less, 10'⁸M or less, 5 x 10'⁹M or less, or between 10'⁸ M and IO'¹⁰ M or less, but does not bind with high affinity to unrelated antigens. An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity to the given antigen, for example, if it exhibits at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the sequence of the given antigen. By way of example, an antibody that binds specifically to human PAD4 can, in some aspects, also have cross-reactivity with PAD4antigens from certain primate species (e.g., cynomolgus PAD4), but cannot cross-react with PAD4 antigens from other species or with an antigen other than PAD4.

[0067] An immunoglobulin can be from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. The IgG isotype is divided in subclasses in certain species: IgGl, IgG2, IgG3 and IgG4 in humans, and IgGl, IgG2a, IgG2b and IgG3 in mice. In some aspects, the anti-PAD4 antibodies described herein are of the IgGl subtype. Immunoglobulins, e.g., IgGl, exist in several allotypes, which differ from each other in at most a few amino acids. "Antibody" includes, by way of example, both naturally-occurring and non- naturally-occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and nonhuman antibodies and wholly synthetic antibodies.

[0068] The term "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human PALM). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding portions encompassed within the term "antigen-binding portion" of an antibody, e.g., an anti-PAD4 antibody described herein, include (i) a Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of the VL, VH, LC and CHI domains; (ii) a F(ab')2 fragment (fragment from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; (vi) an isolated complementarity determining region (CDR) and (vii) a combination of two or more isolated CDRs which can optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chePAD41 cleavage of intact immunoglobulins. [0069] A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0070] The term "monoclonal antibody," as used herein, refers to an antibody from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprised in the population are substantially similar and bind the same epitope(s) (e.g., the antibodies display a single binding specificity and affinity), except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. 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. The term "human monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies that display(s) a single binding specificity and which has variable and optional constant regions derived from human germline immunoglobulin sequences. In some aspects, human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0071] The term "recombinant human antibody," as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies comprise variable and constant regions that utilize particular human germline immunoglobulin sequences encoded by the germline genes, but include subsequent rearrangements and mutations which occur, for example, during antibody maturation. As known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9): 1117- 1125), the variable region contains the antigen binding domain, which is encoded by various genes that rearrange to form an antibody specific for a foreign antigen. In addition to rearrangement, the variable region can be further modified by multiple single amino acid changes (referred to as somatic mutation or hypermutation) to increase the affinity of the antibody to the foreign antigen. The constant region will change in further response to an antigen (i.e., isotype switch). Therefore, the rearranged and somatically mutated nucleic acid molecules that encode the light chain and heavy chain immunoglobulin polypeptides in response to an antigen cannot have sequence identity with the original nucleic acid molecules, but instead will be substantially identical or similar i.e., have at least 80% identity).

[0072] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The anti-PAD4 antibodies described herein can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibodies and "fully human" antibodies are used synonymously.

[0073] A "humanized" antibody refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In some aspects of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen. A "humanized" antibody retains an antigenic specificity similar to that of the original antibody.

[0074] A "chimeric antibody" refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.

[0075] As used herein, "isotype" refers to the antibody class (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibody) that is encoded by the heavy chain constant region genes. [0076] "Allotype" refers to naturally-occurring variants within a specific isotype group, which variants differ in a few amino acids (see, e.g., Jefferis et al. (2009) mAbs 1 : 1). Anti-PAD4 antibodies described herein can be of any allotype. As used herein, antibodies referred to as "IgGlf," "IgGl. If," or "IgG1.3f" isotype are IgGl, effectorless IgGl.l, and effectorless IgGl.3 antibodies, respectively, of the allotype "f," i.e., having 214R, 356E and 358M according to the EU index as in Kabat.

[0077] The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen."

[0078] An "isolated antibody," as used herein, is intended to refer to an antibody which is substantially free of other proteins and cellular material.

[0079] As used herein, an antibody that "binds PAD4" is intended to refer to an antibody that interacts with PAD4, e.g., in binding assays using CHO cells transfected with human PAD4 or PAD4 expressing tumor cells, with an ECso of about 25 pg/mL or less, about 23 pg/mL or less, about 20 pg/mL or less, about 15 pg/mL or less, about 10 pg/mL or less, about 5 pg/mL or less, about 3 pg/mL or less, about 2 pg/mL or less, about 1 pg/mL or less, about 0.5 pg/mL or less, about 0.45 pg/mL or less, about 0.4 pg/mL or less, about 0.35 pg/mL or less, or about 0.3 pg/mL or less, in art-recognized methods, e.g., the FACS-based binding assays described herein. In some aspects, the anti-PAD4 antibody binds human-PAD4 expressed on, e.g., CHO cells, with an ECso of about 200 nM or less, about 175 nM or less, about 160 nM or less, about 150 nM or less, about 125 nM or less, about 110 nM or less, about 100 nM or less about 80 nM or less, about 75 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 35 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 15 nM or less, about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, about 1.9 nM or less, about 1.8 nM or less, about 1.7 nM or less, about 1.6 nM or less, about 1.5 nM or less, about 1.4 nM or less, about 1.3 nM or less, about 1.2 nM or less, about 1.1 nM or less, about 1.0 nM or less, about 0.9 nM or less, about 0.8 nM or less, about 0.7 nM or less, about 0.6 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.2 nM or less, or about 0.1 nM or less. In some aspects, the anti-PAD4 antibody binds human-PAD4 expressed on, e.g., CHO cells, with an ECso of less than about 10 nM. In some aspects, the anti-PAD4 antibody binds human-PAD4 expressed on, e.g., CHO cells, with an ECso of less than about 5 nM. In some aspects, the anti-PAD4 antibody binds human-PAD4 expressed on, e.g., CHO cells, with an ECso of less than about 1.5 nM. In some aspects, the anti-PAD4 antibody binds human-PAD4 expressed on, e.g., CHO cells, with an ECso of about 1 nM or less. In some aspects, the anti-PAD4 antibody binds human-PAD4 expressed on, e.g., CHO cells, with an ECso of about 0.5 nM or less. In some aspects, the anti-PAD4 antibody binds human-PAD4 expressed on, e.g., CHO cells, with an ECso of about 0.3 nM or less. In some aspects, the anti-PAD4 antibody binds human-PAD4 expressed on, e.g., CHO cells, with an ECso of about 0.2 nM or less.

[0080] An "effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or a biochePAD41 event that results therefrom. Exemplary "effector functions" include Clq binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcyR- mediated effector functions such as ADCC and antibody dependent cell-mediated phagocytosis (ADCP), and downregulation of a cell surface receptor (e.g., the B cell receptor; BCR). Such effector functions generally require the Fc region to be combined with a binding domain (e.g, an antibody variable domain).

[0081] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to an IgG antibody comprise receptors of the FcyR family, including allelic variants and alternatively spliced forms of these receptors. The FcyR family consists of three activating (FcyRI, FcyRIII, and FcyRIV in mice; FcyRIA, FcyRIIA, and FcyRIIIA in humans) and one inhibitory (FcyRIIB) receptor. Various properties of human FcyRs are known in the art. The majority of innate effector cell types coexpress one or more activating FcyR and the inhibitory FcyRIIB, whereas natural killer (NK) cells selectively express one activating Fc receptor (FcyRIII in mice and FcyRIIIA in humans) but not the inhibitory FcyRIIB in mice and humans. Human IgGl binds to most human Fc receptors and is considered equivalent to murine IgG2a with respect to the types of activating Fc receptors that it binds to.

[0082] An "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C- terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g, effector cells) or to the first component (Clq) of the classical complement system. Thus, an Fc region comprises the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g, CHI or CL). In IgG, IgA and IgD antibody isotypes, the Fc region comprises two identical protein fragments, derived from the second (CH2) and third (CH3) constant domains of the antibody's two heavy chains; IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region comprises immunoglobulin domains CH2 and CH3 and the hinge between CHI and CH2 domains. Although the definition of the boundaries of the Fc region of an immunoglobulin heavy chain might vary, as defined herein, the human IgG heavy chain Fc region is defined to stretch from an amino acid residue D221 for IgGl, V222 for IgG2, L221 for IgG3 and P224 for IgG4 to the carboxyterminus of the heavy chain, wherein the numbering is according to the EU index as in Kabat. The CH2 domain of a human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is positioned on C-terminal side of a CH2 domain in an Fc region, i.e., it extends from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is absent) or 445 (if the C-terminal glycine and lysine residues are absent) of an IgG. As used herein, the Fc region can be a native sequence Fc, including any allotypic variant, or a variant Fc (e.g., a non-naturally- occurring Fc).

[0083] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human IgGl Fc region; native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally-occurring variants thereof. Native sequence Fc include the various allotypes of Fes (see, e.g., Jefferis et al. (2009) mAbs 1 : 1).

[0084] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g, PAD4) to which an immunoglobulin or antibody specifically binds, e.g., as defined by the specific method used to identify it. Epitopes can be formed both from contiguous amino acids (usually a linear epitope) or noncontiguous amino acids juxtaposed by tertiary folding of a protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from (e.g., from PAD4) are tested for reactivity with a given antibody (e.g., anti-PAD4 antibody). Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography, x-ray co-crystallography, antigen mutational analysis, 2-dimensional nuclear magnetic resonance and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

[0085] The term "epitope mapping" refers to the process of identification of the molecular determinants for antibody-antigen recognition.

[0086] The term "binds to the same epitope" with reference to two or more antibodies means that the antibodies bind to the same segment of amino acid residues, as determined by a given method. Techniques for determining whether antibodies bind to the "same epitope on PAD4" with the antibodies described herein include, for example, epitope mapping methods, such as, x-ray analyses of crystals of antigemantibody complexes which provides atomic resolution of the epitope and hydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of the antibody to antigen fragments or mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. Antibodies having the same VH and VL or the same CDR1, 2 and 3 sequences are expected to bind to the same epitope.

[0087] Antibodies that "compete with another antibody for binding to a target" refer to antibodies that inhibit (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target, can be determined using known competition experiments, e.g., BIACORE® surface plasmon resonance (SPR) analysis. In some aspects, an antibody competes with, and inhibits binding of another antibody to a target by at least 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition or competition can be different depending on which antibody is the "blocking antibody" (i.e., the cold antibody that is incubated first with the target). Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.1101/pdb.prot4277 or in Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Two antibodies "cross-compete" if antibodies block each other both ways by at least 50%, z.e., regardless of whether one or the other antibody is contacted first with the antigen in the competition experiment.

[0088] Competitive binding assays for determining whether two antibodies compete or crosscompete for binding include: competition for binding to cells expressing PAD4, e.g., by flow cytometry, such as described in the Examples. Other methods include: SPR (e.g., BIACORE®), solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al. , Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel etal., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung etal., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer etal., Scand. J. Immunol. 32:77 (1990)).

[0089] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds," refer to antibody binding to an epitope on a predetermined antigen. Typically, the antibody (i) binds with an equilibrium dissociation constant (KD) of approximately less than 10'⁷ M, such as approximately less than 10'⁸ M, 10'⁹ M or 10'¹⁰ M or even lower when determined by, e.g., surface plasmon resonance (SPR) technology in a BIACORE® 2000 instrument using the predetermined antigen, e.g., recombinant human PAD4, as the analyte and the antibody as the ligand, or Scatchard analysis of binding of the antibody to antigen positive cells, and (ii) binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. Accordingly, an antibody that "specifically binds to human PAD4" refers to an antibody that binds to soluble or cell bound human PAD4 with a KD of 10'⁷M or less, such as approximately less than 10'⁸M, 10'⁹M or 10'¹⁰M or even lower. An antibody that "cross-reacts with cynomolgus PAD4" refers to an antibody that binds to cynomolgus PAD4 with a KD of 10'⁷ M or less, such as approximately less than 10'⁸ M, 10'⁹ M or 10'¹⁰ M or even lower. In some aspects, such antibodies that do not cross-react with PAD4 from a non-human species exhibit essentially undetectable binding against these proteins in standard binding assays. [0090] The term "kassoc" or "k_a", as used herein, is intended to refer to the association rate of a particular antibody- antigen interaction, whereas the term "kdis" or "kd," as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of kd to k_a (z.e.,. kd/ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. Available methods for determining the KD of an antibody include surface plasmon resonance, a biosensor system such as a BIACORE® system or flow cytometry and Scatchard analysis.

[0091] As used herein, the term "high affinity" for an IgG antibody refers to an antibody having aKo of 1 O'⁸ M or less, 1 O'⁹ M or less, or 1 O'¹⁰ M or less for a target antigen. However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody having a KD of IO'¹⁰ M or less, or 10'⁸ M or less.

[0092] The term "ECso" in the context of an in vitro or in vivo assay using an antibody or antigen binding portion thereof, refers to the concentration of an antibody or an antigen-binding portion thereof that induces a response that is 50% of the maximal response, i.e., halfway between the maximal response and the baseline.

[0093] The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.

[0094] A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in the protein can contain a modification such as, but not limited to, glycosylation, phosphorylation or disulfide bond formation. A "protein" can comprise one or more polypeptides.

[0095] The term "nucleic acid molecule," as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single- stranded or double- stranded, and can be cDNA.

[0096] Conservative amino acid substitutions" refer to substitutions of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g, threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some aspects, a predicted nonessential amino acid residue in an anti-PAD4 antibody is replaced with another amino acid residue from the same side chain family. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879- 884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0097] For nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, at least about 90% to 95%, or at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.

[0098] For polypeptides, the term "substantial homology" indicates that two polypeptides, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate amino acid insertions or deletions, in at least about 80% of the amino acids, at least about 90% to 95%, or at least about 98% to 99.5% of the amino acids.

[0099] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0100] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0101] The nucleic acid and protein sequences described herein can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worl d wi de web . neb i . nl m . ni h . gov .

[0102] The nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., the other parts of the chromosome) or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0103] Nucleic acids, e.g., cDNA, can be mutated, in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, can affect amino acid sequence as desired. In particular, DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where "derived" indicates that a sequence is identical or modified from another sequence).

[0104] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, also included are other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0105] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell that comprises a nucleic acid that is not naturally present in the cell, and can be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny cannot, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0106] An "immune response" is as understood in the art, and generally refers to a biological response within a vertebrate against foreign agents or abnormal which response protects the organism against these agents and diseases caused by them. An immune response is mediated by the action of one or more cells of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell, a Th cell, a CD4⁺ cell, a CD8⁺ T cell, or a Treg cell, or activation or inhibition of any other cell of the immune system, e.g., NK cell.

[0107] An "immunomodulator" or "immunoregulator" refers to an agent, e.g., an agent targeting a component of a signaling pathway that can be involved in modulating, regulating, or modifying an immune response. "Modulating," "regulating," or "modifying" an immune response refers to any alteration in a cell of the immune system or in the activity of such cell e.g., an effector T cell, such as a Thl cell). Such modulation includes stimulation or suppression of the immune system which can be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other changes which can occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which can have enhanced function in a tumor microenvironment. In some aspects, the immunomodulator targets a molecule on the surface of a T cell. An "immunomodulatory target" or "immunoregulatory target" is a molecule, e.g., a cell surface molecule, that is targeted for binding by, and whose activity is altered by the binding of, a substance, agent, moiety, compound or molecule. Immunomodulatory targets include, for example, receptors on the surface of a cell ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands").

[0108] "Immunotherapy" refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying the immune system or an immune response.

[0109] "Potentiating an endogenous immune response" means increasing the effectiveness or potency of an existing immune response in a subject. This increase in effectiveness and potency can be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0110] An increased ability to stimulate an immune response or the immune system, can result from an enhanced agonist activity of T cell co-stimulatory receptors and/or an enhanced antagonist activity of inhibitory receptors. An increased ability to stimulate an immune response or the immune system can be reflected by a fold increase of the ECso or maximal level of activity in an assay that measures an immune response, e.g., an assay that measures changes in cytokine or chemokine release, cytolytic activity (determined directly on target cells or indirectly via detecting CD 107a or granzymes) and proliferation. The ability to stimulate an immune response or the immune system activity can be enhanced by at least 10%, 30%, 50%, 75%, 2 fold, 3 fold, 5 fold or more.

[OHl] As used herein, the term "linked" refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage also can be genetic (i.e., recombinantly fused). Such linkages can be achieved using a wide variety of art recognized techniques, such as chePAD41 conjugation and recombinant protein production. [0112] As used herein, "administering" refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Different routes of administration for the anti-PAD4 antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, an antibody described herein can be administered via a non- parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

[0113] The terms "treat," "treating," and "treatment," as used herein, refer to any type of intervention or process performed on, or administering an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease or enhancing overall survival. Treatment can be of a subject having a disease or a subject who does not have a disease (e.g., for prophylaxis).

[0114] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, an increase in overall survival (the length of time from either the date of diagnosis or the start of treatment for a disease that patients diagnosed with the disease are still alive), or a prevention of impairment or disability due to the disease affliction. A therapeutically effective amount or dosage of a drug includes a " prophy lactically effective amount" or a "prophylactically effective dosage", which is any amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or of suffering a recurrence of disease, inhibits the development or recurrence of the disease. The ability of a therapeutic agent to promote disease regression or inhibit the development or recurrence of the disease can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0115] By way of example for the treatment of tumors, a therapeutically effective amount or dosage of the drug inhibits cell growth or tumor growth by at least about 20%, by at least about 40%, by at least about 60%, or by at least about 80% relative to untreated subjects. In some aspects, a therapeutically effective amount or dosage of the drug completely inhibits cell growth or tumor growth, i.e., inhibits cell growth or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be evaluated using the assays described infra. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit cell growth, such inhibition can be measured in vitro by assays known to the skilled practitioner. In some aspects described herein, tumor regression can be observed and continue for a period of at least about 20 days, at least about 40 days, or at least about 60 days.

[0116] The term "patient" includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[0117] As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions described herein can be used to treat a subject having RA. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.

[0118] Various aspects described herein are described in further detail in the following subsections.

II. Anti-human PAD4 antibodies

[0119] Described herein are antibodies, e.g., fully human antibodies, which are capable of specifically binding to PAD4, and inhibiting, preventing or reducing PAD4. For example, the antibodies specifically bind human PAD4, and more specifically, a particular epitope of human PAD4. In some aspects, the antibodies specifically bind to PAD4, thereby reducing the PAD4 level in serum. In some aspects, anti-PAD4 antibodies cross-react with PAD4 from one or more non- human primates, such as cynomolgus PAD4.

[0120] In some aspects, anti-PAD4 antibodies described herein bind to human PAD4 with high affinity, for example, with a KD of 10'⁶M or less, 10'⁷M or less, 10'⁸ M or less, 10'⁹M or less, 10’ ¹⁰M or less, 10’¹¹ M or less, 10’¹²M or less, 10’¹²M to 10’⁷M, lO^ M to 10’⁷M, 10’¹⁰M to 10’⁷M, or 10'⁹ M to 10'⁷ M. In some aspects, the anti-PAD4 antibody binds to human PAD4, e.g., as determined by Surface Plasmon Resonance, e.g. using BIACORE™ (e.g., as described in the Examples), with a KD of 10'⁶ M or less, 10'⁷M or less, 10'⁸M or less, 10'⁹ M (1 nM) or less, IO'¹⁰ M or less, 10’¹²M to 10’⁷M, lO^ M to 10’⁷M, 10’¹⁰M to 10’⁷M, 10’⁹M to 10’⁷M, or 10’⁸M to 10’ ⁷ M. In some aspects, an anti-PAD4 antibody binds to human sPAD4, e.g., as determined by ELISA, e.g., a PAD4 ELISA kit, e.g., ABCAM ab 100592, with an ECso of ECso of 100 nM or less, 10 nM or less, 1 nM or less, 100 nM to 0.01 nM, 100 nM to 0.1 nM, 100 nM to 1 nM, or 10 nM to 1 nM, or 10 ug/mL or less, 5 ug/mL or less, 1 ug/mL or less, 0.9 ug/mL or less, 0.8 ug/mL or less, 0.7 ug/mL or less, 0.6 ug/mL or less, 0.5 ug/mL or less, 0.4 ug/mL or less, 0.3 ug/mL or less, 0.2 ug/mL or less, 0.1 ug/mL or less, 0.05 ug/mL or less, or 0.01 ug/mL or less. In some aspects, anti- PAD4 antibodies described herein bind to cyno PAD4, for example, with a KD of 10'⁶ M or less, 10'⁷M or less, 10'⁸ M or less, 10'⁹ M or less, 10'¹⁰ M or less, 10'¹¹ M or less, 10'¹² M or less, 10'¹² M to 10'⁷ M, 10^-11 M to 10'⁷ M, 10'¹⁰ M to 10'⁷ M, or 10'⁹ M to 10'⁷ M. In some aspects, an anti- PAD4 antibody binds to soluble cyno PAD4, e.g., as determined by BIACORE™ (e.g., as described in the Examples), with a KD of 10'⁶ M or less, 10'⁷ M or less, 10'⁸ M or less, 10'⁹ M (1 nM) or less, IO’¹⁰ M or less, 10’¹² M to 10’⁷M, lO^ M to 10’⁷M, 10’¹⁰M to 10’⁷M, 10’⁹ M to IO’⁷ M, or 10'⁸ M to 10'⁷M. Anti-PAD4 antibodies can bind to cynomolgus sPAD4, e.g., with an ECso of 100 nM or less, 10 nM or less, 100 nM to 0.01 nM, 100 nM to 0.1 nM, 100 nM to 1 nM, or 10 nM to 1 nM, e.g., as measured by ELISA (e.g., as described in the Examples).

[0121] In some aspects, the anti-PAD4 antibody specifically binds to human PAD4 with a KD of about 5 x 10'⁴ M or less, about 1 x 10'⁴ M or less, 5 x 10'⁵ M or less, about 1 x 10'⁵ M or less, about 1 x 10'⁶ M or less, about 1 x 10'⁷ M or less, or about 1 x 10'⁸ M or less, wherein KD is measured by surface plasmon resonance (Biacore) analysis. In some aspects, the anti-PAD4 antibody specifically binds to human PAD4 allele PAD4*002 with a KD of about 1 x 10'⁴ M or less, about 1 x 10'⁵ M or less, 1 x 10'⁶ M or less, about 1 x 10'⁷ M or less, or about 1 x 10'⁸ M or less, wherein KD is measured by surface plasmon resonance (Biacore) analysis. In some aspects, the anti-PAD4 antibody specifically binds to human PAD4 allele PAD4*004 with a KD of about 1 x 10'⁴ M or less, about 1 x 10'⁵ M or less, 1 x 10'⁶ M or less, about 1 x 10'⁷ M or less, or about 1 x 10'⁸ M or less, wherein KD is measured by surface plasmon resonance (Biacore) analysis. In some aspects, the anti-PAD4 antibody specifically binds to human PAD4 allele PAD4*008 with a KD of about 1 x 10'⁴ M or less, about 1 x 10'⁵ M or less, 1 x 10'⁶ M or less, about 1 x 10'⁷ M or less, or about 1 x 10'⁸ M or less, wherein KD is measured by surface plasmon resonance (Biacore) analysis. In some aspects, the anti-PAD4 antibody specifically binds to human PAD4 allele PAD4*009 with a KD of about about 1 x 10'⁴ M or less, about 1 x 10'⁵ M or less, 1 x 10'⁶ M or less, about 1 x 10'⁷ M or less, or about 1 x 10'⁸ M or less, wherein KD is measured by surface plasmon resonance (Biacore) analysis. In some aspects, the anti-PAD4 antibody specifically binds to human MICB allele MICB*005 with a KD of about 1 x 10'⁴ M or less, about 1 x 10'⁵ M or less, 1 x 10'⁶ M or less, about 1 x 10'⁷ M or less, or about 1 x 10'⁸ M or less, wherein KD is measured by surface plasmon resonance (Biacore) analysis.

[0122] In some aspects, the anti-PAD4 antibody specifically binds human PAD4 with an association constant (k_a) rate of at least about 1 x 10³ ms’¹, at least about 5 x 10³ ms’¹, at least about 1 x 10⁴ms^-1, at least about 5 x 10⁴ms^-1, at least about 1 x 1 Corns’¹, at least about 5 x 1 Co rns’¹, or at least about 1 x 10⁶ ms^-1, wherein k_a is measured by surface plasmon resonance (Biacore) analysis. [0123] In some aspects, the anti-PAD4 antibody specifically binds human PAD4 with a dissociation constant (kd) rate of about 0.1 s'¹ or less, 0.05 s'¹ or less, 0.01 s'¹ or less, 5 x 10'³ s'¹ or less, 1 x 10'³ s'¹ or less, 5 x 10'⁴ s'¹ or less, 1 x 10'⁴ s'¹ or less, 5 x 10'⁵ s'¹ or less, or 1 x 10'⁵ s'¹ or less, wherein KD is measured by surface plasmon resonance (Biacore) analysis.

[0124] In some aspects, the anti-PAD4 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises a VH complementarity determining region (CDR) 1 (VH-CDR1), a VH-CDR2, and a VH-CDR3 and the VL comprises a VL-CDR1, a VL-CDR2, and a VL-CDR3; wherein the VH-CDR3 comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 3, 13, 23, 33, or 43. In some aspects, the anti-PAD4 antibody comprises a VH-CDR3 comprising an amino acid sequence set forth in SEQ ID NOs: 3, 13, 23, 33, or 43. In one aspect, the anti-PAD4 antibody comprises a VH-CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 3. In one aspect, the anti-PAD4 antibody comprises a VH-CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 13. In one aspect, the anti-PAD4 antibody comprises a VH-CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 23. In one aspect, the anti-PAD4 antibody comprises a VH-CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 33. In one aspect, the anti-PAD4 antibody comprises a VH-CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 43.

[0125] In some aspects, the anti-PAD4 antibody comprises a VH-CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 2, 12, 22, 32, or 42. In some aspects, the anti-PAD4 antibody comprises a VH-CDR2 comprising an amino acid sequence set forth in SEQ ID NOs: 2, 12, 22, 32, or 42. In one aspect, the anti-PAD4 antibody comprises a VH-CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 2. In one aspect, the anti-PAD4 antibody comprises a VH-CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 12. In one aspect, the anti-PAD4 antibody comprises a VH-CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 22. In one aspect, the anti-PAD4 antibody comprises a VH-CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 32. In one aspect, the anti-PAD4 antibody comprises a VH- CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 42.

[0126] In some aspects, the anti-PAD4 antibody comprises a VH-CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 1, 11, 21, 31, or 41. In some aspects, the anti-PAD4 antibody comprises a VH-CDR1 comprising an amino acid sequence set forth in SEQ ID NOs: 1, 11, 21, 31, or 41. In one aspect, the anti-PAD4 antibody comprises a VH-CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1. In one aspect, the anti-PAD4 antibody comprises a VH-CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 11. In one aspect, the anti-PAD4 antibody comprises a VH-CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 21. In one aspect, the anti-PAD4 antibody comprises a VH-CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 31. In one aspect, the anti-PAD4 antibody comprises a VH- CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 41.

[0127] In some aspects, the anti-PAD4 antibody comprises a VL-CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 6, 16, 26, 36, or 46. In some aspects, the anti-PAD4 antibody comprises a VL-CDR1 comprising an amino acid sequence set forth in SEQ ID NOs: 6, 16, 26, 36, or 46. In one aspect, the anti-PAD4 antibody comprises a VL-CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 6. In one aspect, the anti-PAD4 antibody comprises a VL-CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 16. In one aspect, the anti-PAD4 antibody comprises a VL-CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 26. In one aspect, the anti-PAD4 antibody comprises a VL-CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 36. In one aspect, the anti-PAD4 antibody comprises a VL- CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 46.

[0128] In some aspects, the anti-PAD4 antibody comprises a VL-CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, or 47. In some aspects, the anti-PAD4 antibody comprises a VL-CDR2 comprising an amino acid sequence set forth in SEQ ID NOs: 7, 17, 27, 37, or 47. In one aspect, the anti-PAD4 antibody comprises a VL-CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 7. In one aspect, the anti-PAD4 antibody comprises a VL-CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 17. In one aspect, the anti-PAD4 antibody comprises a VL-CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 27. In one aspect, the anti-PAD4 antibody comprises a VL-CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 37. In one aspect, the anti-PAD4 antibody comprises a VL-CDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 47.

[0129] In some aspects, the anti-PAD4 antibody comprises a VL-CDR3 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 8, 18, 28, 38, or 48. In some aspects, the anti-PAD4 antibody comprises a VL-CDR3 comprising an amino acid sequence set forth in SEQ ID NOs: 8, 18, 28, 38, or 48. In one aspect, the anti-PAD4 antibody comprises a VL-CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 8. In one aspect, the anti-PAD4 antibody comprises a VL-CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 18. In one aspect, the anti-PAD4 antibody comprises a VL-CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 28. In one aspect, the anti-PAD4 antibody comprises a VL-CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 38. In one aspect, the anti-PAD4 antibody comprises a VL- CDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 48.

[0130] In some aspects, the anti-PAD4 antibody comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:6, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:8. In some aspects, the anti-PAD4 antibody is non- fucosylated. In some aspects, the anti-PAD4 antibody is hypo-fucosylated. In some aspects, the anti-PAD4 antibody cross competes for binding to human PAD4 with a reference antibody comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH- CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:6, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:8. In some aspects, the anti-PAD4 antibody binds to the same epitope as a reference antibody comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:6, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8.

[0131] In some aspects, the anti-PAD4 antibody comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18. In some aspects, the anti-PAD4 antibody is non- fucosylated. In some aspects, the anti-PAD4 antibody is hypo-fucosylated. In some aspects, the anti-PAD4 antibody cross competes for binding to human PAD4 with a reference antibody comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a VH- CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18. [0132] In some aspects, the anti-PAD4 antibody comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:21, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:22, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:23, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:26, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:27, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:28. In some aspects, the anti-PAD4 antibody is non- fucosylated. In some aspects, the anti-PAD4 antibody is hypo-fucosylated. In some aspects, the anti-PAD4 antibody cross competes for binding to human PAD4 with a reference antibody comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:21, a VH- CDR2 comprising the amino acid sequence set forth in SEQ ID NO:22, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:23, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:26, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:27, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:28. [0133] In some aspects, the anti-PAD4 antibody comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:31, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:32, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:36, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:37, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:38. In some aspects, the anti-PAD4 antibody is non- fucosylated. In some aspects, the anti-PAD4 antibody is hypo-fucosylated. In some aspects, the anti-PAD4 antibody cross competes for binding to human PAD4 with a reference antibody comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:31, a VH- CDR2 comprising the amino acid sequence set forth in SEQ ID NO:32, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:36, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:37, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:38. [0134] In some aspects, the anti-PAD4 antibody comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:46, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:47, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:48. In some aspects, the anti-PAD4 antibody is non- fucosylated. In some aspects, the anti-PAD4 antibody is hypo-fucosylated. In some aspects, the anti-PAD4 antibody cross competes for binding to human PAD4 with a reference antibody comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:41, a VH- CDR2 comprising the amino acid sequence set forth in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:46, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:47, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:48. [0135] In some aspects, the anti-PAD4 antibody comprises a VH and a VL; wherein the VH comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 of the VH comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 4, 14, 24, 34, or 44, and the VL comprises a VL-CDR1, a VL- CDR2, and a VL-CDR3 of the VL comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, or 49. In some aspects, the anti-PAD4 antibody comprises a VH and a VL; wherein the VH comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 of the VH comprising an amino acid sequence set forth in SEQ ID NOs: 4, 14, 24, 34, or 44, and the VL comprises a VL-CDR1, a VL- CDR2, and a VL-CDR3 of the VL comprising an amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, or 49. In some aspects, the anti-PAD4 antibody comprises a VH and a VL; wherein the VH comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 of the VH comprising an amino acid sequence set forth in SEQ ID NO: 4 and the VL comprises a VL-CDR1, a VL-CDR2, and a VL-CDR3 of the VL comprising an amino acid sequence set forth in SEQ ID NO: 9. In some aspects, the anti-PAD4 antibody comprises a VH and a VL; wherein the VH comprises a VH- CDR1, a VH-CDR2, and a VH-CDR3 of the VH comprising an amino acid sequence set forth in SEQ ID NO: 14 and the VL comprises a VL-CDR1, a VL-CDR2, and a VL-CDR3 of the VL comprising an amino acid sequence set forth in SEQ ID NO: 19. In some aspects, the anti-PAD4 antibody comprises a VH and a VL; wherein the VH comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 of the VH comprising an amino acid sequence set forth in SEQ ID NO: 24 and the VL comprises a VL-CDR1, a VL-CDR2, and a VL-CDR3 of the VL comprising an amino acid sequence set forth in SEQ ID NO: 29. In some aspects, the anti-PAD4 antibody comprises a VH and a VL; wherein the VH comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 of the VH comprising an amino acid sequence set forth in SEQ ID NO: 34 and the VL comprises a VL-CDR1, a VL-CDR2, and a VL-CDR3 of the VL comprising an amino acid sequence set forth in SEQ ID NO: 39. In some aspects, the anti-PAD4 antibody comprises a VH and a VL; wherein the VH comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 of the VH comprising an amino acid sequence set forth in SEQ ID NO: 44 and the VL comprises a VL-CDR1, a VL-CDR2, and a VL- CDR3 of the VL comprising an amino acid sequence set forth in SEQ ID NO: 49.

[0136] In some aspects, the anti-PAD4 antibody comprises a VH comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 4, 14, 24, 34, or 44. In some aspects, the VH comprises an amino acid sequence set forth in SEQ ID NOs: 4, 14, 24, 34, or 44.

[0137] In some aspects, the anti-PAD4 antibody comprises a VL comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, or 49. In some aspects, the VL comprises an amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, or 49.

[0138] In some aspects, the anti-PAD4 antibody comprises a VH comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:4. In some aspects, the VH comprises the amino acid sequence set forth in SEQ ID NO:4. In some aspects, the anti-PAD4 antibody comprises a VL comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOV. In some aspects, the VL comprises the amino acid sequence set forth in SEQ ID NOV. In one aspect, the VH comprises the amino acid sequence set forth in SEQ ID NO:4 and the VL comprises the amino acid sequence set forth in SEQ ID NOV.

[0139] In some aspects, the anti-PAD4 antibody comprises a VH comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 14. In some aspects, the VH comprises the amino acid sequence set forth in SEQ ID NO: 14. In some aspects, the anti-PAD4 antibody comprises a VL comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 19. In some aspects, the VL comprises the amino acid sequence set forth in SEQ ID NO: 19. In ansome aspect, the VH comprises the amino acid sequence set forth in SEQ ID NO: 14 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 19.

[0140] In some aspects, the anti-PAD4 antibody comprises a VH comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:24. In some aspects, the VH comprises the amino acid sequence set forth in SEQ ID NO:24. In some aspects, the anti-PAD4 antibody comprises a VL comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:29. In some aspects, the VL comprises the amino acid sequence set forth in SEQ ID NO:29. In ansome aspect, the VH comprises the amino acid sequence set forth in SEQ ID NO:24 and the VL comprises the amino acid sequence set forth in SEQ ID NO:29.

[0141] In some aspects, the anti-PAD4 antibody comprises a VH comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:34. In some aspects, the VH comprises the amino acid sequence set forth in SEQ ID NO:34. In some aspects, the anti-PAD4 antibody comprises a VL comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:39. In some aspects, the VL comprises the amino acid sequence set forth in SEQ ID NO:39. In ansome aspect, the VH comprises the amino acid sequence set forth in SEQ ID NO:34 and the VL comprises the amino acid sequence set forth in SEQ ID NO:39. [0142] In some aspects, the anti-PAD4 antibody comprises a VH comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:44. In some aspects, the VH comprises the amino acid sequence set forth in SEQ ID NO:44. In some aspects, the anti-PAD4 antibody comprises a VL comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:49. In some aspects, the VL comprises the amino acid sequence set forth in SEQ ID NO:49. In ansome aspect, the VH comprises the amino acid sequence set forth in SEQ ID NO:44 and the VL comprises the amino acid sequence set forth in SEQ ID NO:49.

[0143] In some aspects, the anti-PAD4 antibody comprises a heavy chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 5, 15, 25, 35, or 45. In some aspects, the anti- PAD4 antibody comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 5, 15, 25, 35, or 45. In some aspects, the heavy chain amino acid sequence comprises one or more deletions, substitutions, or mutations within the immunoglobulin constant region, e.g., within the CHI domain, the CH2 domain, the CH3 domain, or the hinge region.

[0144] In some aspects, the anti-PAD4 antibody comprises a light chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 52, 54, 56, 58, or 60. In some aspects, the anti-PAD4 antibody comprises a light chain comprising an amino acid sequence selected from SEQ ID NOs: 10, 20, 30, 40, or 50.

[0145] In some aspects, the anti-PAD4 antibody comprises a heavy chain Fab comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the anti-PAD4 antibody comprises a heavy chain Fab comprising an amino acid sequence set forth in SEQ ID NO:5. In some aspects, the anti-PAD4 antibody comprises a light chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. In some aspects, the anti-PAD4 antibody comprises a light chain Fab comprising an amino acid sequence set forth in SEQ ID NO: 10. In ansome aspect, the anti-PAD4 antibody comprises a heavy chain Fab comprising the amino acid sequence set forth in SEQ ID NO: 5 and a light chain Fab comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0146] In some aspects, the anti-PAD4 antibody comprises a heavy chain Fab comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some aspects, the anti-PAD4 antibody comprises a heavy chain Fab comprising an amino acid sequence set forth in SEQ ID NO: 15. In some aspects, the anti-PAD4 antibody comprises a light chain Fab comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20. In some aspects, the anti-PAD4 antibody comprises a light chain Fab comprising an amino acid sequence set forth in SEQ ID NO: 20. In ansome aspect, the anti-PAD4 antibody comprises a heavy chain Fab comprising the amino acid sequence set forth in SEQ ID NO: 15 and a light chain Fab comprising the amino acid sequence set forth in SEQ ID NO: 20.

[0147] In some aspects, the anti-PAD4 antibody comprises a heavy chain Fab comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25. In some aspects, the anti-PAD4 antibody comprises a heavy chain Fab comprising an amino acid sequence set forth in SEQ ID NO: 25. In some aspects, the anti-PAD4 antibody comprises a light chain Fab comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30. In some aspects, the anti-PAD4 antibody comprises a light chain Fab comprising an amino acid sequence set forth in SEQ ID NO: 30. In ansome aspect, the anti-PAD4 antibody comprises a heavy chain Fab comprising the amino acid sequence set forth in SEQ ID NO: 25 and a light chain Fab comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0148] In some aspects, the anti-PAD4 antibody comprises a heavy chain Fab comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35. In some aspects, the anti-PAD4 antibody comprises a heavy chain Fab comprising an amino acid sequence set forth in SEQ ID NO: 35. In some aspects, the anti-PAD4 antibody comprises a light chain Fab comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40. In some aspects, the anti-PAD4 antibody comprises a light chain Fab comprising an amino acid sequence set forth in SEQ ID NO: 40. In ansome aspect, the anti-PAD4 antibody comprises a heavy chain Fab comprising the amino acid sequence set forth in SEQ ID NO: 35 and a light chain Fab comprising the amino acid sequence set forth in SEQ ID NO: 40.

[0149] In some aspects, the anti-PAD4 antibody comprises a heavy chain Fab comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 45. In some aspects, the anti-PAD4 antibody comprises a heavy chain Fab comprising an amino acid sequence set forth in SEQ ID NO: 45. In some aspects, the anti-PAD4 antibody comprises a light chain Fab comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 50. In some aspects, the anti-PAD4 antibody comprises a light chain Fab comprising an amino acid sequence set forth in SEQ ID NO: 50. In ansome aspect, the anti-PAD4 antibody comprises a heavy chain Fab comprising the amino acid sequence set forth in SEQ ID NO: 45 and a light chain Fab comprising the amino acid sequence set forth in SEQ ID NO: 50.

[0150] In some aspects, the anti-PAD4 antibody comprises a heavy chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 51, 53, 55, 57, or 59. In some aspects, the anti-PAD4 antibody comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 51, 53, 55, 57, or 59. In some aspects, the heavy chain amino acid sequence comprises one or more deletions, substitutions, or mutations within the immunoglobulin constant region, e.g., within the CHI domain, the CH2 domain, the CH3 domain, or the hinge region.

[0151] In some aspects, the anti-PAD4 antibody comprises a light chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 52, 54, 56, 58, or 60. In some aspects, the anti-PAD4 antibody comprises a light chain comprising an amino acid sequence selected from SEQ ID NOs: 52, 54, 56, 58, or 60.

[0152] In some aspects, the anti-PAD4 antibody comprises a heavy chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 51. In some aspects, the anti-PAD4 antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO:4. In some aspects, the anti-PAD4 antibody comprises a light chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52. In ansome aspect, the anti-PAD4 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 51 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 52.

[0153] In some aspects, the anti-PAD4 antibody comprises a heavy chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53. In some aspects, the anti-PAD4 antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 53. In some aspects, the anti-PAD4 antibody comprises a light chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54. In some aspects, the anti-PAD4 antibody comprises a light chain comprising an amino acid sequence set forth in SEQ ID NO: 54. In ansome aspect, the anti- PAD4 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 53 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 54.

[0154] In some aspects, the anti-PAD4 antibody comprises a heavy chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 55. In some aspects, the anti-PAD4 antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 55. In some aspects, the anti-PAD4 antibody comprises a light chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 56. In some aspects, the anti-PAD4 antibody comprises a light chain comprising an amino acid sequence set forth in SEQ ID NO: 56. In ansome aspect, the anti- PAD4 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 55 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 56. [0155] In some aspects, the anti-PAD4 antibody comprises a heavy chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 57. In some aspects, the anti-PAD4 antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 57. In some aspects, the anti-PAD4 antibody comprises a light chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 58. In some aspects, the anti-PAD4 antibody comprises a light chain comprising an amino acid sequence set forth in SEQ ID NO: 58. In ansome aspect, the anti- PAD4 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 57 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 58.

[0156] In some aspects, the anti-PAD4 antibody comprises a heavy chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 59. In some aspects, the anti-PAD4 antibody comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 59. In some aspects, the anti-PAD4 antibody comprises a light chain comprising an amino acid sequence having at least about at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 60. In some aspects, the anti-PAD4 antibody comprises a light chain comprising an amino acid sequence set forth in SEQ ID NO: 60. In ansome aspect, the anti- PAD4 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 59 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 60.

[0157] In some aspects, the anti-PAD4 antibody described herein is an IgGl, an IgG2, an IgG3, an IgG4 or a variant thereof. In some aspect, the anti-PAD4 described herein antibody is an IgGl antibody. In some aspects, the anti-PAD4 antibody described herein is non-fucosylated. In some aspects, the anti-PAD4 antibody described herein is hypofucosylated. [0158] In some aspects, the anti-PAD4 antibody described herein is a human antibody. In some aspects, the anti-PAD4 antibody described herein is a humanized antibody. In some aspects, the anti-PAD4 antibody described herein is a chimeric antibody.

A. Human PAD4 Epitopes

[0159] In some aspects, the anti-PAD4 antibody binds a specific epitope on human PAD4. The term "epitope," as used herein, includes any determinant capable being bound by an antigen binding protein, such as an antibody. An epitope is a region of an antigen that is bound by an antigen binding protein that targets that antigen, and when the antigen is a protein, includes specific amino acids that directly contact the antigen binding protein. Epitope determinants can include chePAD411y active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and/or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

[0160] In some aspects, the epitope comprises at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, or at least ten amino acids. Where the epitope comprises more than one amino acid, the more than one amino acid can be sequential or separated by more than one amino acid that does not directly interact with the anti-PAD4 antibody or which is not required for anti-PAD4 binding. In some aspects, the epitope comprises two sequential amino acids, three sequential amino acids, four sequential amino acids, five sequential amino acids, six sequential amino acids, seven sequential amino acids, eight sequential amino acids, nine sequential amino acids, ten sequential amino acids, or more than ten sequential amino acids. In some aspects, the epitope comprises at least two amino acids which are not sequentially location, but which are positioned in proximity when the human PAD4 is present in its three dimensional confirmation.

[0161] An epitope on an antigen, e.g., an epitope on human PAD4 that is bound by an anti- PAD4 antibody, can be identified using any method known in the art. In some aspects, the epitope is determined using yeast surface display. In some aspects, the epitope is determined by hydrogen/deuterium exchange mass spectrometry (HDX-MS).

[0162] A VH domain, or one or more CDRs thereof, described herein can be linked to a constant domain for forming a heavy chain, e.g., a full-length heavy chain. Similarly, a VL domain, or one or more CDRs thereof, described herein can be linked to a constant domain for forming a light chain, e.g., a full-length light chain. A full-length heavy chain (optionally with the exception of the C-terminal lysine (K) or with the exception of the C-terminal glycine and lysine (GK), which can be absent) and full length light chain may combine to form a full length antibody.

[0163] A VH domain described herein can be fused to the constant domain of a human IgG, e.g., IgGl, IgG2, IgG3 or IgG4, which are either naturally-occurring or modified, e.g., as further described herein. For example, a VH domain can comprise the amino acid sequence of any VH domain described herein fused to a human IgG, e.g., an IgGl, constant region, such as the following wild-type human IgGl constant domain amino acid sequence:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 63) or that of an allotypic variant of SEQ ID NO: 63 and have the following amino acid sequences: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 64; allotype specific amino acid residues are in bold and underlined).

[0164] A VH domain of an anti-PAD4 antibody can comprise the amino acid sequence of any VH domain described herein fused to an effectorless constant region, e.g., the following effectorless human IgGl constant domain amino acid sequences:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 65; "IgGl.lf," comprising substitutions L234A, L235E, G237A, A330S and P331S, which are underlined) or

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 66; "IgG1.3f", comprising substitutions L234A, L235E and G237A, which are underlined).

[0165] For example, an allotypic variant of IgGl comprises K97R, D239E, and/or L241M (underlined and bolded above) as numbered in SEQ ID NOs: 63. Within the full-length heavy chain constant region and according to EU numbering, these amino acid substitutions are numbered K214R, D356E, and L358M. In some aspects, the constant region of an anti-PAD4 antibody can further comprises one or more mutations or substitutions at amino acids LI 17, Al 18, G120, A213, and P214 (underlined above) as numbered in SEQ ID NO: 64, 65, and 66, or L234, A235, G237, A330 and P331, per EU numbering. In some aspects, the constant region of an anti-PAD4 antibody comprises one or more mutations or substitutions at amino acids LI 17A, Al 18E, G120A, A213S, and P214S of SEQ ID NO: 63, or L234A, L235E, G237A, A330S and P331S, per EU numbering. The constant region of an anti-PAD4 antibody may also comprise one or more mutations or substitutions L117A, A118E and G120A of SEQ ID NO: 63, or L234A, L235E and G237A, per EU numbering.

[0166] Alternatively, a VH domain of an anti-PAD4 antibody can comprise the amino acid sequence of any VH domain described herein fused to a human IgG4 constant region, e.g., the following human IgG4 amino acid sequence or variants thereof: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 67, comprising S228P). [0167] In some aspects, the heavy chain constant region comprises a lysine or another amino acid at the C-terminus. In some aspects, the heavy chain constant region is lacking one or more amino acids at the C-terminus.

[0168] The amino acid sequences of exemplary heavy and light chains are described herein.

[0169] Provided herein are isolated anti-human PAD4 antibodies, or antigen-binding portion thereof, comprising:

(al) heavy and light chain sequences comprising SEQ ID NOs: 51 and 52, respectively;

(a2) heavy and light chain sequences comprising SEQ ID NOs: 53 and 54, respectively;

(a3) heavy and light chain sequences comprising SEQ ID NOs: 55 and 56, respectively;

(a4) heavy and light chain sequences comprising SEQ ID NOs: 57 and 58, respectively; or

(a5) heavy and light chain sequences comprising SEQ ID NOs: 59 and 60, respectively. wherein the antibody specifically binds to human PAD4.

[0170] In some aspects, an anti-PAD4 antibody comprises a combination of a heavy and light chain sequences set forth herein, e.g., in the preceding paragraph, wherein the antibody comprises two heavy chains and two light chains, and can further comprise at least one disulfide bond linking the two heavy chains together. The antibodies can also comprise disulfide bonds linking each of the light chains to each of the heavy chains.

[0171] Heavy and light chains comprising an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75% or 70% identical to any of the heavy or light chains set forth herein (or their variable regions) can be used for forming anti -human PAD4 antibodies having the desired characteristics, e.g., those further described herein. Exemplary variants are those comprising an allotypic variation, e.g., in the constant domain, and/or a mutation in the variable or constant region, such as the mutations disclosed herein. Heavy and light chains comprising an amino acid sequence that differs in at most 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1 amino acid (by substitution, addition or deletion) from any of the heavy or light chains set forth herein (or their variable regions) can be used for forming anti-human PAD4 antibodies having the desired characteristics, e.g., those further described herein.

[0172] As used herein, a human antibody comprises heavy and light chain variable regions that are "the product of' or "derived from" a particular germline sequence if the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with the antigen of interest or screening a human immunoglobulin gene library displayed on phage with the antigen of interest. A human antibody that is "the product of or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., greatest % identity) to the sequence of the human antibody. A human antibody that is "the product of or "derived from" a particular human germline immunoglobulin sequence can contain amino acid differences as compared to the germline sequence, due to, for example, naturally- occurring somatic mutations or intentional introduction of site-directed mutation. However, a selected human antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as being human when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In some cases, a human antibody can be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody can display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0173] Anti-PAD4 antibodies can comprise a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences comprise specified amino acid sequences based on the anti-PAD4 antibodies described herein, or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the anti-PAD4 antibodies described herein.

[0174] Conservative amino acid substitutions can be made in portions of the antibodies other than, or in addition to, the CDRs. For example, conservative amino acid modifications can be made in a framework region or in the Fc region. A variable region or a heavy or light chain can comprise 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 conservative amino acid substitutions relative to the anti-PAD4 antibody sequences provided herein. In some aspects, an anti-PAD4 antibody comprises a combination of conservative and non-conservative amino acid modification. [0175] Also provided are engineered and modified antibodies that can be prepared using an antibody having one or more of the VH and/or VL sequences disclosed herein as starting material to engineer a modified antibody, which modified antibody can have altered properties from the starting antibody. An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody.

[0176] One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally- occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally-occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321 :522-525; Queen, C. et al. (1989) Proc. Natl. Acad. Set. U.S.A. 86: 10029- 10033; U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al).

[0177] Accordingly, some aspects described herein pertain to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences described herein, and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences described herein. Thus, such antibodies contain the VH and VL CDR sequences of monoclonal antibodies described herein yet can contain different framework sequences from these antibodies.

[0178] Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al. (1992) "The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of VH Segments with Different Hypervariable Loops" / . Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) "A Directory of Human Germ-line VH Segments Reveals a Strong Bias in their Usage" Eur. J. Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference.

[0179] In some aspects, the framework sequences for use in the anti-PAD4 antibodies described herein are those that are structurally similar to the framework sequences used by the anti- PAD4 antibodies described herein. The VH CDR1, CDR2 and CDR3 sequences, and the VL CDR1, CDR2 and CDR3 sequences, can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derive, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences. For example, it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queen et al.).

[0180] Engineered anti-PAD4 antibodies described herein include those in which modifications have been made to framework residues within VH and/or VL, e.g., to improve the properties of the antibody. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR- mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed. Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

[0181] Another type of variable region modification is to mutate amino acid residues within the VH and/or VL CDR1, CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein and provided in the Examples. In some aspects, conservative modifications (as discussed above) are introduced. The mutations can be amino acid substitutions, additions or deletions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.

[0182] Methionine residues in CDRs of antibodies can be oxidized, resulting in potential chePAD41 degradation and consequent reduction in potency of the antibody. Accordingly, also provided are anti-PAD4 antibodies which have one or more methionine residues in the heavy and/or light chain CDRs replaced with amino acid residues which do not undergo oxidative degradation. In some aspects, the methionine residues in the CDRs of antibodies PAD4.36, PAD4.52, PAD4.54, PAD4.2, and 71C2 are replaced with amino acid residues which do not undergo oxidative degradation.

[0183] Similarly, deamidation sites can be removed from anti-PAD4 antibodies, particularly in the CDRs.

[0184] Anti-PAD4 variable regions described herein can be linked (e.g., covalently linked or fused) to an Fc, e.g., an IgGl, IgG2, IgG3 or IgG4 Fc, which can be of any allotype or isoallotype, e.g., for IgGl : Glm, Glml(a), Glm2(x), Glm3(f), Glml7(z); for IgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(gl), G3m28(g5), G3ml l(b0), G3m5(bl), G3ml3(b3), G3ml4(b4), G3ml0(b5), G3ml5(s), G3ml6(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v); and for K: Km, Kml, Km2, Km3 (see, e.g., Jefferies et al. (2009) mAbs 1 : 1).

[0185] Generally, variable regions described herein can be linked to an Fc comprising one or more modification, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, antigen-dependent cellular cytotoxicity, and/or antibody-dependent cellular phagocytosis. Furthermore, an antibody described herein can be chePAD411y modified (e.g.,, one or more chePAD41 moi eties can be attached to the antibody) or be modified to alter its glycosylation, to alter one or more functional properties of the antibody. Each of these aspects is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

[0186] The Fc region encompasses domains derived from the constant region of an immunoglobulin, including a fragment, analog, variant, mutant or derivative of the constant region. Suitable immunoglobulins include IgGl, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE and IgM, The constant region of an immunoglobulin is defined as a naturally- occurring or synthetically-produced polypeptide homologous to the immunoglobulin C-terminal region, and can include a CHI domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain, separately or in combination.

[0187] Ig molecules interact with multiple classes of cellular receptors. For example IgG molecules interact with three classes of Fey receptors (FcyR) specific for the IgG class of antibody, namely FcyRI, FcyRII, and FcyRIII. The important sequences for the binding of IgG to the FcyR receptors have been reported to be located in the CH2 and CH3 domains. The serum half-life of an antibody is influenced by the ability of that antibody to bind to an Fc receptor (FcR).

[0188] In some aspects, the Fc region is a variant Fc region, e.g., an Fc sequence that has been modified (e.g., by amino acid substitution, deletion and/or insertion) relative to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to generate a variant), to provide desirable structural features and/or biological activity,

[0189] Generally, variants of the constant region or portions thereof, e.g., CHI, CL, hinge, CH2 or CH3 domains can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, and/or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutation, or 1-10 or 1-5 mutations, or comprise an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of the corresponding wild-type region or domain (CHI, CL, hinge, CH2, or CH3 domain, respectively), provided that the heavy chain constant region comprising the specific variant retains the necessary biological activity.

[0190] For example, one can make modifications in the Fc region in order to generate an Fc variant that (a) mediates increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP), (b) mediates increased or decreased complement mediated cytotoxicity (CDC), (c) has increased or decreased affinity for Clq and/or (d) has increased or decreased affinity for a Fc receptor relative to the parent Fc. Such Fc region variants will generally comprise at least one amino acid modification in the Fc region. Combining amino acid modifications is thought to be particularly desirable. For example, the variant Fc region can include two, three, four, five, etc. substitutions therein, e.g., of the specific Fc region positions identified herein.

[0191] A variant Fc region can also comprise a sequence alteration wherein amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removal can avoid reaction with other cysteine-containing proteins present in the host cell used to produce the anti-PAD4 antibodies described herein. Even when cysteine residues are removed, single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently. In some aspects, the Fc region can be modified to make it more compatible with a selected host cell. For example, one can remove the PA sequence near the N-terminus of a typical native Fc region, which can be recognized by a digestive enzyme in E. coli such as proline iminopeptidase. In some aspects, one or more glycosylation sites within the Fc domain can be removed. Residues that are typically glycosylated (e.g., asparagine) can confer cytolytic response. Such residues can be deleted or substituted with unglycosylated residues (e.g., alanine). In some aspects, sites involved in interaction with complement, such as the Clq binding site, can be removed from the Fc region. For example, one can delete or substitute the EKK sequence of human IgGl. In some aspects, sites that affect binding to Fc receptors can be removed, preferably sites other than salvage receptor binding sites. In some aspects, an Fc region can be modified to remove an ADCC site. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgGl . Specific examples of variant Fc domains are disclosed for example, in WO 97/34631, WO 96/32478 and W007/041635.

[0192] In some aspects, the hinge region of Fc is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. PatentNo. 5,677,425 by Bodmer etal. The number of cysteine residues in the hinge region of Fc is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In some aspects, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc- hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[0193] In some aspects, 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 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.

[0194] In another example, 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.

[0195] In another example, 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.

[0196] In another example, the Fc region can be modified to enhance affinity for an Fey and increase macrophage-mediated phagocytosis. See, e.g., Richard et al., Mo. Cancer. Ther. 7(8):2517-27 (2008), which is incorporated by reference herein in its entirety. In some aspects, the Fc region can be modified to increase affinity for FcyRIIa relative to inhibitory FcyRIIb. One particular point mutation, G236A (whose numbering is according to the EU index), has been identified as having increased affinity for FcyRIIa relative to inhibitory FcyRIIb. This increased affinity for FcRIIa correlated with increased macrophage-mediated phagocytosis, relative to native IgGl. In some aspects, the Fc region of the anti-PAD4 antibody comprises one or more mutation or combination of mutations selected from G236A, I332E, S239/I332E, I332E/G236A, and S239D/I332E/G236A. Other modifications to the Fc region can increase antibody dependent cellular cytotoxicity (ADCC), e.g., by increasing affinity for activating receptors such as FcyRI and/or FcyRIIIa. For example, the G236A substitution, and combination of the G236A substitution with modifications that improve affinity for activating receptors (e.g., FcyRI and/or FcyRIIIa), for example including but not limited to substitutions at 332 and 239, provide substantially improved ADCC relative to the parent WT antibody. See U.S. Patent No. 9,040,041, which is incorporated by reference herein in its entirety.

[0197] In another example, 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. 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. Other modifications for enhancing FcyR and complement interactions include but are not limited to substitutions 298A, 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.

[0198] 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 Kabat (WO00/42072).

[0199] 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; 9,040,041; 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).

[0200] The affinities and binding properties of an Fc region for its ligand can be determined by a variety of in vitro assay methods (biochePAD41 or immunological based assays) known in the art including but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent 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.

[0201] In some aspects, 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 et al., 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/311S (Dall Acqua et al. Journal of Immunology, 2002, 169:5171-5180, Dall'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.

[0202] In some aspects, 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 aspects 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.

[0203] 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 cd., 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 etal., 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.

[0204] Specific mutations at positions 234, 235, 236, 239, 267, 268, 293, 295, 324, 327, 328, 330, and 332 were shown to improve binding to FcyRIIa and/or reduce binding to FcyRIIb, resulting in enhanced ADCC and/or ADCP activity (Richards et al., Mol. Cancer Ther. 7(8):2517- 2527; U.S. Patent No. 9,040,041). In particular, Fc variants that selectively improve binding to one or more human activating receptors relative to FcyRIIb, or selectively improve binding to FcyRIIb relative to one or more activating receptors, may comprise a substitution selected from the group consisting of 234G, 2341, 235D, 235E, 2351, 235Y, 236A, 236S, 239D, 267D, 267E, 267Q, 268D, 268E, 293R, 295E, 324G, 3241, 327H, 328A, 328F, 3281, 3301, 330L, 330Y, 332D, and 332E. Additional substitutions that may also be combined include other substitutions that modulate FcyR affinity and complement activity, including but not limited to 298A, 298T, 326A, 326D, 326E, 326W, 326Y, 333 A, 333 S, 334L, and 334A (U.S. Pat. No. 6,737,056; Shields et al, lournal of Biological Chemistry, 2001, 276(9):6591-6604; U.S. Pat. No. 6,528,624; Idusogie et al., 2001, I. Immunology 166:2571-2572). Preferred variants that may be particularly useful to combine with other Fc variants include those that comprise the substitutions 298A, 326A, 333A, and 334A. Additional substitutions that may be combined with the FcyR selective variants include 247L, 255L, 270E, 392T, 396L, and 421K (U.S. Ser. No. 10/754,922; U.S. Ser. No. 10/902,588); and 280H, 280Q, and 280Y (U.S. Ser. No. 10/370,749).

[0205] When using an IgG4 constant domain, it can include the substitution S228P, which mimics the hinge sequence in IgGl and thereby stabilizes IgG4 molecules. III. Nonfucosylation, hypofucosylation and reduced fucosylation

[0206] In some aspects, the glycosylation of an antibody may be 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.

[0207] 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.

[0208] The interaction of antibodies with FcyRs can also be enhanced by modifying the glycan moiety attached to each Fc fragment at the N297 residue. In particular, the absence of core fucose residues enhances ADCC via improved binding of IgG to activating FcyRIIIA without altering antigen binding or CDC. Natsume et al. (2009) Drug Des. DeveL Ther. 3:7. There is convincing evidence that afucosylated tumor-specific antibodies translate into enhanced therapeutic activity in mouse models in vivo. Nimmerjahn & Ravetch (2005) Science 310:1510; Mossner et al. (2010) Blood 115:4393. Modification of antibody glycosylation 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 of this disclosure to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (a-(l,6) fucosyltransferase (see U.S. Pat. App. Publication No. 20040110704; Yamane-Ohnuki etal. (2004) BiotechnoL Bioeng. 87: 614), such that antibodies expressed in these cell lines lack fucose on their carbohydrates. As another example, EP 1176195 also describes a cell line with a functionally disrupted FUT8 gene as well as cell lines that have little or no activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody, for example, the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 describes a variant CHO cell line, Lecl3, 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 et al. (2002) J. Biol. Chem. 277 :26733. Antibodies with a modified glycosylation profile can also be produced in chicken eggs, as described in PCT Publication No. WO 2006/089231. Alternatively, antibodies with a modified glycosylation profile can be produced in plant cells, such as Lemna. See e.g. U.S. Publication No. 2012/0276086. PCT Publication No. WO 99/54342 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 etal. (1999) Nat. Biotech. 17: 176. Alternatively, the fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the enzyme alpha-L-fucosidase removes fucosyl residues from antibodies. Tarentino et al. (1975) Biochem. 14:5516. Antibodies with reduced fucosylation may also be produced in cells harboring a recombinant gene encoding an enzyme that uses GDP-6-deoxy-D-lyxo-4-hexylose as a substrate, such as GDP-6-deoxy-D-lyxo-4-hexylose reductase (RMD), as described at U.S. Pat. No. 8,642,292. Alternatively, cells may be grown in medium containing fucose analogs that block the addition of fucose residues to the N-linked glycan or a glycoprotein, such as antibody, produced by cells grown in the medium. U.S. Pat. No. 8,163,551; WO 09/135181.

[0209] Because nonfucosylated antibodies exhibit greatly enhanced ADCC and/or ADCP compared with fucosylated antibodies, antibody preparations need not be completely free of fucosylated heavy chains to be useful in the present invention. Residual levels of fucosylated heavy chains will not significantly interfere with the ADCC and/or ADCP activity of a preparation substantially of nonfucosylated heavy chains. Antibodies produced in conventional CHO cells, which are fully competent to add core fucose to N-glycans, may nevertheless comprise from a few percent up to 15% nonfucosylated antibodies. Nonfucosylated antibodies may exhibit ten-fold higher affinity for CD16, and up to 30- to 100-fold enhancement of ADCC and/or ADCP activity, so even a small increase in the proportion of nonfucosylated antibodies may drastically increase the ADCC and/or ADCP activity of a preparation. Any preparation comprising more nonfucosylated antibodies than would be produced in normal CHO cells in culture may exhibit some level of enhanced ADCC and/or ADCP. Such antibody preparations are referred to herein as preparations having reduced fucosylation. Depending on the original level of nonfucosylation obtained from normal CHO cells, reduced fucosylation preparations may comprise 80%, 70%, 60% 50%, 30%, 20%, 10% and even 5% nonfucosylated antibodies. Reduced fucosylation may be functionally defined as preparations exhibiting 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, two-fold, three-fold or greater enhancement of ADCC and/or ADCP compared with antibodies prepared in normal CHO cells, and not with reference to any fixed percentage of nonfucosylated species.

[0210] The level of nonfucosylation may be structurally defined. As used herein, “nonfucosylated” or “afucosylated” (terms used synonymously) refers to antibody preparations in which over 95% of heavy chains lack fucose, such as over 96%, over 97%, over 98%, over 99%, or 100%. The term “hypofucosylated” refers to antibody preparations in which more than 80% and less than or equal to 95% heavy chains lack fucose, e.g. antibody preparations in which between 85 and 95%, between 80 and 85%, between 80 and 90%, between 85 and 90%, or between 90 and 95% of heavy chains lack fucose. The term “hypofucosylated or nonfucosylated” refers to antibody preparations in which 80% or more of heavy chains lack fucose. The term “reduced fucosylation” refers to antibody preparations in which between 10 and 80% of heavy chains lack fucose, such as 20-80%, 30-80%, 40-80%, 50-80%, 60-80%, 70-80%, 20-70%, 30-70%, 40-70%, 50-70%, 60-70%, 20-60%, 30-60%, 40-60%, 50-60%, 20-50%, 30-50%, 40-50%, 20-40%, 30- 40%, 10-20%, 10-30%, or 20-30%.

[0211] In some aspects, hypofucosylated or nonfucosylated anti-PAD4 antibodies may be produced in cells lacking an enzyme essential to fucosylation, such as FUT8 (e.g. U.S. Pat. No. 7,214,775), or in cells in which an exogenous enzyme partially depletes the pool of metabolic precursors for fucosylation (e.g. U.S. Pat. No. 8,642,292), or in cells cultured in the presence of a small molecule inhibitor of an enzyme involved in fucosylation (e.g. WO 09/135181).

[0212] The level of fucosylation in an antibody preparation may be determined by any method known in the art, including but not limited to gel electrophoresis, capillary electrophoresis, liquid chromatography, and mass spectrometry. In some aspects, the level of fucosylation in an antibody preparation may be determined by hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC). In some aspects, to determine the level of fucosylation of an antibody preparation, samples are denatured and treated with PNGase F to cleave N-linked glycans, which are then analyzed for fucose content. LC/MS of full-length antibody chains is an alternative method to detect the level of fucosylation of an antibody preparation, but mass spectroscopy is inherently less quantitative.

[0213] In some aspects, the anti-PAD4 antibodies described herein may have reduced fucosylation, or be hypofucosylated or nonfucosylated. In some aspects, the anti-PAD4 antibodies described herein may have reduced fucosylation. In some aspects, the anti-PAD4 antibodies described herein may be hypofucosylated. In some aspects, the anti-PAD4 antibodies described herein may be nonfucosylated.

[0214] In some aspects, the anti-PAD4 antibodies described herein may comprise i) one or more amino acid mutations to the Fc region to alter FcyR binding and optionally ii) reduced or eliminated fucosylation. In some aspects, the anti-PAD4 antibodies described herein may comprise one or more amino acid mutations to the Fc region to alter FcyR binding and be hypofucosylated or nonfucosylated. In some aspects, the anti-PAD4 antibodies described herein may comprise one or more amino acid mutations to the Fc region to alter FcyR binding and be nonfucosylated [0215] Another modification of the anti-PAD4 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 aspects, 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 some aspects, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the anti-PAD4 antibodies described herein. See, for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

IV Antibody Physical Properties

[0216] Anti-PAD4 antibodies, e.g., those described herein, have some or all of the physical characteristics of the specific anti-PAD4 antibodies described herein, such as the characteristics described in the Examples.

[0217] Anti-PAD4 antibodies described herein can contain one or more glycosylation sites in either the light or heavy chain variable region. Such glycosylation sites can result in increased immunogenicity of the antibody or an alteration of the pK of the antibody due to altered antigen binding (Marshall et al., (1972) Anna Rev Biochem 41 :673-702; Gala and Morrison (2004) J. Immunol 172:5489-94; Wallick e 109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al., (1985) Nature 316:452-7; Mimura et al., (2000) Mol Immunol 37:697- 706). Glycosylation has been known to occur at motifs containing an N-X-S/T sequence. In some instances, an anti-PAD4 antibody does not contain variable region glycosylation. This can be achieved either by selecting antibodies that do not contain the glycosylation motif in the variable region or by mutating residues within the glycosylation region.

[0218] In some aspects, the anti-PAD4 antibodies described herein do not contain asparagine isomerism sites. The deamidation of asparagine can occur on N-G or D-G sequences and result in the creation of an isoaspartic acid residue that introduces a kink into the polypeptide chain and decreases its stability (isoaspartic acid effect).

[0219] Each antibody will have a unique isoelectric point (pi), which generally falls in the pH range between 6 and 9.5. The pi for an IgGl antibody typically falls within the pH range of 7-9.5 and the pi for an IgG4 antibody typically falls within the pH range of 6-8. There is speculation that antibodies with a pi outside the normal range can have some unfolding and instability under in vivo conditions. Thus, an anti-PAD4 antibody can contain a pi value that falls in the normal range. This can be achieved either by selecting antibodies with a pi in the normal range or by mutating charged surface residues.

[0220] Each antibody will have a characteristic melting temperature, with a higher melting temperature indicating greater overall stability in vivo (Krishnamurthy R and Manning M C (2002) Curr Pharm Biotechnol 3:361-71). Generally, the Txii (the temperature of initial unfolding) can be greater than 60°C, greater than 65°C, or greater than 70°C. The melting point of an antibody can be measured using differential scanning calorimetry (Chen et al., (2003) Pharm Res 20: 1952-60; Ghirlando et al., (1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al., (2002) J. Chromatogr Sci 40:343-9).

[0221] In some aspects, antibodies are selected that do not degrade rapidly. Degradation of an antibody can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995) Anal Chem 67:3626-32).

[0222] In some aspects, antibodies are selected that have minimal aggregation effects, which can lead to the triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties. Generally, antibodies are acceptable with aggregation of 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. Aggregation can be measured by several techniques, including size-exclusion column (SEC), high performance liquid chromatography (HPLC), and light scattering. In some aspects, the antibodies display a desirable solubility, e.g., solubility that allows commercial manufacturing. [0223] In some aspects, the antibodies described herein have higher stability than a reference antibody. In some aspects, the antibodies described herein have a higher melting temperature than a reference antibody. In some aspects, the antibodies described herein have a lower tendency for aggregation than a reference antibody. In some aspects, the antibodies described herein have a higher solubility than a reference antibody. In some aspects, the antibodies described herein have a higher rate of absorption, lower toxicity, higher biological activity and/or target selectivity, better manufacturability, and/or lower immunogenicity than a reference antibody. The reference antibody can be another antibody or fragments thereof, or conjugate thereof, that binds to PAD4.

V. Methods of Engineering Antibodies

[0224] As discussed above, the anti-PAD4 antibodies having VH and VL sequences disclosed herein can be used to create new anti-PAD4 antibodies by modifying the VH and/or VL sequences, or the constant region(s) attached thereto. Thus, in ansome aspect described herein, the structural features of an anti-PAD4 antibody described herein are used to create structurally related anti- PAD4 antibodies that retain at least one functional property of the anti-PAD4 antibodies described herein, such as binding to human PALM and cynomolgus PALM. For example, one or more CDR regions of hI281, hI364, or hI365 can be combined recombinantly with known framework regions and/or other CDRs to create additional, recombinantly-engineered, anti-PAD4 antibodies described herein, as discussed above. Other types of modifications include those described in the previous section. The starting material for the engineering method is one or more of the VH and/or VL sequences provided herein, or one or more CDR regions thereof. To create the engineered antibody, it is not necessary to actually prepare (i.e., express as a protein) an antibody having one or more of the VH and/or VL sequences provided herein, or one or more CDR regions thereof. Rather, the information contained in the sequence(s) is used as the starting material to create a "second generation" sequence(s) derived from the original sequence(s) and then the "second generation" sequence(s) is prepared and expressed as a protein.

[0225] Accordingly, provided herein are methods for preparing an anti-PAD4 antibody described herein.

[0226] The altered antibody can exhibit at least one of the functional properties set herein. The functional properties of the altered antibodies can be assessed using standard assays available in the art and/or described herein, such as those set forth in the Examples (e.g., ELIS As, FACS). [0227] In some aspects of the methods of engineering the anti-PAD4 antibodies described herein, mutations can be introduced randomly or selectively along all or part of an anti-PAD4 antibody coding sequence and the resulting modified anti-PAD4 antibodies can be screened for binding activity and/or other functional properties as described herein. Mutational methods have been described in the art. For example, PCT Publication WO 02/092780 by Short describes methods for creating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, PCT Publication WO 03/074679 by Lazar et al. describes methods of using computational screening methods to optimize physiochemical properties of antibodies.

VI. Nucleic Acid Molecules

[0228] Ansome aspect described herein pertains to nucleic acid molecules that encode the anti- PAD4 antibodies described herein. The nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., the chromosomal DNA that is linked to the isolated DNA in nature) or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, etal., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid described herein can be, for example, DNA or RNA and can or cannot contain intronic sequences. In some aspects, the nucleic acid is a cDNA molecule.

[0229] Nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acid encoding the antibody can be recovered from the library. VII. C. Generation of Transfectomas Producing Monoclonal Antibodies to PAD4

[0230] Antibodies can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (Morrison, S. (1985) Science 229: 1202).

[0231] For example, to express antibodies, or antibody fragments thereof, DNAs encoding partial or full-length light and heavy chains, can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma that expresses the antibody of interest) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector(s) by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). The light and heavy chain variable regions of the anti-PAD4 antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector.

[0232] Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0233] In addition to the antibody chain genes, recombinant expression vectors can carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g, the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, nonviral regulatory sequences can be used, such as the ubiquitin promoter or P-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

[0234] In addition to the antibody chain genes and regulatory sequences, recombinant expression vectors can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g, origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g,, U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

[0235] For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.

[0236] It is possible to express the anti-PAD4 antibodies described herein in either prokaryotic or eukaryotic host cells, such as mammalian cells.

[0237] Certain mammalian host cells for expressing the recombinant anti-PAD4 antibodies described herein include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 759:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

VIII. Immunoconjugates, Antibody Derivatives and Diagnostics

[0238] Anti-PAD4 antibodies described herein can be used for diagnostic purposes, including sample testing and in vivo imaging, and for this purpose the antibody (or binding portion thereof) can be conjugated to an appropriate detectable agent, to form an immunoconjugate. For diagnostic purposes, appropriate agents are detectable labels that include radioisotopes, for whole body imaging, and radioisotopes, enzymes, fluorescent labels and other suitable antibody tags for sample testing.

[0239] The detectable labels that can be linked to any anti-PAD4 antibody described herein can be any of the various types used currently in the field of in vitro diagnostics, including particulate labels including metal sols such as colloidal gold, isotopes such as I¹²⁵ or Tc" presented for instance with a peptidic chelating agent of the N2S2, N3S or N4 type, chromophores including fluorescent markers, luminescent markers, phosphorescent markers and the like, as well as enzyme labels that convert a given substrate to a detectable marker, and polynucleotide tags that are revealed following amplification such as by polymerase chain reaction. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase and the like. For instance, the label can be the enzyme alkaline phosphatase, detected by measuring the presence or formation of chemiluminescence following conversion of 1,2 dioxetane substrates such as adamantyl methoxy phosphoryloxy phenyl dioxetane (AMPPD), disodium 3-(4-(methoxyspiro{l,2-dioxetane-3,2'-(5'- chloro)tricyclo{3.3.1.1 3,7}decan}-4-yl) phenyl phosphate (CSPD), as well as CDP and CDP- STAR® or other luminescent substrates well-known to those in the art, for example the chelates of suitable lanthanides such as Terbium(III) and Europium(III). The detection means is determined by the chosen label. Appearance of the label or its reaction products can be achieved using the naked eye, in the case where the label is particulate and accumulates at appropriate levels, or using instruments such as a spectrophotometer, a luminometer, a fluorimeter, and the like, all in accordance with standard practice.

[0240] In some aspects, conjugation methods result in linkages which are substantially (or nearly) non-immunogenic, e.g., peptide- (i.e., amide-), sulfide-, (sterically hindered), disulfide-, hydrazone-, and ether linkages. These linkages are nearly non-immunogenic and show reasonable stability within serum (see e.g., Senter, P. D., Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009/059278; WO 95/17886).

[0241] In general, site specific reaction and covalent coupling is based on transforming a natural amino acid into an amino acid with a reactivity which is orthogonal to the reactivity of the other functional groups present. For example, a specific cysteine within a rare sequence context can be enzymatically converted in an aldehyde (see Frese, M. A., and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain a desired amino acid modification by utilizing the specific enzymatic reactivity of certain enzymes with a natural amino acid in a given sequence context (see, e.g., Taki, M. et al., Prot. Eng. Des. Sei. 17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136; and Protease-catalyzed formation of C — N bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403). Site specific reaction and covalent coupling can also be achieved by the selective reaction of terminal amino

[0242] The moiety can also be a synthetic peptide or peptide mimic. In case a polypeptide is chePAD411y synthesized, amino acids with orthogonal chePAD41 reactivity can be incorporated during such synthesis (see e.g., de Graaf, A. J. et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since a great variety of orthogonal functional groups is at stake and can be introduced into a synthetic peptide, conjugation of such peptide to a linker is standard chemistry.

[0243] In order to obtain a mono-labeled polypeptide, the conjugate with 1 : 1 stoichiometry can be separated by chromatography from other conjugation side-products. This procedure can be facilitated by using a dye labeled binding pair member and a charged linker. By using this kind of labeled and highly negatively charged binding pair member, mono conjugated polypeptides are easily separated from non-labeled polypeptides and polypeptides which carry more than one linker, since the difference in charge and molecular weight can be used for separation. The fluorescent dye can be useful for purifying the complex from un-bound components, like a labeled monovalent binder.

[0244] In some aspects the moiety attached to an anti-PAD4 antibody is selected from the group consisting of a binding moiety, a labeling moiety, and a biologically active moiety. [0245] In some aspects, the therapeutic agent is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive agent, a second antibody, an enzyme, an anti- neoplastic agent, and any combination thereof.

[0246] In some aspects, the immunoconjugate comprises an anti-PAD4 antibody and a radioactive agent. In some aspects, the radioactive agent is a radionucleotide. In some aspects, the radioactive agent comprises radioactive iodine. In particular aspects, the radioactive agent comprises 131-iodine. In some aspects, the radioactive agent comprises the radioactive isotope Yttrium-90.

[0247] In some aspects, the immunoconjugate comprises an anti-PAD4 antibody and an enzyme. In some aspects, the enzyme comprises glucose oxidase. In some aspects, the enzyme comprises a peroxidase. In some aspects, the enzyme comprises myeloperoxidase. In some aspects, the enzyme comprises glucose oxidase. In some aspects, the enzyme comprises horseradish peroxidase.

[0248] Anti-PAD4 antibodies, e.g., those described herein, can also be used for detecting PAD4, such as human PAD4, e.g., human PAD4 on the surface of a cell or soluble PAD4 in serum. The antibodies can be used, e.g., in an ELISA assay or in flow cytometry. In some aspects, an anti- PAD4 antibody is contacted with cells or serum for a time appropriate for specific binding to occur, and then a reagent, e.g., an antibody that detects the anti-PAD4 antibody, is added. Exemplary assays are provided in the Examples. Exemplary methods for detecting PAD4, e.g., surface expressed PAD4 or soluble PAD4 (sPAD4) in a sample (serum) comprise (i) contacting a sample with an anti-PAD4 antibody, for a time sufficient for allowing specific binding of the anti-PAD4 antibody to PAD4 in the sample, and (2) contacting the sample with a detection reagent, e.g., an antibody, that specifically binds to the anti-PAD4 antibody, such as to the Fc region of the anti- PAD4 antibody, to thereby detect PAD4 bound by the anti-PAD4 antibody. Wash steps can be included after the incubation with the antibody and/or detection reagent. Anti-PAD4 antibodies for use in these methods do not have to be linked to a label or detection agents, as a separate detection agent can be used.

[0249] Other uses for anti-PAD4 antibodies, e.g., as monotherapy or combination therapy, are provided elsewhere herein, e.g., in the section pertaining to combination treatments.

IX. Bispecific Molecules

[0250] Anti-PAD4 antibodies described herein can be used for forming bispecific molecules. An anti-PAD4 antibody, or antigen-binding portions thereof, can be derivatized or linked to another functional molecule, e.g., another peptide or protein e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. For example, an anti-PAD4 antibody can be linked to an antibody or scFv that binds specifically to any protein that can be used as potential targets for combination treatments, such as the proteins described herein. The antibody described herein can in fact be derived or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule described herein, an antibody described herein can be functionally linked to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results.

[0251] Accordingly, provided herein are bispecific molecules comprising at least one first binding specificity for PALM and a second binding specificity for a second target epitope. In some aspects described herein in which the bispecific molecule is multispecific, the molecule can further include a third binding specificity.

[0252] In some aspects, the bispecific molecules described herein comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an Fab, Fab', F(ab')2, Fv, or a single chain Fv (scFv). The antibody can also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. U.S. Patent No. 4,946,778.

[0253] While human monoclonal antibodies are preferred, other antibodies which can be employed in the bispecific molecules described herein are murine, chimeric and humanized monoclonal antibodies.

[0254] The bispecific molecules described herein can be prepared by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or crosslinking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2- pyridyldithio)propi onate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1- carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Some conjugating agents are SATA and sulfo-SMCC, both available from Pierce ChePAD41 Co. (Rockford, IL).

[0255] When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In some aspects, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation. [0256] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific molecule is a mAb x mAb, mAb x Fab, mAb x (scFv)2, Fab x F(ab')2 or ligand x Fab fusion protein. A bispecific antibody can comprise an antibody comprising an scFv at the C- terminus of each heavy chain. A bispecific molecule described herein can be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules can comprise at least two single chain molecules. Methods for preparing bispecific molecules are described for example in U.S. Patent Number 5,260,203; U.S. Patent Number 5,455,030; U.S. Patent Number 4,881,175; U.S. Patent Number 5,132,405; U.S. Patent Number 5,091,513; U.S. Patent Number 5,476,786; U.S. Patent Number 5,013,653; U.S. Patent Number 5,258,498; and U.S. Patent Number 5,482,858.

[0257] Binding of the bi specific molecules to their specific targets can be confirmed using art- recognized methods, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

X. Compositions

[0258] Further provided are compositions, e.g., a pharmaceutical compositions, containing one or a combination of anti-PAD4 antibodies or combination with antibodies to other targets, or antigen-binding portion(s) thereof, described herein, formulated together with a pharmaceutically acceptable carrier. Such compositions can include one or a combination of (e.g., two or more different) antibodies, or immunoconjugates or bispecific molecules described herein. For example, a pharmaceutical composition described herein can comprise a combination of antibodies (or immunoconjugates or bispecifics) that bind to different epitopes on the target antigen or that have complementary activities.

[0259] In some aspects, the composition of the invention further comprises a bulking agent. A bulking agent can be selected from the group consisting of NaCl, mannitol, glycine, alanine, and any combination thereof. In some aspects, the composition of the invention comprises a stabilizing agent. The stabilizing agent can be selected from the group consisting of sucrose, trehalose, raffinose, arginine, and any combination thereof. In some aspects, the composition of the invention comprises a surfactant. The surfactant can be selected from the group consisting of polysorbate 80 (PS80), polysorbate 20 (PS20), and any combination thereof. In some aspects, the composition further comprises a chelating agent. The chelating agent can be selected from the group consisting of diethylenetriaminepentaacetic acid (DTP A), ethylenediaminetetraacetic acid, nitrilotri acetic acid, and any combination thereof.

[0260] In one aspect, the composition further comprises NaCl, mannitol, pentetic acid (DTP A), sucrose, PS80, and any combination thereof.

[0261] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some aspects, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). An option for subcutaneous injection is based on Halozyme Therapeutics’ ENHANZE® drug-delivery technology, involving a co-formulation of an Ab with recombinant human hyaluronidase enzyme (rHuPH20) that removes traditional limitations on the volume of biologies and drugs that can be delivered subcutaneously due to the extracellular matrix (U.S. Patent No. 7,767,429). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound. [0262] The pharmaceutical compounds described herein can include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N- methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[0263] A pharmaceutical composition described herein can also include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alphatocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0264] Examples of suitable aqueous and nonaqueous carriers that can be employed in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0265] These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms can be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0266] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions described herein is contemplated. A pharmaceutical composition can comprise a preservative or can be devoid of a preservative. Supplementary active compounds can be incorporated into the compositions.

[0267] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, the compositions can include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0268] A composition described herein can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for the anti-PAD4 antibodies described herein can include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0269] Alternatively, an antibody described herein could potentially be administered via a non- parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0270] The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0271] In some aspects, the anti-PAD4 antibodies described herein can be formulated to ensure proper distribution in vivo.

XL Uses and Methods

[0272] Some aspects of the present disclosure are directed to method of treating a subject, comprising administering to the subject an anti-PAD4 antibody disclosed herein, a polynucleotide encoding the anti-PAD4 antibody, a vector comprising the polynucleotide, a host cell comprising the polynucleotide, an immunoconjugate comprising an anti-PAD4 antibody, or any combination thereof.

[0273] Some aspects of the present disclosure are directed to a method of treating a rheumatoid arthritis in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein (e.g., an antibody, a polynucleotide, a vector, a host cell, an immunoconjugate, or a pharmaceutical composition).

[0274] Some aspects of the present disclosure are directed to a method of treating Parkinson's disease in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein (e.g., an antibody, a polynucleotide, a vector, a host cell, an immunoconjugate, or a pharmaceutical composition).

[0275] Some aspects of the present disclosure are directed to a method of treating a multiple sclerosis in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein (e.g., an antibody, a polynucleotide, a vector, a host cell, an immunoconjugate, or a pharmaceutical composition).

[0276] Some aspects of the present disclosure are directed to a method of treating Alzheimer's disease in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein (e.g., an antibody, a polynucleotide, a vector, a host cell, an immunoconjugate, or a pharmaceutical composition).

[0277] Some aspects of the present disclosure are directed to a method of treating lupus in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein (e.g., an antibody, a polynucleotide, a vector, a host cell, an immunoconjugate, or a pharmaceutical composition).

[0278] Some aspects of the present disclosure are directed to a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein (e.g., an antibody, a polynucleotide, a vector, a host cell, an immunoconjugate, or a pharmaceutical composition).

[0279] The compositions of the present disclosure can be administered using any pharmaceutically acceptable route. In some aspects, the composition (e.g., antibody, polynucleotide, vector, host cell, immunoconjugate, or pharmaceutical composition) is administered intravenously, intraperitoneally, intramuscularly, intraarterially, intrathecally, intralymphaticly, intralesionally, intracapsularly, intraorbitally, intracardiacly, intradermally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly, intraspinally, epidurally, intrasternally, topically, epidermally, mucosally, or any combination thereof. In some aspects, the composition is administered intravenously. In some aspects, the composition is administered subcutaneously.

XI.P Combination Therapies

[0280] In addition to the combinations therapies provided above, anti-PAD4 antibodies, e.g., those described herein, can also be used in combination therapy as described below.

[0281] Provided herein are methods of combination therapy in which an anti-PAD4 antibody is coadministered with one or more additional agents, e.g., small molecule drugs, antibodies or antigen binding portions thereof, that are effective in stimulating immune responses to thereby further enhance, stimulate or upregulate immune responses in a subject.

Table 1. Sequences¹.

¹ The CDRs are determined by the Kabat numbering system.

[0282] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) ImmunochePAD41 Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); ); Crooks, Antisense drug Technology: Principles, strategies and applications, 2^nd Ed. CRC Press (2007) and in Ausubel et al. (1989)

Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0283] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1.

[0284] Unlocking the potential of protein arginine deiminase 4 (PAD4) as a drug target for rheumatoid arthritis requires a deeper understanding of its regulation. In this study, we use unbiased antibody selections to identify functional antibodies capable of either activating or inhibiting PAD4 activity. Through cryo-electron microscopy, we characterized the structures of these antibodies in complex with PAD4 and revealed insights on their mechanisms of action. Remarkably, rather than steric occlusion of the substrate-binding catalytic pocket, the antibodies modulate PAD4 activity through interactions with allosteric binding sites adjacent to the catalytic pocket. These binding events lead to either alteration of the active site conformation or the enzyme oligomeric state, resulting in modulation of PALM activity. Our study uses antibody engineering to reveal new mechanisms for enzyme regulation and highlights the potential of using PAD4 agonist and antagonist antibodies for studying PAD4-dependency in disease models and future therapeutic development.

[0285] Antibodies are powerful tools for capturing the dynamic nature of proteins by binding to both active sites and allosteric sites of enzymes and interrogating different protein conformations.¹⁵ For example, deliberately selecting for antibodies to natural on- or off-states in proteins can be achieved by first “trapping” those conformations and then using differential in vitro antibody selection to isolate state-specific binders.^16,17,18 Instead of trapping the enzymatic state prior to selection, we hypothesized that using unbiased antibody selections on a native enzyme sampling various conformations in solution will allow us to identify novel conformations that activate or inhibit the enzyme. PALM is ideally suited for such an approach to better understand the mechanisms and protein states associated with activation and inhibition of the enzyme.

[0286] Moreover, antibodies are an ideal modality for targeting extracellular PALM since, unlike small molecules, they do not penetrate cells and would not interfere with normal homeostatic activities of intracellular PAD enzymes. Structural studies show each PAD4 binds five Ca²⁺ ions in two different pockets to become fully activated, and calcium-bound PAD4 is 10,000x as active as apo-PAD4.^19,20 PAD4’s calcium-dependent activation may be relevant when released into extracellular milieu where calcium concentration reaches millimolar (mM) levels compared to micromolar (pM). However, it’s extracellular regulation remains ambiguous given the high oxidizing environment outside of cells (https://www.sigmaaldrich.com/US/en/technical- documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell- culture/calcium-in-cell-culture). Also contributing to PAD4 extracellular regulation is the presence of autoantibodies that bind and further activate PAD4, which are found in 20-45% of RA patients. The presence of these autoantibodies is typically associated with a more severe and erosive disease phenotype.^21,22’²³’²⁴’²⁵’²⁶ The binding epitopes of several patient-derived anti-PAD4 autoantibodies have been characterized, but their mechanisms of action remain unclear.25,26 The existence of endogenous anti-PAD4 antibodies lead us to believe that we can use in vitro methods to identify anti-PAD4 antibodies also capable of modulating extracellular PAD4 activity.

[0287] Here, we utilized an unbiased antibody selection strategy, coupled with functional screening and Cryo-EM structural analysis, to identify new conformations and mechanisms for inhibiting or activating both murine and human PAD4 in the presence of high Ca²⁺. We discovered that PAD4 activity can be enhanced through antibody binding to an interface loop which promotes PAD4 dimerization while reducing disorder in the substrate-binding loop. We also discovered an inhibitory antibody that binds and re-structures a helix in the Ca+2 binding pocket that mediates a conformational change in the active site, preventing calcium ion and substrate binding. These engineered antibodies form a versatile toolkit for studying PAD4-dependence of disease states in both mouse models and patient samples. Through structural analysis of the antibody-PAD4 complexes, we shed light on previously unknown PAD4 regulatory mechanisms, providing new opportunities for pharmacological targeting of the enzyme.

Results:

[0288] Identifying PAD4 antibodies by phage display

[0289] As a first step toward antibody generation, it was critical to produce highly purified native forms of PAD4 suitable for in vitro phage antibody selections. N-terminal biotinylated forms of human PAD4 (hPAD4) and mouse PAD4 (mPAD4) were constructed and expressed in BirA- expressing E. coli for facile one-step purification and immobilization on streptavidin magnetic beads for phage selection (FIG. IF). Biotinylation was validated by a gel shift assay (FIG. 1G). Because PAD4 has free cysteines on the surface, 0.5 mM of Tris(2-carboxyethyl)phosphine (TCEP) was supplemented to the protein purification and phage selection buffers to prevent protein aggregation and loss of function (FIG. 1H). As antibodies are held together by a series of disulfide bonds, we confirmed that the Fab scaffold used in this phage library does not lose the antigenbinding capability even in the presence of the TCEP needed to stabilize PAD4 (FIG. II and FIG.

1J) [0290] We carried out two phage selection campaigns with a synthetic antibody Fab-phage library either in the presence of 10 mM free calcium or EDTA to sample the active and inactive states of PAD4 as PAD4’s activity is dependent on calcium (FIG. 1A and FIG. IK ). We observed that the thermostability of the Ca²⁺ bound state is significantly higher than the apo-state, shown by a 20oC upward shift in the melting temperature, demonstrating the enzyme exhibits two distinct conformational states with or without calcium (FIG. 1L-M) Four rounds of selections were performed with either 10 mM Ca²⁺ or 1 mM EDTA, respectively. To increase the stringency of selection, we systematically decreased the PAD4 concentration in every subsequent round of selection. At the end of the fourth round of selection, 95 individual clones were screened for binding by Fab-phage enzyme-linked immunosorbent assay (ELIS As). The top 10 binders from both the Ca²⁺ and EDTA selections were sequenced and expressed. We then performed a second hPAD4 selection to identify PAD4 activators and inhibitors that targeted different epitopes than the ones discovered in our first selection campaign. To do this, we added hI281 in excess during selection to block this previously identified epitope. This strategy allowed us to discover novel binders and create a toolkit of diverse PAD4 antibody modulators. The top binding clones from our second selection Fab-phage ELISAs were sequenced, affording 16 new anti-hPAD4 clones.

[0291] We were also interested in identifying antibodies against mouse PAD4, as these binders can be used as tools for studying the role of PAD4 in mouse models of RA and mimic the development of anti-PAD4 autoantibodies in RA patients.²⁷ We carried out an additional phage selection campaign against mPAD4 and after four rounds of selection, 12 antibodies that exclusively bind to mPAD4 and 16 antibodies that were hPAD4/mPAD4 cross-reactive were identified (FIG. 2F).

[0292] Characterization of PAD4 antibodies

[0293] We utilized two established PAD4 activity assays for initial characterization of the functional consequences of antibody binding. The first is an end-point immunoblot assay that detects citrullination of a large natural protein substrate, histone H3. The second is a spectrophotometric assay utilizing a small molecule trypsin-fluorogenic substrate pair in which the substrate citrullination by PAD4 prevents hydrolysis by trypsin, so that a reduced fluorescence readout is proportional to higher PAD4 activity (FIG. 1B-C). As expected, PAD4 activity significantly increases with higher concentrations of Ca²⁺ as measured by each assay. We used the more rapid and higher-throughput try p sin/ fluor ogenic peptide assay to screen the function of antibodies identified through our phage selection strategy. [0294] The naming of our antibodies was as follows: the first letter (h or m) stands for human or mouse PAD4 to which the antibody binds. If the antibody is cross-reactive to human and mouse, we designate it as hm. The second letter (I, A or N) means inhibitory, activating, or neutral, respectively. The letters are followed by the plasmid ID number. We found inhibitory, activating, and neutral binders towards both hPAD4 and mPAD4 and summarize their binding and functional properties in Table 2.

[0295] Table 2. Summary table of all anti-human/mouse PAD4 antibodie detailing binding affinity, functional properties, mechanism of action, and associated PDB IDs.

[0296] We identified five antibodies to hPAD4 (hI281, hA288, hA362, hI364 and hI365) which showed either antagonistic or agonistic effects (FIG. 1C). hI281 and hA288 were identified as the strongest inhibitors and activators from the first selection, respectively, and these binders were then used in a second selection against hPAD4 to mask their epitopes for explore others. This epitope blocking strategy allowed us to identify activating antibody hA362, and inhibitory antibodies hI364, and hI365 that bind to different epitopes than the masking hI281 and hA288 (FIG. 1C). Our PAD4 modulating binders from the second selection also inhibited or activated PAD4-mediated histone H3 citrullination, indicating that these PAD4-binders can modulate citrullination of protein substrates as well as small synthetic substrates (FIG. 1D-E).

[0297] Interestingly, some of our most potent inhibitors (hI364, hI365) exhibited selective binding to the Ca²⁺-bound state of PAD4 and showed either weaker or no binding to the apoenzyme as measured by biolayer interferometry (BLI) in the presence or absence of Ca²⁺ (FIG. 2G). These results suggest that there are epitopes formed in only the Ca²⁺ bound form of PAD4 that are critical to enzyme activity. We believe that our calcium-dependent antibodies are targeting these critical epitopes, thus inhibiting activity. [0298] Our top binders were also tested for cross-reactivity between mPAD4 and hPAD4 as this would be a useful feature for studying PAD4 between mouse and human. However, most binders identified from single species selections lacked cross-reactivity when terminally evolved for five rounds. This may be attributed to the fact that hPAD4 and mPAD4 share only 73% sequence homology and contain notable amino acid differences near important structural regions. For example, the N-terminal domain calcium-binding pocket contains several significant mutations from human to mouse PAD4 (D157E, E170K, and D388N). We hypothesize that these changes may prevent the identification of cross-reactive binders to this important of region of enzyme regulation. In a further effort to identify pan-binding clones we took the eluted phage pool from round three of selection on mPAD4, and continued to select an additional two rounds on hPAD4. This selection on dual antigens significantly enriched for cross-reactive binders and we found one clone (hmI400) to inhibit both hPAD4 and mPAD4 (FIG. 3E).

[0299] Binding of antibodies to human and mouse PAD4

[0300] We characterized the binding affinity of several inhibiting and activating antibodies to human or mouse PAD4 using BLI. The binding affinity of each antibody was investigated under varying concentrations of calcium (0, 2, 10 mM). Of our inhibitory lead clones, hI364 and hI365 both exhibited no binding to PAD4 in calcium-free conditions but showed high affinity to PAD4 in 2 and 10 mM Ca²⁺ (KD for hI364 = 0.64 nM; KD for hI365 = 1.2 nM). The binding of other clones (hI281, hA362) remained unaffected by calcium concentration (FIG. 2G).

[0301] We similarly characterized the binding of our mPAD4 binders and identified mA342 to be a potent activator of mPAD4. Interestingly, this clone also exhibited calcium selectivity as its KD improves from 200 uM to < 1 nM upon addition of 10 mM calcium (FIG. 3F). However, mA342 tended to aggregate in solution and suffered from poor biophysical properties characterized by a low intensity, widely dispersed peak on SEC (FIG. 3G). We hypothesized that the poor solubility was due to several hydrophobic residues in the complementarity determining regions (CDRs). To test this, we performed an alanine scan of the CDRs to identify hotspots of hydrophobicity to improve the solubility of mA342.

[0302] The alanine scan identified four hydrophobic CDR residues that were dispensable as they did not impact binding of mA342 to mPAD4. These four residues (light chain 197; heavy chain Y101, Y111, Ml 14) were mutated to alanine to generate mA342-c3, which showed a clean, monodispersed peak on SEC, and improved binding and activation of mPAD4 measured by BLI (FIG. 3G-H) We believe that mA342-c3 could be a promising candidate for mouse models mimicking the pathology of activating anti-PAD4 autoantibodies.

[0303] Several antibodies modulate hPAD4 enzyme dimerization

[0304] We utilized negative stain EM (NSEM) to screen the overall shapes of PAD4 and the PAD4/antibody complexes, which are schematically represented in FIG. 2A. As expected from X- ray structures of PAD4, the enzyme formed a C-shaped monomeric species and a globular, dimeric species of hPAD4 (FIG. 2B). Previous reports have shown that PAD4 exists as both low activity monomers and highly active dimeric states. We next incubated hPAD4 with our functional antibody Fab fragments in equimolar amounts and characterized the stoichiometry of the protein complexes by NSEM. The antibody Fabs showed the typical donut-shaped structures in the EM micrographs. Interestingly, the 1 : 1 complex of hPAD4 to antibody was seen for hPAD4 in complex with the inhibitory antibody, hI281, while a 2:2 complex of hPAD4 to antibody was observed for hPAD4 in complex with both activating antibodies, hA288 and hA362 (FIG. 2B). Taken together, these data suggest that the inhibitor (hI281) may be blocking PAD4 dimerization as the antibody is only seen interacting with monomeric PAD4, while activators (hA288 and hA362) are promoting PAD4 dimerization and promoting activity.

[0305] We confirmed the stoichiometries for 1 : 1 inhibitor/PAD4 (120 kDa) and 2:2 activator/PAD4 (240 kDa) complexes by analytical size exclusion chromatography (SEC) (FIG 2C). The free hPAD4 shows a major dimeric peak and a minor monomeric peak on SEC reflecting its dimer to monomer equilibrium. The hPAD4/hA288 complex runs as a homogeneous species at its expected MWs of a 2:2 complex, whereas the hPAD4/hI281 complex runs at the MW of a 1 : 1 complex. These data confirm that hI281 stabilizes the hPAD4 monomer, forming a 1 : 1 complex with hPAD4, while hA288 promotes hPAD4 dimerization to a 2:2 complex.

[0306] PAD4 dimerization influences activity and antibody function

[0307] To further study how dimerization influences enzyme activity, we applied computation- guided mutational analysis to disrupt and test the PAD4 dimerization interface. Briefly, after relaxing and minimizing the structures in Rosetta (PDB: 1WDA), we used PyMOL to select interacting residues within 5 A from the two subunits. The interface energy function in Rosetta was applied to calculate an interaction score of different residues. Residues R8, Y435, F541, and W548 stood out as the top four residues contributing to interface association with large negative interaction scores (FIG. 4E). We chose R8 and Y435 located at the two ends of the interface for a mutational analysis (FIG. 4F). The interface energies of single mutant R8E, Y435A and double mutant R8E/Y435A were calculated to be significantly higher than the wildtype, indicating these mutations would be effective in breaking dimerization. R8 and Y435 were also reported in a previous mutational study to influence PAD4 dimerization constant.29 Additionally, PAD1 is the only PAD isoform that exists as a monomer in solution. This finding is likely explained by an R8Q substitution in the N-terminal domain of PAD1, again supporting the importance of R8 in enzyme dimerization.³⁰

[0308] Based on these analyses, we cloned and expressed the R8E, Y435A and double mutant R8E/Y435A of PAD4. We also expressed two additional mutants as negative controls, N438A and N438R, as residue N438 is predicted to minimally contribute to the interface energy. Indeed, we found that R8E, Y435A, and R8E/Y435A run as monomers on SEC while the WT, N438A, and N438R form dimers (FIG. 4G). All monomeric mutants were virtually inactive in the peptide citrullination assay while N438A and N438R had comparable activities to WT hPAD4 (FIG. 2D). These data confirmed that dimerization is important for PAD4 activity. We also found the monomerizing mutations slightly destabilized the enzyme, reducing the melting temperature of apo-PAD4 from 45°C to 40-42°C, and Ca²⁺-bound PAD4 from 65°C to 64°C (FIG. 4H-I).

[0309] We next studied how WT and mutant PAD4s bind to the activating antibody hA362 that promotes enzyme dimerization. The binding affinity of hA362 was decreased by about 3-, 10- , and 160-fold for R8E, Y435A and the R8E/Y435 double mutant, respectively; their ability to activate PAD4 decreased in rough proportion to their reduction in binding affinity. While the Y435A mutation directly affects the hA362 binding epitope, the R8E mutation alters a series of electrostatic interactions that also exist near the epitope, thus explaining the observed reduction in binding affinity and antibody functionality. In contrast, the null mutants, N438A and N438R, did not significantly impact binding (FIG. 2E and FIG. 4J). These results further support that the binding and activation efficacy of hA362 is influenced by dimerization of PAD4.

[0310] hA362 activates hPAD4 by stabilizing dimerization

[0311] To further understand the molecular mechanism of hA362 activation of PAD4 we obtained the cryo-EM structure of hPAD4 in complex with hA362 at 3.5 A resolution. The overall structure is a 2:2 complex, containing two copies of hA362 bound to a homodimeric PAD4 (FIG. 3). Consistent with previous structures of the enzyme alone, PAD4 forms an anti-parallel head-to- tail homodimer in C2 symmetry. Each PAD4 monomer adopts an elongated fold with a N-terminal domain in immunoglobulin-like structure and a C-terminal domain in an a/p propeller structure, and it binds to a total of five Ca²⁺ ions in two pockets (FIG. 5G-5H). [0312] Although each hA362 Fab predominantly interacts with the N-terminal domain of each PAD4 (Buried Surface Area (BSA): 1196.3 A2), the Fab HC spans across the PAD4 monomer to make an additional interaction with the C-terminal domain of a second PALM, close to the substrate binding site (BSA: 183.9 A2) (FIG. 3B). This readily explains why the binding affinity of hA362 is lower to the monomeric PAD4 mutants as monomerization eliminates a portion of the binding epitope. Importantly, this additional contact with the PALM in trans is with the interface loop (the I-loop, shown in red), a known regulatory motif (FIG. 3C-D and FIG. 51). The specific interaction of hA362 with the I-loop mechanistically explains why PALM activity is enhanced, as the substrate binding loop (S-loop, shown in purple) neighbors the I-loop. The I-loop engages the S-loop through an electrostatic interaction between R441 and D465 and this interaction is important for catalytic activity.31 Consistently, previous molecular dynamics simulations have found that the I-loop in a monomeric PALM W548 mutants exhibits high flexibility, accounting for the increased flexibility of the S loop and low activity of monomeric PAD4.31 Our structural results further show that the I-loop and the associated S-loop can be stabilized through antibody binding. Our data suggest that hA362 activates PALM by stabilizing the dimer and by directly facilitating organization of the active site.

[0313] hPAD4 inhibition by re-structuring binding pockets

[0314] Fabs hI364 and hI365 are two of the most potent inhibitors we identified. Both Fabs only recognize the Ca²⁺-bound form of PALM. We determined the cryo-EM structure of hPAD4 in complex with hI365 at 3.2 A resolution to understand the antibody calcium dependency and inhibitory mechanism (FIG. 4B). Similar to hA362, hPALM adopts a homodimeric structure and forms a 2:2 complex with hI365 (FIG 4A-B), but here each antibody only directly interacts with one PALM monomer (BSA: 1122.9 A2). The CDRH1 and H2 of hI365 binds to a region that is structured only upon binding of three Ca²⁺ ions in the N-terminal domain, explaining why the binding of hI365 is Ca²⁺-dependent (FIG. 4B).

[0315] Interestingly, each hPALM monomer in the hI365/hPAD4 complex only contains three of the five Ca²⁺ ions that are typically bound in PALM structures and the hA362/PAD4 complex (FIG. 5J). A closer look of the structure reveals that binding of hI365 to hPALM leads to the occlusion of both Ca²⁺ ions in C-terminal domain while the ions in the N-terminal domain remain bound (FIG. 4C and FIG. 5K). Additionally, the Y106 residue in CDRH3 loop of hI365 interacts with Trp347 in hPALM (FIG. 4C, pink), pulling the 340-352 loop out of the substrate binding site and disrupting a small helix usually formed in the active structures (residues 374-383).³² In the original hPAD4 structure (PDB: 1WDA) with substrate benzoyl-L-arginine amide (BAA), the 340- 352 loop contains key residues for interactions with both the C-terminal Ca²⁺ ions and the hPAD4 substrate.32 The 347-350 region also stabilizes R374, which binds substrate, along with the surrounding Ca²⁺ binding region (purple). R374 and the nearby helical region are also no longer resolved with hI365 binding (FIG. 4C, right panel, FIG. 5K). Therefore, structural alteration of these residues diminishes the ability of the protein to bind calcium in its C-terminal domain and the substrate in this pocket. Specifically, the negatively charged D350 residue is known to stabilize binding of the arginine substrate, and mutation of D350 results in loss of enzyme activity.³² Our structure shows that binding of hI365 to PALM results in D350 flipping away from the substrate pocket, thus leading to enzyme inhibition (FIG. 4C). Additionally, hI365 forms many hydrophobic interactions with one predominant PAD4 monomer though loop H3 also interacts with the second PALM monomer (FIG. 4D). These results reveal another mechanism for blocking PALM activity, namely pulling out loop 340-352 and preventing Ca²⁺ and substrate from binding.

[0316] Optimized hI365 shows potent inhibition of hPAD4 in vitro

[0317] While hI365 exhibited inhibitory properties against PALM in preliminary activity assays, the antibody performed inconsistently when attempting to obtain an IC50. In addition, we observed late elution of the antibodies on SEC, suggesting presence of sticky, hydrophobic residues that may have contributed to assay variability by causing antibody aggregation (FIG. 5B). We thus used structural information to inform in silico and experimental methods to improve the solubility of hI365. First, using a Rosetta-based pairwise interaction analysis, we identified a G58D mutation that improved antibody solubility and SEC behavior (FIG. 6A-C). Next, we used Rosetta Antibody Design (RAbD) algorithm to engineer light chain CDR loop 3 (L3). As seen in the cyro-EM structure, L3 is present at the binding interface but does not currently form any appreciable contacts to PAD4 (FIG. 5A and FIG. 6D). We predicted that optimizing the length of L3, which is composed of 9 amino acids in parental hI365, could promote additional contacts to PAD4. Computational screening of various randomized L3 grafts showed that the optimal length of L3 is 9 to 10 amino acids (FIG. 6E-H). However, though our L3 mutants bound PAD4 with similar affinity to WT hI365, all clones identified from our L3 engineering exhibited poor SEC profiles and did not approve the ability of the antibody to inhibit PAD4 (FIG. 6G-H).

[0318] Lastly, a library-based randomization approach based on the knowledge gained by structure-informed in silico designs was carried out to simultaneously optimize binding and biophysical properties. We created two libraries. In Library 1, all CDR H3 residues were targeted for soft randomization using a 70-10-10-10 formula.³³ In Library 2, an RVK degenerate codon strategy was employed at certain positions to bias mutagenesis to more hydrophilic residues (FIG. 5C). In both libraries, we incorporated information gained from our rAbD engineering of CDRL3 and randomized this loop to contain both 9 and 10 amino acids. After four rounds of panning, we identified six unique clones that improved binding to hPAD4 compared to the parent hI365 (FIG. 7A-D). Binding was initially measured via ELISA at 20 nM and 5 nM hPAD4 antigen, and results show that library-based clones outperformed the parental hI365, especially at 5 nM hPAD4 (FIG. 7C). To estimate antibody off-rates, we pre-formed an Ab/hPAD4 complex before adding the mixture to hPAD4-coated ELISA plates. While hI365 dissociated from the initial Ab/PAD4 complex and readily bound plate-bound hPAD4 antigen, the newly identified library clones remained associated to hPAD4 in the pre-formed complex, suggesting an improvement in affinity (FIG. 7D)

[0319] In particular, clones E3 and E6 showed significantly improved SEC profiles, binding kinetics measured via BLI, and a marked increase in PAD4 inhibition compared to hI365 (IC50 values of 94 nM and 13 nM, respectively; FIG. 5D-5F and FIG. 7E). E3 and E6 both incorporate the G58D mutation previously shown to improve antibody solubility (FIG. 5D). In agreement with our computational CDR L3 studies, parental hI365 and E3 have 9 amino acid L3 loop length, while clone E6 has a 10 amino acid long loop length. Thus, the affinity and the developability profiles of these antibodies were successfully improved by structure-informed antibody optimization, making them more suitable probes of PAD4 function.

[0320] Antibody-PAD4 specificity and broader substrate profiling

[0321] While current small molecule inhibitors show poor specificity for PAD4 over other PAD isoforms, we believe that our antibodies may offer the advantage of improved specificity that is commonly noted with biologies. We tested binding of our functional human antibodies (hI281, hA288, hA362, hI364, hI365, E3, and E6) against PAD4 alongside PAD2 and PAD3 via BLI, and all antibodies exhibited binding to PAD4 exclusively (FIG. 8A).

[0322] In addition to developing PAD4 specific binders, we wanted to ensure that our antibodies inhibited PAD4 in biologically relevant environments. As extracellular PAD4 activity is hypothesized to be pathogenic in RA, we performed a PAD4 activity assay against whole cell lysate containing cytosolic, nuclear, and membrane-bound proteins. While PAD4 alone citrullinated a variety of proteins present in whole cell lysate, our functional antibodies retained the ability to inhibit PAD4 (FIG. 8B). Table 3. Cryo-EM data collection, processing, and refinement statistics.

PAD4/hA362 PAD4/hl365

Data collection

Grids Quantifoil R1 .2/1 .3 Au 300 mesh

Vitrification method FEI Vitrobot FEI Vitrobot

Microscope Titan Krios Titan Krios

Magnification 105,000x 105,000x

Voltage (kV) 300 300

Stage tilt (°) 0, (15, 30) 0, (15, 30)

Detector K3 K3

Recording mode Counting Counting

Dose rate (e-Zpix/sec) 8, (15) 8, (8)

Total electron exposure (e-/A2) 70, (74) 77, (68)

Number of frames 140, (116) 140, (120)

Defocus range (pm) -1 to -2 -1 to -2

Pixel size (A) 0.835 0.835

Number of micrographs 4,882 4,559

Initial particle images (no.) 1 ,593,955 1 ,215,874

Data processing: C2 symmetry Final particle images (no.) 92,424 45,834

Symmetry C2 C2

Map resolution (A) 3.5 3.3

Refinement Initial model used (PDB code) 1WD9, 6OTC, 1 N8Z 1WD9, 6OTC, 1 N8Z

Symmetry C2 C2

PDB code EMDB code Model resolution (A) 3.5 3.3

FSC threshold 0.143 0.143

Map sharpening B factor (A2) -67 -100

Model composition Nonhydrogen atoms 30762 24640

Protein residues 2000 1594

Ligands 10 6

B factors (A2) Protein (min/max/mean) 0.00/578.92/95.83 37.44/357.03/79.88

Ligand 0.00/52.03/33.01 53.31/103.13/74.46

RMS deviations

Bond lengths (A) 0.011 (12) 0.010 (8)

Bond angles (°) 1 .450 (8) 1 .211 (4)

Validation

MolProbity score 0.64 0.81

Clash score 0.39 0.85

Rotamer outliers (%) 0 0.14

Ramachandran plot

Favored (%) 98.41 97.8

Allowed (%) 1.59 2.08

Disallowed (%) 0 0.13

Antibody Sequences: Fab-hl281

Light Chain (AA (SEQ ID NO: 68)):

DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC

QQSYYRTLFTFGQGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS

LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Heavy Chain (AA) (SEQ ID NO: 69):

EVQLVESGGGLVQPGGSLRLSCAASGFNVSYSSIHWVRQAPGKGLEWVASIYPYYGSTSYADSVKGRFTISADTSKNTAYLQMNSLR

AEDTAVYYCARQMYYWMFSKLALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

Fab-hl364

Light Chain (AA) (SEQ ID NO: 70):

DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC

QQSSSSUTFGQGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS

STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Heavy Chain (AA) (SEQ ID NO: 71 ):

EVQLVESGGGLVQPGGSLRLSCAASGFNVYSSSIHWVRQAPGKGLEWVASISSSSGSTSYADSVKGRFTISADTSKNTAYLQMNSLR

AEDTAVYYCARYSDHYYYWSYWSSWYSGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS

GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

Fab-hl365

Light Chain (AA) (SEQ ID NO: 72):

DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC

QQSSSSLVTFGQGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL

SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Heavy Chain (AA) (SEQ ID NO: 73):

EVQLVESGGGLVQPGGSLRLSCAASGFNFYYSIHWVRQAPGKGLEWVASISPYSGYTSYADSVKGRFTISADTSKNTAYLQMNSLRA

EDTAVYYCARKHPGSYPFWGWALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

Fab-hl365-E3

Light Chain (AA) (SEQ ID NO: 74):

DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC

QQSSSSLVTFGQGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL

SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Heavy Chain (AA) (SEQ ID NO: 75):

EVQLVESGGGLVQPGGSLRLSCAASGFNFFYSIHWVRQAPGKGLEWVASISPYTDYTSYADSVKGRFTISADTSKNTAYLQMNSLRA

EDTAVYYCARKHPGSYPFWGFALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

Fab-hl365-E6

Light Chain (AA) (SEQ ID NO: 76):

DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC

QQSMSSQLVTFGQGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS

LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Heavy Chain (AA) (SEQ ID NO: 77):

EVQLVESGGGLVQPGGSLRLSCAASGFNFYYSIHWVRQAPGKGLEWVASISPYTDRTSYADSVKGRFTISADTSKNTAYLQMNSLRA

EDTAVYYCARKHPGRYPNWGFALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

Discussion:

[0323] New therapeutic mechanisms for autoimmune diseases like RA are in significant demand as current approved treatments are limited in scope or susceptible to broad-spectrum resistance.³⁴ Upregulation of the expression and/or activity of PAD4, one of the five PAD isozymes expressed in the nucleus of bloodstream granulocytes, is associated with various autoimmune diseases including rheumatoid arthritis (RA), Alzheimer’s disease, multiple sclerosis, lupus, Parkinson’s disease, and cancer.³⁵’³⁶ Genetic studies have also found an association between polymorphisms in PAD4 gene expression and RA disease risk, while PAD4 knockout or pharmacological inhibition through small molecules have proven to reduce disease severities in several RA mouse models.³⁷ These results have inspired a recent rise in interest for developing PAD4 inhibitors as a new generation of RA therapeutics. [0324] Blocking PAD4-mediated extracellular citrullination is a promising strategy for autoimmune intervention, but further validation and mechanistic interrogation needs to be performed and requires new tools.³⁸ Antibodies are established protein tools and have been commonly employed to bind under specific conditions including pH and high calcium concentrations to target different protein conformations to interrogate their function.^39,40 Here, using an unbiased, Fab-phage display approach, we discovered conformation-selective antibodies and built a toolkit of both inhibitory and activating antibodies to influence PAD4 activity. We believe these function-modulating antibodies can aid the study of PAD4’s involvement in disease pathology and ultimately reveal insights for designing alternative and unique treatments of RA.

[0325] Our study reports the first structures of antibody binders in complex with PAD4, and our findings identify several allosteric mechanisms that may be harnessed for PAD4 activation or inhibition. The first mechanism is modulation of dimerization. Previous studies have discussed the impact of PAD4 oligomerization state on enzyme activity as structural changes along the dimerization interface heavily impact formation of the enzyme’s substrate binding loop.^3,28,30 Though the monomeric form remains active, kcat values of previously reported PALM monomerization mutants are decreased as much as 4-fold compared to the wild type enzyme. These results provide a rationale for developing PALM inhibitors that function as dimerization blockers. In our study we identified several binders, hI281, hA288, and hA362, that affect PALM dimerization and PALM function. In particular, hI281 blocks enzyme dimerization. Blocking enzyme dimerization with hI281 effectively inhibits enzyme activity, just as PALM monomerization mutants are also less active. On the other hand, the activating antibodies, hA288 and hA362, promoted dimerization and catalytic activity. Through cryo-EM analysis of the Fab/PALM complexes, hA362 was found to bind and stabilize the interface loop, which has previously been shown to stabilize the substrate binding loop upon PALM dimer formation.³⁰ These findings both reinforce previously known PALM regulation mechanisms afforded by enzyme oligomerization state and show that functional antibodies can be used to both activate and inhibit PALM activity through this mechanism.

[0326] Along with dimerization, calcium binding also plays an important role in PALM function. Ca²⁺ binding in the C-terminal domain is vital in structuring the enzyme catalytic pocket, while Ca²⁺ binding in the N-terminal domain plays an important role in proper protein folding.1 As PALM’s catalytic pocket is partially disordered in the absence of C-terminal Ca²⁺ ions, this acidic pocket presents an allosteric site that directly affects PAD4’s ability to catalyze citrullination. As determined through cryo-EM, our antibody hI365 binds to a structural fragment in the C-terminal Ca²⁺ pocket. Once hI365 engages PAD4, several key residues in this pocket are rearranged, inhibiting Ca²⁺ binding and subsequently leading to disruption of substrate binding and loss of enzymatic activity. Interestingly, hI365 also recognizes the folded Ca²⁺ site in the N- terminal domain, thus exhibiting selective binding to the PAD4 only in the presence of Ca²⁺. Specifically targeting calcium-bound PAD4 presents an intriguing strategy for RA treatment as a calcium-form selective binder will ignore inactive, apo-PAD4. This could hypothetically improve the therapeutic index of an anti-PAD4 drug, as we would only target protein actively contributing to disease pathogenesis in the joints.

[0327] Additionally, our functional antibodies show high specificity for PAD4 over other PAD isoforms. Of the 4 other PAD isoforms ( -1, -2, -3, and -6) we thought it was most relevant to test binding of our antibodies against PAD2 and PAD3, both of which share significant structural similarities to PAD4.^41,42 PAD2 is also implicated in various autoimmune diseases and ubiquitously expressed across all tissue, so there is a need for PAD4-specific tools to address its role separately from PAD2 in disease pathology. As for PAD3, it is known that a subset of RA patients has PAD3/PAD4 cross-reactive autoantibodies.^21,22 Given the precedence for PAD3/PAD4 antibody cross-reactivity, we thought it would be important to show that our antibodies are still PAD4 specific. To address this, we tested the binding of our antibodies against commercially available PAD2 and PAD3 via BLI and found that none of our antibodies exhibit binding to PAD2 or PAD3. These PAD4 specific antibodies should be useful for isolating the functional effects of extracellular PAD4 from PAD2/3.

[0328] Taken together, we discovered functional antibody modulators of PAD4 through unbiased phage selection methods. These antibodies were key to reveal alternative mechanisms, direct and allosteric, for both PAD4 activation and inhibition. In the future, these highly specific and functional PAD4 binders may be used to investigate PAD4 activity in mouse arthritis models and human samples and shed light on how PAD4-mediated citrullination impacts RA disease progression. Given the increasing evidence that PAD4 is a feasible anti-RA therapeutic target, using biologies to target extracellular PAD4 may provide a safer and more potent alternative for RA treatment.

Methods:

[0329] Vector design and construction [0330] We used a previously described vector for expression of Fabs in E. coli.⁴³ The pFUSE- hlgGl-Fc (InvivoGen) vector was used for expression of IgGs wherein the heavy chain was genetically fused to the hlgGl-Fc and the light chain was expressed on a separate copy of the vector without Fc. The vector used to express hPAD4 and mPAD4 was generated by Gibson cloning into the same vector for Fab expression. Each PAD4 was fused to an N-terminal His6-AviTag- PreScission or TEV cleavage site.

[0331] Expression and purification of PAD4

[0332] C43 (DE3) Pro+ or BL21 Gold (DE3) E. coli containing PAD4 expression vectors were grown in 2xYT at 37 °C to an OD-600 of 0.4-0.8 and then protein expression was induced by the addition of 0.5-1.0 mM IPTG. Incubation temperature was subsequently reduced to 18°C and the cultures were allowed to shake for 16-20 h. Cells were harvested by centrifugation and lysed using sonication. The lysate was centrifuged to remove inclusion bodies. The enzymes were purified by Ni-NTA resin with 0.5 mM TCEP supplemented to all buffers to prevent PAD4 oxidation. The purified enzyme was buffer exchanged to 50 mM Tris (pH 8), 400 mM sodium chloride, and supplemented with 0.5 mM TCEP. Purification steps were performed on ice to maintain high PAD4 enzymatic activity. Purified enzyme was aliquoted and flash frozen.

[0333] Phage display selections

[0334] All phage selections were done according to previously established protocols. Briefly, selections with antibody phage Library E were performed using biotinylated antigens captured with streptavidin-coated magnetic beads (Promega) ⁴³ Prior to each selection, the phage pool was incubated with streptavidin beads in order to deplete the library of any binders to the beads or sticky antibodies. In total, 3-5 rounds of selection were performed with decreasing amounts of PAD4 antigens (100 nM, 50 nM, 10 nM, 10 nM, 10 nM). 10 mM CaC12 or 1 mM EDTA was added for Ca²⁺ or calcium-free selection schemes. 0.5 mM TCEP was used in all buffers to keep thiols reduced.

[0335] Phage library-based affinity maturation

[0336] Kunkel mutagenesis was used to incorporate mutagenized oligo pools into CDR regions as previously described ^{44, 45}‘ ⁴⁶ Briefly, each soft randomization library was first generated by producing ssDNA in dut-/ung- E. coli cells. Phosphorylated oligos were annealed to the ssDNA and subsequently amplified to generate cccDNA which was electroporated into SS320 E. coli cells, grown up, and infected with M13K07 helper phage to generate our Fab-phage library. Selections using this library were then performed against PAD4 as described previously. [0337] Expression of Fabs

[0338] Fabs were expressed as previously described.43 Briefly, C43 (DE3) Pro+ E. coli containing expression plasmids were grown in TB at 37°C in an autoinduction media for 6 hrs and incubation temperature was subsequently reduced to 30°C where the cultures were allowed to grow for an additional 16-18 h. Cells were harvested by centrifugation, lysed, and Fabs were purified by Ni-NTA resin. Fab purity and integrity were assessed by SDS-PAGE. Fab sequences are provided herein.

[0339] Expression of IgGs

[0340] Expi293 (Life Technologies) cells were transiently co-transfected with two pFUSE (InvivoGen) vectors harboring either the IgG heavy chain and the IgG light chain at a mass ratio of 1 : 1. The ExpiFectamine 293 transfection kit (Life Technologies) was used for transfections as per manufacturer’s instructions. Cells were incubated for 5 days at 37°C in an 8% CO2 environment before the supernatants were harvested by centrifugation. IgGs were purified by Protein A affinity chromatography or Ni-NTA resin and assessed for quality and integrity by SDS- PAGE. IgG sequences are provided in the Supplementary Note 1.

[0341] Phage ELISAs

[0342] ELISAs were performed according to standard protocols. Briefly, 96-well Maxisorp plates were coated with NeutrAvidin (10 pg/ml) overnight at 4°C and subsequently blocked with BSA (2% w/v) for 1 h at 20°C. 20 nM of biotinylated PAD4 was captured on the NeutrAvidin- coated wells for 30 minutes followed by the addition phage supernatants diluted 1 :5 in ELISA buffer (TBS, pH 7.4, 0.05% Tween-20, 0.2% BSA) for 30 minutes. 10 mM calcium or 1 mM EDTA were supplemented to buffers to determine binding with either the calcium bound form or the apo form of PAD4. The bound phage was then detected using a horseradish peroxidase (HRP)- conjugated anti-phage monoclonal antibody (GE Lifesciences 27-9421-01) and imaged on a Tecan i-control (v3.4.3.0) plate reader. For competition ELISAs, diluted phage supernatants and PAD4 were incubated for 30 minutes at 20°C before addition to the NeutrAvidin coated plates.

[0343] DSF antibody stability assay

[0344] Fab or IgG samples in PBS were mixed with Sypro Orange dye (20 X stock) to make a final antibody concentration of 2, 4, 8, or 16 pM and a 4X dye concentration. 10 pL of each mixture was transferred to a Biorad 384-well PCR plate and covered by a qPCR Sealing Tape. The assay was preformed over a temperature range of 25°C to 95°C with a temperature ramping rate of approximately 0.5°C/30 seconds, and fluorescence was detected using a Roche LC480 Light Cycler. Due to instrumentation constraints, the ramp rate is set based on the number of data points acquired per °C. Currently this is set at 20 acquisitions per °C equating to a ramp rate of 0.03 °C/sec.

[0345] PAD4 293 Flp-in cells

[0346] To construct the PAD4 expressing HEK293 cell lines, Flp-In HEK293 (ThermoFisher) cells were co-transfected with the pOG44 vector (ThermoFisher) and a construct encoding PAD4, PAD4 D350A, or PAD4 with a green fluorescent protein Dronpa tag in the pcDNA5/FRT Mammalian Expression vector (ThermoFisher). Cells expressing PAD4 constructs were selected for in DMEM supplemented with 10% FBS, 100 pg/mL zeocin, and 100 pg/mL Hygromycin B (ThermoFisher). Protein expression was confirmed by fluorescence microscopy detection of the Dronpa tag or PAD4 activity assays. To prepare cell lysates for the PAD4 activity assays, cells were lysed in RIPA cell lysis buffer (50 million cells/mL) and supplemented with protease inhibitor cocktail (Sigma- Aldrich). After rotating at 4°C for 15 min, cell lysates were sonicated and spun at 16,000 g to remove cell debris.

[0347] Fluorescence-based PAD4 activity assay

[0348] PAD4 activities in the absence or presence of antibodies were assessed in a fluorescence-based assay with a pro-fluorescence substrate analog.28 1 pM of PAD4 was mixed with various concentrations of antibodies and calcium at 4°C for 45 min, followed by the addition of 25 pM substrate ZRcoum, an arginine mimetic that releases a fluorophore once cleaved by trypsin. The reaction was incubated at 37°C for 110 min. Upon addition of excess trypsin/EDTA to the solution, the fluorophore was liberated from uncitrullinated ZRcoum, but remained quenched and unmodified when the substrate was citrullinated. The reaction was read on a Tecan i-control (v3.4.3.0) fluorescence plate reader with an excitation wavelength of 345 nm and an emission wavelength of 465 nm.

[0349] Citrullinated histone H3 PAD4 activity assay

[0350] PAD4 activities in the absence or presence of antibodies were also assessed using a citrullinated histone H3 Western assay. 10-100 nM recombinant PAD4 were mixed with antibodies with various concentrations of calcium at 4°C for 45 min, and then incubated with 760 nM recombinant histone H3.1 (New England Biolabs). The reaction was incubated at 37°C for 110 min, followed by western analysis using an anti-citrullinated H3 primary antibody (Abeam Ab5103) and an anti-rabbit HRP secondary antibody. For the PAD4 293-Flp-in cell lysate assay, 10 pL of cell lysate was mixed with varying concentrations of antibodies and calcium in a final volume of 19 pL at 4°C for 45 min. The reactions were then incubated at 37°C for 110 min and subsequently analyzed by western blot using an anti-citrullinated H3 antibody (Ab5103) and antirabbit HRP secondary antibody. Images were acquired in Image Lab (v5.0) and processed with Image Studio Software (v5.2). IC50 measurements were obtained with technical triplicates and quantified using Fiji.⁴⁷

[0351] Modified citrulline western blot assay

[0352] Lysate was harvested from Expi293T cells using a lx RIPA + protease inhibitor solution. Antibody -PALM complexes were pre-formed at 4°C for 45 min. The complexes were then incubated at 37°C for 110 min and subsequently analyzed by western blot using an anti-modified citrulline detection kit (EMD Millipore). PVDF membrane was incubated with anti-citrulline probe overnight, then blocked and stained with a primary anti-modified citrulline antibody and secondary HRP linked anti-IgG antibody. HRP signal was detected using a BioRad ChemiDoc imager.

[0353] Size exclusion chromatography

[0354] SEC analysis was performed using an Agilent HPLC 1260 Infinity II LC system equipped with an AdvanceBio SEC column (300 A, 2.7 pm, Agilent). Each analyte was injected at 10 pM and run with a constant mobile phase of TBS high salt buffer (50 mM Tris, pH = 8, 400 mM NaCl) for 15 minutes. Fluorescence (excitation 285 nm, emission 340 nm) and absorbance were measured and analyzed with Agilent OpenLab CDS ChemStation software.

[0355] Biolayer interferometry

[0356] BLI measurements were made using an Octet RED384 (ForteBio) instrument. Biotinylated PAD4 was immobilized on optically transparent streptavidin biosensors (ForteBio) and loaded until a 1 nm signal was achieved. After blocking with 10 pM biotin, purified binders in solution were used as the analyte. TBSTB was used for all buffers. Data were analyzed using the ForteBio Octet analysis software, and kinetic parameters were determined using a 1 : 1 monovalent binding model (https://www.sartorius.com/en/products/protein-analysis/octet-bli-detection/octet- sy stem s- software) .

[0357] Negative-stain TEM

[0358] 2.5 uL of PAD4 samples at 40 pg/mL were applied to a glow-discharged Cu grid covered by continuous carbon film, then stained with 0.75% (w/v) uranyl formate.48 A Tecnai T12 microscope (ThermoFisher FEI Company) operated at 120 kV was used to analyze negatively stained grids. Images were recorded using an UltraScan 4000 camera (Gatan) at normal magnification of 52,000x corresponding to a pixel size of 2.21 A on the specimen.

[0359] Cryo-EM studies of PAD4 in complex with hA362 and hI365

[0360] hPAD4/hA362: Sample concentrations used for cryo-EM were between 2-3 uM in 50 mM Tris pH 8.0, 150 mM NaCl, 10 mM CaC12, 0.5 mM TCEP. Grids were frozen at 7 sec blot time, 5 °C, 95% humidity on a Vitrobot. Movies were collected on a Titan Krios at 300 kV and 0.835 A /pix, 74 e- total dose. 2214 images were collected untilted; 1620 movies were collected at 15° tilt; 1048 were collected at 30° tilt. CTF (constant transfer function) was estimated using Patch CTF in cryoSPARC2.49 1593955 particles were template picked after curation. 3D classification was performed in cryoSPARC2. One class at 3.⁴¹ A resolution with 274910 particles was chosen for further refinement. These particles underwent 2 rounds of ab initio reconstruction into 2 models. After this point, C2 symmetry was imposed. The resulting stack of 134775 particles went into one round of ab initio (2 classes) and one round of heterogeneous refinement (2 classes). One class with 92424 particles underwent homogeneous refinement to 3.5 A. These particles and corresponding map were imported into cisTEM and auto refined to 2 classes.50 Four rounds of local refinement into 2 classes were performed at increasing starting resolutions (10 A, 7 A, 5 A, 4.5 A), leading to a final local refinement where 1 class with 59.78% (55251) of the particles at 3.3 A was chosen. Generate3D was then used to generate half maps. Local and global resolution maps were generated in cryoSPARC2, and directional resolution was determined with the 3DFSC server.⁵¹

[0361] Model building: PDB 1WD9 was fit, SWISS-MODEL was used with model Fab 6OTC (light chain) and 1N8Z (heavy chain) to build a homology model from the hA362 Fab sequence. This model then underwent Phenix Real-Space Refinement,⁵² followed by Rosetta relaxation in torsion space. Loops that were not visible in the density were deleted with Coot. Isolde was then used for local adjustments to fix Ramachandran outliers and manual placement into the map density.⁵³ A final Rosetta torsion relax with C2 symmetry imposed was used to create the final model. Validation was performed in Phenix, and model-map correlation was performed with the MapQ plugin for UCSF Chimera. Buried surface area was calculated with the PISA server (FIG. 9).

[0362] hPAD4/hI365: Sample concentrations used for cryo-EM were between 2-3 uM in 50 mM Tris pH 8.0, 150 mM NaCl, 10 mM CaC12, 0.5 mM TCEP. Grids were frozen at 7 sec blot time, 5 °C, 95% humidity on a Vitrobot. As initial processing of untilited micrographs resulted in anisotropic resolution reconstruction, additional tilted data sets were collected. Movies were collected on a Titan Krios at 300 kV and 0.835 A /pix, 70 e- total dose. 1773 images were collected untilted; 1844 movies were collected at 15° tilt; 842 were collected at 30° tilt. CTF was estimated with Patch CTF in cryoSPARC2. Bad micrographs were removed with the exposure curation tool, leading to 1225 untilted, 1366 at 15° tilt, and 648 at 30° tilt. These underwent template picking, and 1215874 particles were picked. These underwent ab Initio reconstruction into 3 classes; the best class with 780324 particles was chosen. This class underwent ab Initio into 2 classes; the best class had 391776 particles. These were re-extracted and particles were placed into 5 ab initio classes. 241 of these then went through 3 classes of heterogeneous refinement; the best class with 143413 particles at 6.3 A was chosen. These particles then underwent 2 rounds of heterogeneous refinement with C2 symmetry imposed, leading to a stack of 73648 particles, which were then imported into cisTEM for further processing. These were classified in 2 classes with Auto Refinement; both classes resulted in 3.9 A maps. All particles then underwent four rounds of local refinement into 2 classes at increasing starting resolutions (8 A, 6 A, 5 A, 4.5 A).

[0363] In the final round, class 1 was chosen with 62.22% (45824) of the particles at 2.98 A. Generate3D was then used to generate half maps. Local and global resolution maps were generated in cryoSPARC2, and directional resolution was determined with the 3DFSC server.

[0364] Model building: PDB 1WD9 was fit, SWISS-MODEL was used with model Fab 6OTC (light chain) and 1N8Z (heavy chain) to build a homology model with the 365 Fab sequence. Fab loops were deleted and RosettaES was used to rebuild the loops. This model was flexibly fit into the density with Rosetta torsion relax. Loops and domains that were not visible in the density were deleted with Coot. Manual placement of resolved regions that did not match the homology models into map density was performed in Isolde. Finally, Rosetta torsion relax was performed again with C2 symmetry imposed to produce a final model. Validation was performed in Phenix, and modelmap correlation was performed with the MapQ plugin for UCSF Chimera. Buried surface area was calculated with the PISA server (FIG. 10).

[0365] Statistics and Reproducibility

[0366] All representative SDS-PAGE protein gels and western blots shown were reproduced three times before inclusion in text.

[0367] Computational methods: command lines and input files:

Example Parameters. xml file <ROSETTASCRIPTS>

<SCOREFXNS>

<ScoreFunction name="beta" weights="beta_novl6" />

</SCOREFXNS>

<MOVERS>

<InterfaceAnalyzerMover fixedchains="F_E_D" name="int_ddG" scorefxn="beta" />

<MutateResidue name="mutate_residue 1" new_res="HIS" target="58F" />

<MinMover bb="l" chi="l" name=" minimize" scorefxn- 'beta" tolerance="0.005" />

<RepackMinimize design_partnerl="O" design_partner2="0" interface_cutoff_distance="6.0" minimize_bb="l" minimize_sc="l" name="repack_interface" optimize_fold_tree='T" repack_non_ala="l" repack_partnerl="l" repack_partner2="l" scorefxn_minimize="beta" scorefxn_repack="beta" /> </MOVERS>

<PROTOCOLS>

<Add mover name- 'mutate residue 1" />

<Add mover name- 'repack interface" />

<Add mover name- 'minimize" />

<Add mover name- 'int ddG" />

</PROTOCOLS>

</ROSETTASCRIPTS>

Example shell script for name in 58 do filename= " $ { name }F.xml" echo $filename for amino in ARG LYS ASP GLU SER THR ASN GLN GLY PRO ALA ILE LEU MET PHE TRP TYR VAL HIS do

/usr/bin/python3 change file.py $filename $amino suffix= "_$ {name } $amino"

/home/shared/Rosetta/main/source/bin/rosetta_scripts.linuxgccrelease -parserprotocol $filename -in:file:s Ab365_PAD4_rm_noCA.pdb @flags.txt -database /home/shared/Rosetta/main/database -out:path:pdb mutant_pdb/ - out:path:score mutant score/ -outsuffix Ssuffix done done

Example change file.py import xml.etree.ElementTree as ET import sys

# amino = [ALA ILE LEU MET PHE TRP TRP TYR VAL] file name = sys.argv[l] amino name = sys.argv[2]

# amino name = amino [idx] file = ET.parse(file name) root = file.getrootQ res = root.findall('MOVERS/MutateResidue') res[O].set('new_res', amino name) file.write(file name)

Example flags.txt

-packing

-exl

-exlaro

-extrachi cutoff 0

-ex2

-nstruct 5

-overwrite

-mute core.util.prof

-mute core.io.database

-corrections : :beta_nov 16

Example script for Rosetta Antibody design of CDR L3 antibody designer.linuxgccrelease -s PAD4-FAB_relaxed_renumbered_min_no_calcium.pdb -graft design cdrs LI L3 -seq_design_cdrs LI L3 -light chain kappa -mc optimize dG -do dock -use epitope constraints -nstruct 1000 - scorefile format json -outprefix PAD4_

OR mpirun -np 16 antibody designer.mpi.linuxgccrelease @ common

Example common file for mpirun

#Input

-s relaxed_calcium.pdb

#-ignore_unrecognized_res

#-ignore_zero_occupancy false

#-load_PDB_components false

#design

-graft design cdrs L3

-seq_design_cdrs L3

-light chain kappa

#number of designs

-nstruct 1000

-random start

-allow omega mismatches for north clusters

#cdr instructions

#-cdr_instructions cdr_instructions.txt

#optimize

-mc optimize dG

#-mc_total_weight .001

#-mc_interface_weight .999

#-mintype relax

#Output

-scorefile format j son

#-pdb_comments

#-skip_connect_info

-outprefix PAD4

#docking & constraints

#-do_dock #-use_epitope_constraints

#Rotamers/packing (Generally recommended, but will slow us down here)

-exl

-ex2

-use input sc

#RAbD Options for speed

-outer cycle rounds 25

-inner cycle rounds 2

References:

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[0368] The foregoing description of the specific aspects will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0369] Some aspects of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0370] All publications, patents, and patent applications disclosed herein are incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed:

1. An antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises a VH complementarity determining region (CDR)

1, a VH-CDR2, and a VH-CDR3 and the VL comprises a VL-CDR1, a VL-CDR2, and a VL- CDR3; wherein the VH-CDR3 comprises an amino acid sequence set forth in SEQ ID NOs: 3, 13, 23, 33, or 43; the VH-CDR2 comprises an amino acid sequence set forth in SEQ ID NOs: 2, 12, 22, 32, or 42; the VH-CDR1 comprises an amino acid sequence set forth in SEQ ID NOs: 1, 11, 21, 31, or

41; the VL-CDR1 comprises an amino acid sequence set forth in SEQ ID NOs: 6, 16, 26, 36, or

46; the VL-CDR2 comprises an amino acid sequence set forth in SEQ ID NOs: 7, 17, 27, 37, or

47; and the VL-CDR3 comprises an amino acid sequence set forth in SEQ ID NOs: 8, 18, 28, 38, or 48.

2. The antibody or antigen binding portion thereo, of claim 2, wherein

(a) the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, the VH- CDR3 comprises the amino acid sequence set forth in SEQ ID NO:3, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:6, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:7, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:8;

(b) the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, the VH- CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, the VL- CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, the VL- CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17 or antigen binding portion thereof,, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18;

(c) the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:21, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:22, the VH- CDR3 comprises the amino acid sequence set forth in SEQ ID NO:23, the VL- CDR1 comprises the amino acid sequence set forth in SEQ ID NO:26, the VL- CDR2 comprises the amino acid sequence set forth in SEQ ID NO:27, and the VL- CDR3 comprises the amino acid sequence set forth in SEQ ID NO:28;

(d) the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:31, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:32, the VH- CDR3 comprises the amino acid sequence set forth in SEQ ID NO:33, the VL- CDR1 comprises the amino acid sequence set forth in SEQ ID NO:36, the VL- CDR2 comprises the amino acid sequence set forth in SEQ ID NO:37, and the VL- CDR3 comprises the amino acid sequence set forth in SEQ ID NO:38; or

(e) the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:41, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:42, the VH- CDR3 comprises the amino acid sequence set forth in SEQ ID NO:43, the VL- CDR1 comprises the amino acid sequence set forth in SEQ ID NO:46, the VL- CDR2 comprises the amino acid sequence set forth in SEQ ID NO:47, and the VL- CDR3 comprises the amino acid sequence set forth in SEQ ID NO:48.

3. An antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises a VH complementarity determining region (CDR) 1, a VH-CDR2, and a VH-CDR3 of the VH comprising an amino acid sequence set forth in SEQ ID NOs: 4, 14, 24, 34, or 44, and the VL comprises a VL-CDR1, a VL-CDR2, and a VL-CDR3 of the VL comprising an amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, or 49.

4. An antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, the VH- CDR3 comprises the amino acid sequence set forth in SEQ ID NO:3, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:6, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 7, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID N0:8.

5. An antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, the VH- CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17 or antigen binding portion thereof,, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18.

6. An antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:21, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:22, the VH- CDR3 comprises the amino acid sequence set forth in SEQ ID NO:23, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:26, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:27, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:28.

7. An antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:31, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:32, the VH- CDR3 comprises the amino acid sequence set forth in SEQ ID NO:33, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:36, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:37, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:38.

8. An antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:41, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:42, the VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:43, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:46, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:47, and the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:48.

9. The antibody of any one of claims 1 to 8, wherein the antibody has one or more following properties:

(a) the antibody inhibits PAD4 activity;

(b) the antibody inhibits PAD dimerization;

(c) the antibody promotes PAD monomerization;

(d) the antibody disrupts Ca²⁺ and substrate binding; or

(e) any combination thereof.

10. The antibody of any one of claims 1 to 8, wherein the VH comprises an amino acid sequence at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 4, 14, 24, 34, or 44.

11. The antibody of any one of claims 1 to 10, wherein the VH comprises an amino acid sequence set forth in SEQ ID NOs: 4, 14, 24, 34, or 44.

12. The antibody of any one of claims 1 to 11, wherein the VL comprises an amino acid sequence at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, or 49.

13. The antibody of any one of claims 1 to 12, wherein the VL comprises an amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, or 49.

14. The antibody of any one of claims 1 to 13, wherein

(a) the VH comprises the amino acid sequence set forth in SEQ ID NO:4 and the VL comprises the amino acid sequence set forth in SEQ ID NOV;

(b) the VH comprises the amino acid sequence set forth in SEQ ID NO: 14 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 19; (c) the VH comprises the amino acid sequence set forth in SEQ ID NO:24 and the VL comprises the amino acid sequence set forth in SEQ ID NO:29;

(d) the VH comprises the amino acid sequence set forth in SEQ ID NO:34 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 39; or

(e) the VH comprises the amino acid sequence set forth in SEQ ID NO:44 and the VL comprises the amino acid sequence set forth in SEQ ID NO:49.

15. The antibody of any one of claims 1 to 14, wherein:

(a) the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 51, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 52;

(b) the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 53, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 54;

(c) the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 55, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 56;

(d) the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 57, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 58; or

(e) the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 59, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 60.

16. An antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the antibody or antigen binding portion thereof cross competes with the antibody or antigen binding portion thereof of any of the preceding claims.

17. An antibody that specifically binds to human Peptidyl arginine deiminase 4 (PAD4), or antigen binding portion thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the antibody or antigen binding portion thereof binds to the same epitope as the antibody or antigen binding portion thereof of any of the preceding claims.

18. The antibody of any one of claims 1 to 17, wherein the antibody specifically binds human PAD4 with a KD of about 1 x 10'⁶ M M or less, wherein KD is measured by surface plasmon resonance (Biacore) analysis.

19. The antibody of any one of claims 1 to 18, wherein the antibody is an IgGl, an IgG2, an IgG3, an IgG4 or a variant thereof.

20. The antibody of any one of claims 1 to 19, wherein the antibody is an IgGl antibody.

21. The antibody of anyone of claims 1 to 20, which is non-fucosylated.

22. The antibody of any one of claims 1 to 21, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

23. A polynucleotide or a set of polynucleotides encoding the antibody of any one of claims 1 to 22.

24. A vector or a set of vectors comprising the polynucleotide or the set of polynucleotides of claim 23.

25. A cell comprising the antibody of any one of claims 1 to 22, the polynucleotide or the set of polynucleotides of claim 23, or the vector or the set of vectors of claim 24.

26. An immunoconjugate comprising the antibody of any one of claims 1 to 22 and a therapeutic agent.

27. The immunoconjugate of claim 26, wherein the therapeutic agent is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive agent, a second antibody, an enzyme, an anti-neoplastic agent, and any combination thereof.

28. A pharmaceutical composition comprising the antibody of any one of claims 1 to 22, the polynucleotide or the set of polynucleotides of claim 23, or the vector or the set of vectors of claim 24, the cell of claim 25, or the immunoconjugate of any one of claims 26 to 27, and a pharmaceutically acceptable excipient.

29. A pharmaceutical composition comprising the antibody of any one of claims 1 to 22 and a second antibody.

30. The pharmaceutical composition of any one of claims 28 to 29, which is formulated for intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, topical, epidermal, or mucosal administration.

31. A method of treating a disease in a subj ect in need thereof, comprising administering to the subject the antibody of any one of claims 1 to 22, the polynucleotide or the set of polynucleotides of claim 23, or the vector or the set of vectors of claim 24, the cell of claim 25, or the immunoconjugate of any one of claims 26 to 27, or the pharmaceutical composition of any one of claims 28 to 30.

32. A method of reducing shed PAD4 in the serum in a subject in need thereof, comprising administering to the subject the antibody of any one of claims 1 to 22, the polynucleotide or the set of polynucleotides of claim 23, or the vector or the set of vectors of claim 24, the cell of claim 25, or the immunoconjugate of any one of claims 26 to 27, or the pharmaceutical composition of any one of claims 28 to 30.

33. A method of promoting PAD monomerization in a subject in need thereof, comprising administering the antibody of any one of claims 1 to 22, the polynucleotide or the set of polynucleotides of claim 23, or the vector or the set of vectors of claim 24, the cell of claim 25, or the immunoconjugate of any one of claims 26 to 27, or the pharmaceutical composition of any one of claims 28 to 30.

34. A method of disrupting Ca²⁺ and substrate binding in a subject in need thereof, comprising administering the antibody of any one of claims 1 to 22, the polynucleotide or the set of polynucleotides of claim 23, or the vector or the set of vectors of claim 24, the cell of claim 25, or the immunoconjugate of any one of claims 26 to 27, or the pharmaceutical composition of any one of claims 28 to 30.

35. The method of any one of claims 31 to 34, wherein the antibody of any one of claims 1 to 22, the polynucleotide or the set of polynucleotides of claim 23, or the vector or the set of vectors of claim 24, the cell of claim 25, or the immunoconjugate of any one of claims 26 to 27, or the pharmaceutical composition of any one of claims 28 to 30 is administered for intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrastemal, topical, epidermal, or mucosal administration.

36. The method of any one of claims 31 to 35, wherein the subject is a human.

37. The method of any one of claims 31 to 36, further comprising administering to the subject a second therapy.