Anti-cleaved histone H3 monoclonal antibody specifically recognizes neutrophil extracellular traps

Abstract

The present invention relates to a monoclonal antibody 3D9 that specifically binds to a C-terminal fragment of cleaved histone H3 in NETs, ​​and the monoclonal antibody 3D9 can be used to distinguish cleaved histone H3 from chromatin of different origins to specifically detect NETs. The present invention further provides a method for in vitro detection of neutrophil extracellular traps in isolated biological samples and a method for evaluating disease states associated with NET formation. The present invention further relates to an isolated fragment of human histone H3 cleaved at site L48R49 and the use of the cleavage site L48R49 for the specific detection of human neutrophil extracellular traps. The present invention further relates to a recombinant nucleic acid sequence encoding said polypeptide, and a host cell comprising a recombinant nucleic acid sequence encoding said polypeptide.

Description

Detailed Description of the Invention

[0001] The present invention relates to monoclonal antibody 3D9, which specifically binds to the C-terminal fragment of cleaved histone H3 in NETs, ​​and can be used to specifically detect NETs, ​​distinguishing them from chromatin of different origins.The present invention further provides a method for in vitro detection of neutrophil extracellular traps in isolated biological samples, and a method for evaluating disease states associated with NET formation.The present invention further relates to an isolated purified fragment of human histone H3 cleaved at site L48R49, and the use of the cleavage site L48R49 for the specific detection of human neutrophil extracellular traps.The present invention further relates to a recombinant nucleic acid sequence encoding said polypeptide, and a host cell comprising a recombinant nucleic acid sequence encoding said polypeptide.

[0002] [Background of the invention] Neutrophil extracellular traps (NETs) are defined histologically as regions of decondensed DNA and histones that colocalize with neutrophil granule or cytoplasmic proteins.

[0003] NETosis is a controlled form of neutrophil cell death that contributes to host defense against pathogens and was linked to a variety of diseases shortly after its first description in 2004. During NETosis, neutrophils release chromatin decorated with antimicrobial proteins, resulting in sticky NETs that can trap and kill pathogens, preventing them from spreading. This self-sacrificing strategy has initiated a field of research that has progressed in the direction of autoimmunity, as defective clearance in NETs can trigger an immune response against DNA, histones, and other intracellular proteins associated with NETs. 5 Neutrophil extracellular traps (NETs) play a role not only in host defense but also in many non-infectious diseases, including cancer, thrombosis, autoimmune diseases, and neurological disorders.

[0004] Currently, NETs are detected by a combination of anti-neutrophil proteins, e.g., myeloperoxidase (MPO) or neutrophil elastase (NE), and anti-chromatin antibodies, as well as DNA staining. Immunofluorescence microscopy is a useful method for detecting NETs in tissue sections and in vitro experiments. However, this can be challenging, as NET components are dispersed throughout large non-condensed structures, resulting in a weak signal. For example, the signal of antibodies against neutrophil elastase (NE) is significantly weaker in NETs than in granules of resting cells, where this protease is highly concentrated. Conversely, anti-histone antibodies strongly stain NETs but not the nuclei of naive neutrophils, where chromatin is compact and inaccessible. This differential histone staining property is currently exploited for the detection and quantification of NETs (Brinkmann et al., 2012). However, sample preparation, and subsequent image analysis, make it difficult to compare results between different laboratories. Therefore, antibodies against NET antigens need to be identified.

[0005] NETs can also be detected based on post-translational modifications (PTMs) that occur during NETosis. One of these PTMs is histone 3 (H3) citrullination by protein arginine deiminase 4 (PAD4), which converts arginine residues to citrulline, an amino acid that is not incorporated into proteins when they are synthesized. Citrullinated H3 (H3cit) has been widely used as a surrogate marker for NETs in both in vitro and in vivo experiments. It is important to note that citrullination is not specific for NET formation.

[0006] An additional PTM that occurs during and contributes to NETosis is the cleavage (also called clipping) of histones by granule-derived neutrophil proteases. Histone cleavage by cysteine ​​or serine proteases promotes the global removal of multiple, more subtle PTMs in histone tails and is a bona fide histone PTM that is conserved from yeast to mammals.

[0007] Research into the role of NETs in disease and as therapeutic targets is hampered by the lack of reliable and specific methods for the detection of NETs. There are no antibodies available that specifically label NETs. NET-specific antibodies would be useful (1) for researchers to identify NETs and (2) for diagnosis - to determine whether complex tissues contain NETs.

[0008] The object of the present invention is to provide compounds, more specifically antibodies, that specifically recognize neutrophil extracellular traps (NETs), which also facilitate easier NET quantification. The object of the invention is solved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, the drawings and the examples of the present application.

[0009] [Brief Description of the Invention] In searching for specific markers of NETs, ​​we have identified a unique histone H3 cleavage event that occurs in human NET formation, which maps to L48R49 of human H3 SEQ ID NO:25 (Figure 1, Figure 2).

[0010] We took advantage of the specificity of this event to generate a mouse monoclonal antibody, 3D9 (Figure 3), against a de novo histone H3 epitope located at the N-terminus starting at R49 of the C-terminal fragment of cleaved histone H3. In other words, the antibody specifically binds to the C-terminal fragment of cleaved histone H3.

[0011] By microscopy, this antibody distinguishes NETs from chromatin in purified cell samples (Figure 5) and mixed cell samples (Figure 6), facilitating simple NET detection and quantification in research. Importantly, 3D9 recognizes human NETs induced by both microbial and host-derived physiological stimuli (Figure 5). Moreover, this antibody also detects NETs in human tissue sections (Figures 8-10). The antibody can also be used for NET quantification by semi-automated and automated methods (Figure 4). Importantly, antibody 3D9 is able to distinguish between NETosis in neutrophils and other forms of cell death (Figure 7A), whereas the anti-chromatin antibody PL2.3 does not (Figure 7B).

[0012] Thus, we demonstrate that this antibody is a new tool for detecting and quantitating NETs, ​​facilitating comparison of results between laboratories, and for examining biopsies and other complex tissues for NETs.

[0013] The antibody of the present invention recognizes a de novo epitope located at position R49 in histone H3 that appears only during NET formation, and is therefore the first antibody that specifically recognizes NETs.

[0014] To date, histone citrullination H3cit is the only PTM family that has been used for antibody-based detection in NETs. Herein, we present histone cleavage at H3R49 as a new histone PTM that can be used for widespread detection in NETs from human samples. The use of H3cit for NET detection is not without controversy. Not all NETs are citrullinated, and NET formation can occur in the absence or inhibition of citrullinating enzymes. By identifying the exact histone cleavage site, we have further elucidated this. The most commonly used H3cit antibody detects citrullination at R2, R8, and R17. However, histone cleavage at R49 would remove the H3cit epitope, making these NET-defining PTMs mutually exclusive at the single histone level. Indeed, co-staining with anti-H3cit and 3D9 in paraffin sections of inflamed kidney (Figure 8C), gallbladder (Figure 9), and appendix (Figure 10) revealed extensive mutual exclusion of the two marks, and more abundant staining of decondensed chromatin with 3D9. Thus, we show that 3D9 allows widespread detection of NETs, ​​but may preferentially display more mature or proteolytically processed NETs.

[0015] In contrast to the present study, histone cleavage was recently reported as distinguishing between different pathways of NET formation (Pieterse et al., 2018, Annals of the rheumatic diseases 77, 1790-1798). Using a sandwich ELISA approach, Pieterse et al. concluded that N-terminal histone tails are generally removed in NOX-dependent but not NOX-independent NET formation. They used a series of N-terminally directed antibodies against H2B, H3, and H4, with all H3 epitopes located N-terminal to the cleavage site H3R49. However, in the final biological sample testing, the authors did not test H3. Interestingly, the authors observed by immunofluorescence microscopy that all histone N-terminal antibodies were unable to stain NETs at time points following cell lysis.

[0016] The inventors have detected NETs in fixed or denatured human samples from in vitro experiments and histological specimens. Thus, histone cleavage at H3R49 is a novel and as yet undescribed cleavage site in eukaryotes.

[0017] Unusually, the cleavage site, H3R49, is located in the globular rather than unstructured tail region of H3. Accordingly, the present invention relates to an isolated antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of a truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8.

[0018] The present invention is also directed to an isolated antibody or antigen-binding portion thereof that specifically binds to the C-terminal fragment of a truncated histone H3 described above, wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0019] A preferred embodiment of the present invention is directed to an isolated antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of the truncated histone H3 described above, wherein the antigen-binding fragment is selected from the group comprising a single-chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising an epitope-binding region.

[0020] Another preferred embodiment of the present invention is directed to a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or an antigen-binding portion thereof, that specifically binds to the C-terminal fragment of the truncated histone H3 described above.

[0021] A more preferred embodiment of the present invention is directed to a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or an antigen-binding portion thereof, that specifically binds to the C-terminal fragment of truncated histone H3 described above, wherein the nucleic acid composition comprises a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:9 and a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:10.

[0022] A further preferred embodiment of the present invention is directed to a host cell containing the above-described recombinant nucleic acid molecule. Another embodiment of the invention provides a method for in vitro detection of neutrophil extracellular traps in an isolated biological sample, the method comprising: 1) contacting the isolated biological sample with an antibody specific for the C-terminal fragment of the cleaved histone H3, or an antigen-binding portion thereof; 2) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; 3) determining whether an antibody specific for a C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample.

[0023] Further embodiments of the present invention provide the above-mentioned methods, wherein the antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, is linked to a detectable label.

[0024] A preferred embodiment of the present invention provides a method for assessing a disease state in an individual in vitro, comprising: i) contacting an isolated organism obtained from the individual with an antibody specific for the C-terminal fragment of the truncated histone H3, or an antigen-binding portion thereof; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; wherein the disease is associated with NET formation; Here, the disease is selected from the group including thrombotic diseases, neurological diseases, bacterial infections, viral infections, fungal infections, inflammatory diseases, autoimmune diseases, cancer diseases, and cancer metastasis diseases.

[0025] Another preferred embodiment of the present invention provides the above-mentioned method, wherein the autoimmune disease is selected from the group comprising: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; immune thrombocytopenia.

[0026] Another preferred embodiment of the present invention provides the above-mentioned method, wherein the antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, is linked to a detectable label.

[0027] A further preferred embodiment of the present invention provides an isolated fragment of human histone H3 cleaved at site L48R49 of SEQ ID NO25, wherein the fragment of human histone H3 cleaved at site L48R49 consists of the amino acid sequence comprised between residues R49 to A135 of SEQ ID NO25, or a fragment thereof, or consists of the amino acid sequence comprised between residues A1 to L48 of SEQ ID NO25, or a fragment thereof.

[0028] In another aspect, the present invention provides a kit for assessing a disease comprising an antibody, or an antigen-binding portion thereof, specific to the above-mentioned C-terminal fragment, and / or the above-mentioned fragment of human histone H3, wherein the disease is selected from the group including thrombotic diseases, neurological diseases, bacterial infections, viral infections, fungal infections, inflammatory diseases, autoimmune diseases, cancer diseases, and cancer metastasis diseases.

[0029] In a further aspect, the present invention provides a kit as described above, wherein the autoimmune disease is selected from the group comprising: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; and immune thrombocytopenia.

[0030] In a particular embodiment, the present invention provides the use of the cleavage site in histone H3 located at L48R49 of SEQ ID NO25 for the specific detection of neutrophil extracellular traps. [Description of the Invention] The present invention relates to an isolated antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of a truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8.

[0031] [Definition] An "antibody that specifically binds to a C-terminal fragment of cleaved histone H3" refers to an antibody that specifically binds to a de novo histone H3 epitope located at the N-terminus starting at R49 of the C-terminal fragment of histone H3 cleaved at site L48R49 of SEQ ID NO25.

[0032] "L48R49" and "H3R49" are used interchangeably herein when referring to SEQ ID NO:25 to denote the cleavage site. SEQ ID NO25 corresponds to the histone H3 sequence Q71DI3, which does not have the first Met. Thus, the cleavage site L48R49 in SEQ ID NO25 corresponds to the cleavage site L49R50 in Q71DI3 and in other H3 variant sequences P0DPK2, Q6NXT2, Q16695, P84243, and P68431 (Example 9).

[0033] Thus, the cleavage site L48R49 also refers to cleavage sites present in other variants or homologues of histone H3 that contain sequences having at least 90% identity to SEQ ID NO25.

[0034] The cleavage itself at site L48R49 occurs near the N-terminal domain of histone H3. This results in two fragments, one of which is called the "N-terminal fragment" of histone H3. The other, larger fragment is composed of the unstructured "C-terminal domain" and the major "helical domain" of histone H3. The antigenic epitope is located N-terminal to this helical domain and is also referred to herein as the "C-terminal fragment" of histone H3.

[0035] Thus, the present invention also relates to an isolated antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of histone H3 truncated at L48R49 of SEQ ID NO25, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8.

[0036] The present invention is also directed to an isolated antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of a truncated histone H3, wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0037] The present invention is further directed to an isolated antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of histone H3 truncated at L48R49 of SEQ ID NO25, wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0038] Thus, the present invention also relates to an isolated antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of a truncated histone H3, Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) an L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having greater than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; and Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0039] A preferred embodiment of the present invention is directed to an isolated antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of the truncated histone H3 described above, wherein the antigen-binding fragment is selected from the group comprising a single-chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising an epitope-binding region.

[0040] A preferred embodiment of the present invention is directed to an isolated antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) an L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having greater than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; and wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0041] A further preferred embodiment of the present invention is directed to an isolated antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0042] An even more preferred embodiment of the present invention is directed to an isolated antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of truncated histone H3, wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0043] A preferred embodiment of the present invention is directed to an isolated antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of histone H3 truncated at L48R49 of SEQ ID NO25, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0044] A further preferred embodiment of the present invention is directed to an isolated antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of histone H3 truncated at L48R49 of SEQ ID NO25, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0045] An even more preferred embodiment of the present invention is directed to an isolated antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of histone H3 truncated at L48R49 of SEQ ID NO25, wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0046] [Nucleic acid] A preferred embodiment of the present invention is directed to a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8.

[0047] Another preferred embodiment of the present invention is directed to a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of a truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) an L-CDR3 comprising an amino acid sequence of SEQ ID NO:8, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:8, wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0048] Another further preferred embodiment of the present invention is directed to a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of a truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0049] Another further preferred embodiment of the present invention is directed to a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of a truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0050] More preferably, the nucleic acid composition comprises a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:9 and a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:10.

[0051] A preferred embodiment of the present invention is directed to a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Here, the nucleic acid compositions include a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:9, and a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:10.

[0052] Another preferred embodiment of the invention is directed to a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of a truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; Here, the nucleic acid compositions include a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:9, and a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:10.

[0053] Another further preferred embodiment of the present invention is directed to a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of a truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; wherein the antigen-binding fragment is selected from the group comprising a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising an epitope-binding region; Here, the nucleic acid compositions include a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:9, and a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:10.

[0054] Another further preferred embodiment of the present invention is directed to a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of a truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; wherein the antigen-binding fragment is selected from the group comprising a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising an epitope-binding region; Here, the nucleic acid compositions include a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:9, and a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:10.

[0055] [Host cell] The invention also provides a host cell containing a recombinant nucleic acid molecule encoding an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of truncated histone H3.

[0056] Accordingly, another embodiment of the invention is directed to a host cell comprising a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8.

[0057] Another preferred embodiment of the present invention is directed to a host cell comprising a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of a truncated histone H3, Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0058] Another further preferred embodiment of the invention is directed to a host cell comprising a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0059] Another further preferred embodiment of the invention is directed to a host cell comprising a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0060] Accordingly, embodiments of the present invention are directed to a host cell comprising a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Here, the nucleic acid compositions include a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:9, and a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:10.

[0061] Another embodiment of the invention is directed to a host cell comprising a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; Here, the nucleic acid compositions include a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:9, and a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:10.

[0062] Another further preferred embodiment of the invention is directed to a host cell comprising a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; wherein the antigen-binding fragment is selected from the group comprising a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising an epitope-binding region; Here, the nucleic acid compositions include a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:9, and a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:10.

[0063] Another further preferred embodiment of the invention is directed to a host cell comprising a nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; wherein the antigen-binding fragment is selected from the group comprising a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising an epitope-binding region; Here, the nucleic acid compositions include a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:9, and a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:10.

[0064] Methods for in vitro detection of NETs Another embodiment of the invention provides a method for in vitro detection of neutrophil extracellular traps in an isolated biological sample, the method comprising: 1) contacting the isolated biological sample with an antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3; 2) providing conditions sufficient to allow the antibody, or antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; 3) determining whether an antibody specific for a C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8.

[0065] A further embodiment of the present invention provides a method for in vitro detection of neutrophil extracellular traps in an isolated biological sample, the method comprising: 1) contacting the isolated biological sample with an antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3; 2) providing conditions sufficient to allow the antibody, or antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; 3) determining whether an antibody specific for a C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0066] Another preferred embodiment of the present invention provides a method for in vitro detection of neutrophil extracellular traps in an isolated biological sample, the method comprising: 1) contacting the isolated biological sample with an antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3; 2) providing conditions sufficient to allow the antibody, or antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; 3) determining whether an antibody specific for a C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0067] Another more preferred embodiment of the present invention provides a method for in vitro detection of neutrophil extracellular traps in an isolated biological sample, the method comprising: 1) contacting the isolated biological sample with an antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3; 2) providing conditions sufficient to allow the antibody, or antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; 3) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0068] In some embodiments, the antibody, or antigen-binding portion, specific for the C-terminal fragment of cleaved histone H3 is linked to a detectable label. Thus, another embodiment of the present invention provides a method for in vitro detection of neutrophil extracellular traps in an isolated biological sample, the method comprising: 1) contacting the isolated biological sample with an antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3; 2) providing conditions sufficient to allow the antibody, or antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; 3) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Here, an antibody, or antigen-binding portion, specific for the C-terminal fragment of cleaved histone H3 is linked to a detectable label.

[0069] A further embodiment of the present invention provides a method for in vitro detection of neutrophil extracellular traps in an isolated biological sample, the method comprising: 1) contacting the isolated biological sample with an antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3; 2) providing conditions sufficient to allow the antibody, or antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; 3) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; Here, an antibody, or antigen-binding portion, specific for the C-terminal fragment of cleaved histone H3 is linked to a detectable label.

[0070] Another preferred embodiment of the present invention provides a method for in vitro detection of neutrophil extracellular traps in an isolated biological sample, the method comprising: 1) contacting the isolated biological sample with an antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3; 2) providing conditions sufficient to allow the antibody, or antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; 3) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; wherein the antigen-binding fragment is selected from the group comprising a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising an epitope-binding region; Here, an antibody, or antigen-binding portion, specific for the C-terminal fragment of cleaved histone H3 is linked to a detectable label.

[0071] Another more preferred embodiment of the present invention provides a method for in vitro detection of neutrophil extracellular traps in an isolated biological sample, the method comprising: 1) contacting the isolated biological sample with an antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3; 2) providing conditions sufficient to allow the antibody, or antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; 3) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; wherein the antigen-binding fragment is selected from the group comprising a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising an epitope-binding region; Here, an antibody, or antigen-binding portion, specific for the C-terminal fragment of cleaved histone H3 is linked to a detectable label.

[0072] The biological sample is preferably selected from a cell sample, a mixed cell sample, a cell line sample, a primary cell sample, a blood sample, a peripheral blood mononuclear cell (PBMC) sample, a neutrophil sample, a tissue sample, a histological tissue sample, such as a paraffin-embedded tissue sample or a cryopreserved tissue sample.

[0073] Methods for in vitro evaluation of diseases associated with NET formation A preferred embodiment of the present invention provides a method for assessing a disease state in an individual in vitro, comprising: i) contacting an isolated biological sample obtained from said individual with an antibody specific for the C-terminal fragment of truncated histone H3 as described above, or an antigen-binding portion thereof; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; wherein the disease is associated with NET formation; wherein the disease is selected from the group including thrombotic diseases, neurological diseases, bacterial infections, viral infections, fungal infections, inflammatory diseases, autoimmune diseases, cancer diseases, and cancer metastasis diseases; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8.

[0074] The biological sample is preferably selected from a cell sample, a mixed cell sample, a cell line sample, a primary cell sample, a blood sample, a peripheral blood mononuclear cell (PBMC) sample, a neutrophil sample, a tissue sample, a histological tissue sample, such as a paraffin-embedded tissue sample or a cryopreserved tissue sample.

[0075] Thus, a more preferred embodiment of the present invention provides a method of assessing a disease state in an individual in vitro, comprising: i) contacting an isolated biological sample obtained from said individual with an antibody specific for the C-terminal fragment of truncated histone H3 as described above, or an antigen-binding portion thereof; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; wherein the disease is associated with NET formation; wherein the disease is selected from the group including thrombotic diseases, neurological diseases, bacterial infections, viral infections, fungal infections, inflammatory diseases, autoimmune diseases, cancer diseases, and cancer metastasis diseases; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Here, the biological sample is selected from a cell sample, a mixed cell sample, a cell line sample, a primary cell sample, a blood sample, a peripheral blood mononuclear cell (PBMC) sample, a neutrophil sample, a tissue sample, a histological tissue sample, a paraffin-embedded tissue sample, or a cryopreserved tissue sample.

[0076] A preferred embodiment of the present invention provides a method for assessing a disease state in an individual in vitro, comprising: i) contacting an isolated biological sample obtained from said individual with an antibody specific for the C-terminal fragment of truncated histone H3 as described above, or an antigen-binding portion thereof; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; wherein the disease is associated with NET formation; wherein the disease is selected from the group including thrombotic diseases, neurological diseases, bacterial infections, viral infections, fungal infections, inflammatory diseases, autoimmune diseases, cancer diseases, and cancer metastasis diseases; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0077] A more preferred embodiment of the present invention provides a method for assessing a disease state in an individual in vitro, comprising: i) contacting an isolated biological sample obtained from said individual with an antibody specific for the C-terminal fragment of truncated histone H3 as described above, or an antigen-binding portion thereof; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; wherein the disease is associated with NET formation; wherein the disease is selected from the group including thrombotic diseases, neurological diseases, bacterial infections, viral infections, fungal infections, inflammatory diseases, autoimmune diseases, cancer diseases, and cancer metastasis diseases; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0078] An even more preferred embodiment of the present invention provides a method for assessing a disease state in an individual in vitro, comprising: i) contacting an isolated biological sample obtained from said individual with an antibody specific for the C-terminal fragment of truncated histone H3 as described above, or an antigen-binding portion thereof; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; wherein the disease is associated with NET formation; wherein the disease is selected from the group including thrombotic diseases, neurological diseases, bacterial infections, viral infections, fungal infections, inflammatory diseases, autoimmune diseases, cancer diseases, and cancer metastasis diseases; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0079] A preferred embodiment of the present invention provides a method for assessing a disease state in an individual in vitro, comprising: i) contacting an isolated biological sample obtained from said individual with an antibody specific for the C-terminal fragment of truncated histone H3 as described above, or an antigen-binding portion thereof; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; wherein the disease is associated with NET formation; wherein the disease is an autoimmune disease selected from the group including: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; and immune thrombocytopenia; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8.

[0080] A preferred embodiment of the present invention provides a method for assessing a disease state in an individual in vitro, comprising: i) contacting an isolated biological sample obtained from said individual with an antibody specific for the C-terminal fragment of truncated histone H3 as described above, or an antigen-binding portion thereof; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; wherein the disease is associated with NET formation; wherein the disease is an autoimmune disease selected from the group including: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; and immune thrombocytopenia; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0081] A more preferred embodiment of the present invention provides a method for assessing a disease state in an individual in vitro, comprising: i) contacting an isolated biological sample obtained from said individual with an antibody specific for the C-terminal fragment of truncated histone H3 as described above, or an antigen-binding portion thereof; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; wherein the disease is associated with NET formation; wherein the disease is an autoimmune disease selected from the group including: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; and immune thrombocytopenia; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0082] An even more preferred embodiment of the present invention provides a method for assessing a disease state in an individual in vitro, comprising: i) contacting an isolated biological sample obtained from said individual with an antibody specific for the C-terminal fragment of truncated histone H3 as described above, or an antigen-binding portion thereof; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; wherein the disease is associated with NET formation; wherein the disease is an autoimmune disease selected from the group including: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; and immune thrombocytopenia; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; wherein the antigen-binding fragment is selected from the group including a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0083] A preferred embodiment of the present invention provides a method for assessing a disease state in an individual in vitro, comprising: i) contacting an isolated biological sample obtained from said individual with an antibody specific for the C-terminal fragment of truncated histone H3 as described above, or an antigen-binding portion thereof; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; wherein the disease is associated with NET formation; wherein the disease is selected from the group including thrombotic diseases, neurological diseases, bacterial infections, viral infections, fungal infections, inflammatory diseases, autoimmune diseases, cancer diseases, and cancer metastasis diseases; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Here, an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, is linked to a detectable label.

[0084] A preferred embodiment of the present invention provides a method for assessing a disease state in an individual in vitro, comprising: i) contacting an isolated biological sample obtained from said individual with an antibody specific for the C-terminal fragment of truncated histone H3 as described above, or an antigen-binding portion thereof; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; wherein the disease is associated with NET formation; wherein the disease is selected from the group including thrombotic diseases, neurological diseases, bacterial infections, viral infections, fungal infections, inflammatory diseases, autoimmune diseases, cancer diseases, and cancer metastasis diseases; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; Here, an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, is linked to a detectable label.

[0085] A more preferred embodiment of the present invention provides a method for assessing a disease state in an individual in vitro, comprising: i) contacting an isolated biological sample obtained from said individual with an antibody specific for the C-terminal fragment of truncated histone H3 as described above, or an antigen-binding portion thereof; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; wherein the disease is associated with NET formation; wherein the disease is selected from the group including thrombotic diseases, neurological diseases, bacterial infections, viral infections, fungal infections, inflammatory diseases, autoimmune diseases, cancer diseases, and cancer metastasis diseases; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; wherein the antigen-binding fragment is selected from the group comprising a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising an epitope-binding region; Here, an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, is linked to a detectable label.

[0086] An even more preferred embodiment of the present invention provides a method for assessing a disease state in an individual in vitro, comprising: i) contacting an isolated biological sample obtained from said individual with an antibody specific for the C-terminal fragment of truncated histone H3 as described above, or an antigen-binding portion thereof; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, specific for a C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, binds to the isolated biological sample; wherein the disease is associated with NET formation; wherein the disease is selected from the group including thrombotic diseases, neurological diseases, bacterial infections, viral infections, fungal infections, inflammatory diseases, autoimmune diseases, cancer diseases, and cancer metastasis diseases; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; wherein the antigen-binding fragment is selected from the group comprising a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising an epitope-binding region; Here, an antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, is linked to a detectable label.

[0087] Another preferred embodiment of the present invention provides the above-mentioned method, wherein the antibody specific for the C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, is linked to a detectable label.

[0088] Preferably, a "detectable label" as used herein is selected from the group comprising fluorescent molecules, phosphorescent molecules, chemiluminescent molecules, bioluminescent molecules, radioisotopes, chromophores. Fragments and segments of histone H3 cleaved at L48R49.

[0089] The inventors now show that cleavage of histone H3 at site L48R49 generates two major fragments: an "N-terminal fragment" of H3 comprising amino acids A1 to L48 of SEQ ID NO:25, and a "C-terminal fragment" of H3 comprising amino acids R49 to A135 of SEQ ID NO:25.

[0090] Furthermore, the "N-terminal fragment" of H3 cleaved at L48R49 can be further cleaved into smaller fragments during NET formation and NETosis, wherein said fragments are comprised in the amino acid sequence between the 25 amino acids A1 to L48 of SEQ ID NO25. Similarly, the "C-terminal fragment" of H3 cleaved at L48R49 can be further cleaved into smaller fragments during NET formation and NETosis, wherein said fragments are comprised in the amino acid sequence between the amino acids R49 to A135 of SEQ ID NO25.

[0091] Thus, the term "fragment" of histone H3 as used herein refers to a fragment of histone H3 generated by cleavage at site L48R49 of SEQ ID NO:25. Said fragment is an "N-terminal fragment" of H3 or a "C-terminal fragment" of H3.

[0092] The term "segment" of histone H3 as used herein refers to segments of H3 generated by further cleavage of histone H3 or histone H3 fragments before or after cleavage at L48R49 during NET formation or NETosis.

[0093] Thus, the present invention is directed to an "N-terminal fragment" of H3 comprising amino acids A1 to L48 of SEQ ID NO:25, or a fragment thereof, and / or a "C-terminal fragment" of H3 comprising amino acids R49 to A135 of SEQ ID NO:25, or a fragment thereof.

[0094] The "N-terminal fragment" of histone H3 disclosed herein, or a fragment thereof, is preferably 5 to 48 amino acids in length, preferably 10 to 48 amino acids in length, preferably 15 to 48 amino acids in length, preferably 20 to 48 amino acids in length, preferably 25 to 48 amino acids in length, preferably 30 to 48 amino acids in length, preferably 35 to 48 amino acids in length, preferably 5 to 38 amino acids in length, preferably 10 to 38 amino acids in length, preferably 15 to 38 amino acids in length, preferably 20 to 38 amino acids in length, preferably 25 to 38 amino acids in length, preferably 30 to 38 amino acids in length, preferably 5 to 28 amino acids in length, preferably 10 to 28 amino acids in length, preferably 15 to 28 amino acids in length, preferably 20 to 28 amino acids in length, preferably 25 to 28 amino acids in length, preferably 5 to 18 amino acids in length, preferably 10 to 18 amino acids in length, preferably 15 to 18 amino acids in length, preferably 2 to 10 amino acids in length, preferably 5 to 10 amino acids in length.

[0095] The "C-terminal fragment" of histone H3 disclosed herein, or a fragment thereof, is preferably 5 to 87 amino acids long, preferably 10 to 87 amino acids long, preferably 15 to 87 amino acids long, preferably 20 to 87 amino acids long, preferably 25 to 87 amino acids long, preferably 30 to 87 amino acids long, preferably 35 to 87 amino acids long, preferably 45 to 87 amino acids long, preferably 55 to 87 amino acids long, preferably 65 to 87 amino acids long, preferably 75 to 87 amino acids long, preferably 5 to 70 amino acids long, preferably 10 to 70 amino acids long, preferably 15 to 70 amino acids long, preferably 20 to 70 amino acids long, preferably 25 to 70 amino acids long, preferably 30 to 70 amino acids long, preferably 35 to 70 amino acids long, preferably 45 to 70 amino acids long, preferably 55 to 70 amino acids long, preferably 65 to 70 amino acids long, preferably 5 to 60 amino acids long, preferably 10 to 60 amino acids long, preferably 15 to 87 amino acids long, preferably 20 to 70 amino acids long. 60 amino acids long, preferably 25 to 60 amino acids long, preferably 30 to 60 amino acids long, preferably 35 to 60 amino acids long, preferably 45 to 60 amino acids long, preferably 55 to 60 amino acids long, preferably between 5 to 50 amino acids long, preferably 10 to 50 amino acids long, preferably 15 to 50 amino acids long, preferably 20 to 50 amino acids long, preferably 25 to 50 amino acids long, preferably 30 to 50 amino acids long, preferably 35 to 50 amino acids long, preferably 40 to 50 amino acids long, preferably 5 to 40 amino acids long, preferably 10 to 40 amino acids long, preferably 15 to 40 amino acids long, preferably 20 to 40 amino acids long, preferably 25 to 40 amino acids long, preferably 30 to 40 amino acids long, preferably 35 to 40 amino acids long, preferably 5 to 30 amino acids long, preferably 10 to 30 amino acids long, preferably 15 to 30 amino acids long, preferably 20 to 30 amino acids long, preferably 5 to 20 amino acids long, preferably 10 to 20 amino acids long.

[0096] Thus, a preferred embodiment of the present invention provides an isolated fragment of human histone H3 cleaved at L48R49 of SEQ ID NO25, wherein the fragment of human histone H3 cleaved at site L48R49 consists of the amino acid sequence comprised between residues R49 and A135 of SEQ ID NO25, or a fragment thereof, or consists of the amino acid sequence comprised between residues A1 and L48 of SEQ ID NO25, or a fragment thereof.

[0097] The term protein or fragment thereof as used herein also includes "variants" of a protein or of a fragment or segment thereof, and refers to a protein or fragment or segment that shares a particular amino acid sequence identity with a reference protein or fragment or segment, upon alignment by methods known in the art. A variant of a protein or of a fragment or segment thereof can include substitutions, insertions, deletions, frameshifts or rearrangements in other proteins. In some embodiments, a variant shares at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with the native protein or with a fragment or segment thereof.

[0098] Thus, a more preferred embodiment of the present invention provides an isolated fragment of human histone H3 cleaved at L48R49 of SEQ ID NO25, wherein the fragment of human histone H3 cleaved at site L48R49 consists of the amino acid sequence contained between residues R49 to A135 of SEQ ID NO25, or a fragment thereof, or the amino acid sequence contained between residues A1 to L48 of SEQ ID NO25, or a fragment thereof, wherein the fragment of human histone H3 or fragment thereof shares at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with a naturally occurring fragment of human histone H3 or fragment thereof.

[0099] In other words, the N-terminal fragment and the C-terminal fragment can also be derived from other variants or homologues of histone H3 that contain a sequence having at least 90% identity with SEQ ID NO 25. Of note, SEQ ID NO 25 corresponds to the histone H3 sequence Q71DI3 that does not have the first Met.

[0100] Kits for assessing NET formation and associated diseases In another aspect, the present invention provides a kit for assessing a disease comprising an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of cleaved histone H3, wherein the disease is selected from the group comprising a thrombotic disease, a neurological disease, a bacterial infection, a viral infection, a fungal infection, an inflammatory disease, an autoimmune disease, a cancer disease, a cancer metastasis disease; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Here, the disease is associated with NET formation.

[0101] In another aspect, the present invention provides a kit for assessing a disease comprising an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of truncated histone H3, wherein the disease is selected from the group comprising a thrombotic disease, a neurological disease, a bacterial infection, a viral infection, a fungal infection, an inflammatory disease, an autoimmune disease, a cancer disease, or a cancer metastasis disease; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; Here, the disease is associated with NET formation.

[0102] Preferably, the antigen-binding fragment is selected from the group comprising a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0103] In another aspect, the present invention provides a kit for assessing a disease comprising an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of histone H3 cleaved at L48R49 of SEQ ID NO25, wherein the disease is selected from the group including a thrombotic disease, a neurological disease, a bacterial infection, a viral infection, a fungal infection, an inflammatory disease, an autoimmune disease, a cancer disease, or a cancer metastasis disease; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Here, the disease is associated with NET formation.

[0104] In another aspect, the present invention provides a kit for assessing a disease comprising an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of histone H3 cleaved at L48R49 of SEQ ID NO25, wherein the disease is selected from the group including a thrombotic disease, a neurological disease, a bacterial infection, a viral infection, a fungal infection, an inflammatory disease, an autoimmune disease, a cancer disease, or a cancer metastasis disease; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; Here, the disease is associated with NET formation.

[0105] In a further aspect, the present invention provides the aforementioned kit, wherein the autoimmune disease is selected from the group comprising: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; immune thrombocytopenia.

[0106] Thus, the present invention provides a kit for assessing a disease comprising an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of cleaved histone H3, wherein the disease is an autoimmune disease selected from the group including: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; immune thrombocytopenia; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Here, the disease is associated with NET formation.

[0107] In another aspect, the present invention provides a kit for assessing a disease comprising an antibody that specifically binds to a C-terminal fragment of cleaved histone H3, or an antigen-binding portion thereof, wherein the disease is an autoimmune disease selected from the group including: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; immune thrombocytopenia; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; Here, the disease is associated with NET formation.

[0108] Preferably, the antigen-binding fragment is selected from the group comprising a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, a F(ab)2 fragment, a peptide, or a proteolytic fragment comprising the epitope-binding region.

[0109] In another aspect, the present invention provides a kit for assessing a disease comprising an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of histone H3 cleaved at L48R49 of SEQ ID NO25, wherein the disease is an autoimmune disease selected from the group including: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; immune thrombocytopenia; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Here, the disease is associated with NET formation.

[0110] In another aspect, the present invention provides a kit for assessing a disease comprising an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of histone H3 cleaved at L48R49 of SEQ ID NO25, wherein the disease is an autoimmune disease selected from the group including: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; immune thrombocytopenia; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; Wherein the antibody comprises: I) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:2; Here, the disease is associated with NET formation.

[0111] A further embodiment of the present invention provides a kit for assessing a disease comprising a fragment of human histone H3, wherein the disease is selected from the group comprising a thrombotic disease, a neurological disease, a bacterial infection, a viral infection, a fungal infection, an inflammatory disease, an autoimmune disease, a cancer disease, a cancer metastasis disease; wherein the fragment of human histone H3 is a fragment of human histone H3 cleaved at the site L48R49 of SEQ ID NO25, wherein the fragment of human histone H3 cleaved at the site L48R49 comprises an amino acid sequence contained between R49 and A135 of SEQ ID NO25, or a fragment thereof, or comprises an amino acid sequence contained between A1 and L48 of SEQ ID NO25, or a fragment thereof; Here, the disease is associated with NET formation.

[0112] A further preferred embodiment of the present invention provides a kit for assessing a disease comprising a fragment of human histone H3, wherein the disease is an autoimmune disease selected from the group comprising: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; immune thrombocytopenia; wherein the fragment of human histone H3 is a fragment of human histone H3 cleaved at the site L48R49 of SEQ ID NO25, wherein the fragment of human histone H3 cleaved at the site L48R49 comprises an amino acid sequence contained between R49 and A135 of SEQ ID NO25, or a fragment thereof, or comprises an amino acid sequence contained between A1 and L48 of SEQ ID NO25, or a fragment thereof; Here, the disease is associated with NET formation.

[0113] Another embodiment of the present invention provides a kit for assessing a disease comprising a fragment of human histone H3, wherein the disease is selected from the group comprising a thrombotic disease, a neurological disease, a bacterial infection, a viral infection, a fungal infection, an inflammatory disease, an autoimmune disease, a cancer disease, a cancer metastasis disease; wherein the fragment of human histone H3 is a fragment of human histone H3 cleaved at the site L48R49 of SEQ ID NO25, wherein the fragment of human histone H3 cleaved at the site L48R49 comprises an amino acid sequence contained between R49 and A135 of SEQ ID NO25, or a fragment thereof, or comprises an amino acid sequence contained between residues A1 and L48 of SEQ ID NO25, or a fragment thereof; wherein the fragment or fragment of human histone H3 shares at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with a sequence of a naturally occurring fragment or fragment of human histone H3; Here, the disease is associated with NET formation.

[0114] A further preferred embodiment of the present invention provides a kit for assessing a disease comprising a fragment of human histone H3, wherein the disease is an autoimmune disease selected from the group comprising: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; immune thrombocytopenia; wherein the fragment of human histone H3 is a fragment of human histone H3 cleaved at the site L48R49 of SEQ ID NO25, wherein the fragment of human histone H3 cleaved at the site L48R49 comprises an amino acid sequence contained between R49 and A135 of SEQ ID NO25, or a fragment thereof, or comprises an amino acid sequence contained between residues A1 and L48 of SEQ ID NO25, or a fragment thereof; wherein the fragment or segment of human histone H3 shares at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with a sequence of a naturally occurring fragment or segment of human histone H3, and Here, the disease is associated with NET formation.

[0115] An even more preferred embodiment is directed to a kit for assessing a disease comprising an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of cleaved histone H3, wherein the disease is selected from the group comprising a thrombotic disease, a neurological disease, a bacterial infection, a viral infection, a fungal infection, an inflammatory disease, an autoimmune disease, a cancer disease, a cancer metastasis disease, wherein said disease is associated with NET formation; Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; wherein the fragment of human histone H3 is a fragment of human histone H3 cleaved at the site L48R49 of SEQ ID NO25, wherein the fragment of human histone H3 cleaved at the site L48R49 consists of an amino acid sequence contained between residues R49 and A135 of SEQ ID NO25, or a fragment thereof; or consisting of the amino acid sequence contained between residues A1 to L48 of SEQ ID NO:25, or a fragment thereof; Here, the disease is associated with NET formation.

[0116] An even more preferred embodiment is directed to a kit for assessing a disease comprising an antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of cleaved histone H3, wherein the disease is an autoimmune disease selected from the group comprising: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; immune thrombocytopenia; and Wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:5; d) L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:7; f) L-CDR3 comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having more than 90% sequence identity with the amino acid sequence of SEQ ID NO:8; wherein the fragment of human histone H3 is a fragment of human histone H3 cleaved at the site L48R49 of SEQ ID NO25, wherein the fragment of human histone H3 cleaved at the site L48R49 consists of an amino acid sequence contained between residues R49 and A135 of SEQ ID NO25, or a fragment thereof; or consisting of the amino acid sequence contained between residues A1 to L48 of SEQ ID NO:25, or a fragment thereof; Here, the disease is associated with NET formation.

[0117] [Using the cleavage site of histone H3] A further preferred embodiment of the present invention provides the use of the histone H3 cleavage site located at L48R49 of SEQ ID NO25 for the specific detection of neutrophil extracellular traps.

[0118] "Antibodies" (Abs) and "immunoglobulins" (Ig) are glycoproteins with the same structural characteristics. Antibodies exhibit binding specificity to a specific antigen, whereas immunoglobulins include both antibodies and other antibody-like molecules that generally lack antigen specificity. The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (such as full-length or intact monoclonal antibodies), polyclonal antibodies, human antibodies, multispecific (or heteroconjugate) antibodies (such as bispecific antibodies), monovalent antibodies, multivalent antibodies, antigen-binding antibody fragments, such as single-chain variable fragments (scFv), variable domain (Fv) fragments, fragment antigen-binding (Fab) fragments, F(ab)2 fragments, peptides, or proteolytic fragments, Fv fragments, sdAbs, VHHs, antibody fusions, and synthetic antibodies (or antibody mimetics). Antibodies can be chimeric, human, humanized, and / or affinity matured.

[0119] The term "antigen" as used herein is defined as any substance capable of eliciting an immune response. As used herein, the term "immunogenic" refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.

[0120] The term "epitope" as used herein is defined as the portion of an antigen molecule that makes contact with the antigen binding site of an antibody or of a T-cell receptor. As used herein, the terms "specific binding" or "specifically binding" or "binds specifically" refer to the interaction between a binding pair (such as an antibody and an antigen).

[0121] "Full length antibody," "intact antibody" or "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure. "Antigen-binding portion of an antibody" refers to a portion of a full-length antibody that is capable of binding the same antigen as the full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', F(ab')2, bispecific antibodies, linear antibodies, single-chain antibody molecules (e.g., scFv, etc.), single domain antibodies, and multispecific antibodies formed from antibody fragments.

[0122] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The heavy and light chain variable domains (VH and VL, respectively) of a natural antibody generally have a similar structure. A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0123] The term "variable" refers to the fact that certain parts of the variable domains vary widely in sequence between antibodies and are used in the binding and specificity of each particular antibody to a particular antigen. However, variability is concentrated in three fragments called complementarity determining regions (CDRs) or hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework regions (FRs). The variable domains in natural heavy and light chains each contain four FR regions, adopting a mainly beta-sheet structure and connected by three CDRs, which form loops to connect and sometimes form part of the beta-sheet structure. In each chain, the CDRs are held together in close proximity by the FR regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., National Institutes of Health, Bethesda, MD (1991)). The constant domains are not directly involved in binding an antibody to an antigen, but exhibit various effector functions, such as participation in antibody-dependent cellular toxicity.

[0124] As used herein, "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms a structurally defined loop. In general, a natural antibody comprises four chains with six HVRs: three in the heavy chain variable domain, VH (H1, H2, H3), and three in the light chain variable domain, VL (L1, L2, L3). A single domain antibody comprises only three HVRs in the heavy chain variable domain. HVRs generally comprise amino acid residues from the hypervariable loops and / or from the "complementarity determining regions" (CDRs). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues, etc.) in the variable domain are numbered herein according to Kabat et al., 1991.

[0125] As used herein, "complementarity determining region" or "CDR" refers to the region within the hypervariable region of the variable domain that has the highest sequence variability and / or is involved in antigen recognition. Generally, a natural antibody contains four chains with six CDRs: three in the heavy chain variable domain, VH (H1, H2, H3), and three in the light chain variable domain, VL (L1, L2, L3). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are located at amino acid residues 24-34 of L1, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 31-35 of H1, amino acid residues 50-65 of H2, and amino acid residues 95-102 of H3 (Kabat et al., 1991).

[0126] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following sequence in a VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0127] "Native antibody" refers to a naturally occurring immunoglobulin molecule. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence in the constant domain.

[0128] As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, except for possible variant antibodies (e.g., variant antibodies include variants that occur naturally or that arise during the production of monoclonal antibodies and are generally present in small amounts). In contrast to polyclonal antibody preparations that typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the term "monoclonal" refers to the nature of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method.

[0129] A "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0130] "Humanized antibody" refers to a chimeric antibody that comprises amino acid sequences derived from non-human HVRs and amino acid sequences derived from human FRs. In certain embodiments, a humanized antibody comprises a substantially single domain antibody in which all or substantially all of the CDRs correspond to the CDRs of a non-human antibody and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0131] "Human antibody" refers to an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0132] "Fc region" or "Fc antibody fragment" refers to a dimeric complex comprising the C-terminal polypeptide sequence of an immunoglobulin heavy chain, where the C-terminal polypeptide sequence is obtained by papain digestion of an intact antibody. The Fc region may comprise a native or mutated Fc sequence.

[0133] "Fab fragment" refers to an antibody fragment that contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. Papain digestion of an antibody produces two identical "Fab" fragments, each with a single antigen-binding site, and a remaining "Fc" fragment, the name reflecting their ability to crystallize easily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and can also be cross-linked. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus in the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between the F(ab')2 antibody fragments. Other chemical linkages in antibody fragments are also known in the art.

[0134] "Fv fragment" refers to an antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain that are tightly bound, for example, in scFv, one heavy chain variable domain and one light chain variable domain can be essentially covalently linked. In this configuration, the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer.

[0135] Collectively, the six HVRs, or a subset of them, confer antigen-binding specificity to the antibody, however, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although usually with lower affinity than the entire binding site.

[0136] "Single-chain Fv" or "scFv" refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired antigen-binding structure.

[0137] An "isolated" antibody, or protein, or protein fragment, is one that has been identified, separated, and / or recovered from components of its natural environment, which are substances that would interfere with research, diagnostic, or therapeutic uses for the antibody, or protein, or protein fragment, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. Ordinarily, an isolated antibody, or protein, or protein fragment will be prepared by at least one purification step.

[0138] The percentage of "sequence identity" is determined by comparing two optimally aligned nucleic acid or polypeptide sequences over a "comparison window" for the entire length of the reference sequence. As used herein, a "comparison window" refers to the optimal alignment between the reference sequence and the variant sequence after the two sequences are optimally aligned, where the variant nucleic acid or polypeptide sequence within the comparison window may contain 20 percent or less, usually 5 percent to 15 percent, or 10 percent to 12 percent additions or deletions (i.e., gaps) compared to the reference sequence for the optimal alignment (wherein the reference sequence does not contain additions or deletions). The percentage of identity is calculated by determining the number of positions in both sequences where identical nucleic acid bases or amino acid residues are present, obtaining the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the entire length of amino acids or nucleotides), and multiplying the result by 100 to obtain the percentage of sequence identity. Two nucleic acid, or protein, or peptide, or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when optimally aligned as described above. The percentage of "sequence identity" can be determined in the comparison window defined above using the "BLAST 2 Sequences" tool available on the NCBI website using blastp (Tatusova A. et al., FEMS Microbiol Lett. 1999, 174: 247-250).

[0139] Alternatively, the variant sequence may be any amino acid sequence resulting from substitutions allowed at any number of positions in the parent sequence according to the following formula: Ser substituted by Ser, Thr, Gly, and Asn; Arg substituted by one of Arg, His, Gin, Lys, Glu; Leu substituted by one of Leu, Ile, Phe, Tyr, Met, and Val; Pro substituted by one of Pro, Gly, Ala, and Thr; Thr substituted by one of Thr, Pro, Ser, Ala, Gly, His, and Gin; Ala substituted by one of Ala, Gly, Thr, and Pro; Val substituted by one of Val, Met, Tyr, Phe, Ile, and Leu; Gly substituted by one of Gly, Ala, Thr, Pro, and Ser; Ile substituted by one of Ile, Met, Tyr, Phe, Val, and Leu; Phe substituted by one of Phe, Trp, Met, Tyr, Lie, Val, and Leu; Tyr substituted by one of Tyr, Trp, Met, Phe, Ile, Val, and Leu; His substituted by one of His, Glu, Lys, Gin, Thr, and Arg; Gln substituted by one of Gin, Glu, Lys, Asn, His, Thr, and Arg; Asn substituted by one of Asn, Glu, Asp, Gin, and Ser; Lys substituted by one of Lys, Glu, Gln, His, and Arg; Asp substituted by one of Asp, Glu, and Asn; Glu substituted by one of Glu, Asp, Lys, Asn, Gln, His, and Arg; Met substituted by one of Met, Phe, Ile, Val, Leu, and Tyr.

[0140] The number of amino acid deletions or substitutions in the variant sequence compared to the parent sequence is preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

[0141] "Polynucleotide" or "nucleic acid molecule", as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. Polynucleotides can also include modified nucleotides, such as methylated nucleotides, and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "caps," substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications, such as, for example, substitution with an uncharged bond (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), and substitution with a charged bond (e.g., phosphorothioates, phosphorodithioates, etc.), substitutions containing pendant moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), substitutions containing intercalating agents (e.g., acridine, psoralen, etc.), substitutions containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), substitutions containing alkylating agents, substitutions with modified linkages (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. Additionally, any of the hydroxyl groups normally present in the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected or activated by standard protecting groups, or attached to a solid or semi-solid support, to prepare additional bonds to additional nucleotides. The 5'- and 3'-terminal OH may be phosphorylated or replaced with amines or with organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls may be derivatized to standard protecting groups.

[0142] As used herein, "oligonucleotide" refers to a short, generally single-stranded, generally synthetic polynucleotide, generally not necessarily less than about 200 nucleotides in length.The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive.The above description of polynucleotide is equally and completely applicable to oligonucleotide.

[0143] The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is 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 phage vector. Another type of vector is a viral vector, in which additional DNA segments can be ligated to the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced. 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 are thereby replicated along with the host genome. In addition, certain vectors can direct the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors" or "recombinant vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, since the plasmid is the most commonly used form of vector.

[0144] The nucleic acid molecule may comprise a native sequence (ie, an endogenous sequence that encodes an antibody or a portion of an antibody) or may comprise a variant of that sequence. Nucleic acid variants contain one or more substitutions, additions, deletions and / or insertions such that the immunoreactivity of the encoded antibody is not substantially different from the reference native antibody. In some embodiments, the nucleic acid variants exhibit at least about 70% identity, in some embodiments at least about 80% identity, in some embodiments at least about 85% identity, in some embodiments at least about 90% identity, and in some embodiments at least about 95% identity to the parent nucleic acid sequence, where the percentage of sequence identity is determined as described above. These values ​​are not meant to be limiting, and increments between the recited percentages are specifically contemplated as part of the present disclosure.

[0145] The nucleic acid molecules in this disclosure can be obtained using chemical synthesis, recombinant methods, or polymerase chain reaction (PCR). Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. Those skilled in the art can use the sequences provided herein and a commercially available DNA synthesis machine to generate the desired DNA sequence. To prepare a polynucleotide using recombinant methods, a polynucleotide containing a desired sequence can be inserted into a suitable vector, as further discussed herein, and the vector can then be introduced into a suitable host cell for replication and amplification. The polynucleotide can be inserted into the host cell by any method known in the art. The cell is transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating, or electroporation. Once introduced, the exogenous polynucleotide can be maintained in the cell as a non-integrated vector (such as a plasmid) or can be integrated into the host cell genome. The polynucleotide thus amplified can be isolated from the host cell by methods well known in the art.

[0146] Suitable cloning and expression vectors can include various components, such as promoters, enhancers, and other transcriptional regulatory sequences. Vectors can be constructed to allow for subsequent cloning of antibody variable domains into different vectors. Suitable cloning vectors can be constructed according to standard techniques or selected from a large number of cloning vectors available in the art. The cloning vector selected can vary according to the host cell intended to be used, but useful cloning vectors will generally be capable of autonomous replication, may have a single target for a specific restriction endonuclease, and / or may have a gene for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+, etc.) and derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, etc., as well as shuttle vectors, such as pSA3 and pAT28. These, and many other cloning vectors, are available from commercial vendors such as Merck, BioRad, Stratagene, and Invitrogen.

[0147] Furthermore, the present invention also contemplates an expression vector comprising a nucleic acid sequence encoding any of the single domain antibodies or variants thereof disclosed herein, as well as a host cell comprising said expression vector. It is implied that an expression vector must be replicable in a host cell, either as an episome or as an integral part of the chromosomal DNA. Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; appropriate transcription control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements are also usually required, such as a ribosome binding site, a translation initiation site, and a stop codon.

[0148] A vector containing the nucleic acid molecule of interest and / or the nucleic acid molecule itself can be introduced into a host cell by any of a number of suitable methods, including transfection using electroporation, calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent, such as vaccinia virus). The choice of a particular procedure will often depend on the characteristics of the host cell.

[0149] As used herein, the terms "in vitro" or "ex vivo" refer to an artificial environment and processes or reactions that occur within an artificial environment, such as, but not limited to, test tubes and cell cultures. The term "in vivo" refers to the natural environment (e.g., an animal or a cell) and processes or reactions that occur within a natural environment.

[0150] In the context of the present invention, an "autoimmune disease" is a disease or disorder caused by and directed against an individual's own tissues, or co-segregation, or expression, or condition resulting therefrom. This class of disorders is highly diverse between and within different types of autoimmune diseases, which complicates diagnosis and effective treatment. The causes of autoimmune diseases are also poorly understood, which results in a treatment process that focuses primarily on symptoms.

[0151] The disease associated with NET formation is preferably selected from the group comprising thrombotic diseases, neurological diseases, bacterial infections, viral infections, fungal infections, inflammatory diseases, autoimmune diseases, cancer diseases, and cancer metastasis diseases.

[0152] The "autoimmune disease" associated with NET formation is preferably selected from the group including: psoriasis; vasculitis; systemic lupus erythematosus (SLE); rheumatoid arthritis; ulcerative colitis; Crohn's disease; immune-mediated or type 1 diabetes; immune-mediated glomerulonephritis; inflammatory bowel disease (IBD); antiphospholipid syndrome; and immune thrombocytopenia.

[0153] "Systemic lupus erythematosus" (SLE) is a debilitating autoimmune disease that can damage multiple organs, induce chronic renal failure, and cause severe morbidity and mortality. A hallmark of SLE is the presence of antinuclear autoantibodies that form immune complexes with cellular debris, causing end-organ damage. Current treatment regimens are limited to nonspecific immunosuppression and management of inflammatory symptoms.

[0154] The term "lupus" as used herein is an autoimmune disease or disorder that generally involves antibodies that attack connective tissue. The main form of lupus is the systemic form, systemic lupus erythematosus (SLE), including cutaneous SLE and subacute cutaneous SLE, as well as other forms of lupus, including nephritis, extrarenal, encephalitis, pediatric, non-renal, discoid, and alopecia. In certain embodiments, the term "systemic lupus erythematosus" refers to a chronic autoimmune disease that can result in skin lesions, joint pain, and swelling, kidney disease (lupus nephritis), fluid around the heart and / or lungs, and inflammation of the heart, and various other systemic conditions. In certain embodiments, the term "lupus nephritis" refers to inflammation of the kidney that occurs in SLE patients. Lupus nephritis may include, for example, glomerulonephritis and / or interstitial nephritis, and can cause high blood pressure, proteinuria, and kidney failure. Classes of lupus nephritis include class I (micromesangial lupus nephritis), class II (mesangial proliferative lupus nephritis), class I-II (focal lupus nephritis), class IV (diffuse partial (IV-S) or diffuse generalized (IV-G) lupus nephritis), class V (membranous lupus nephritis), and class VI (progressive sclerosing lupus nephritis).

[0155] "Cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by a cell proliferative disorder. Cancer may generally include, but is not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of cancer may include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, leukemia, and other lymphoproliferative disorders, and various types of head and neck cancer.

[0156] As used herein, the terms "individual," "subject," and "patient" are used interchangeably herein and refer to both human and non-human animals. The term "non-human animals" of the present disclosure includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Preferably, the subject is a human patient suffering from an autoimmune disease or a human patient suffering from an autoimmune disease. [Drawing Description] [Brief description of the drawings]

[0157] [Figure 1] FIG. 1 shows that PMA-induced histone H3 cleavage occurs at the N-terminal domain and is blocked by serine protease inhibition. (A) As indicated, neutrophils were preincubated with the serine protease inhibitor AEBSF in microcentrifuge tubes for 30 min and then stimulated with PMA. Lysates were resolved by SDS-PAGE and immunoblotted with N- and C-terminal antibodies against H3. GAPDH was used as a loading control. The data show that serine proteases cleave the N-terminus of H3 at specific amino acids. (B) Immunofluorescence confocal microscopy of NET formation. As indicated, neutrophils were seeded on coverslips and preincubated with AEBSF and then stimulated with PMA (50 nM). Cells were fixed at 150 min and stained with DAPI (4',6-diamidino-2-phenylindole) for neutrophil elastase (NE), chromatin (using H2A-H2B-DNA antibody PL2.3), and DNA. NS: no stimulation. Images were taken at 63×, scale bar is 20 μm. [Figure 2A]Figure 2 shows the identification of histone H3 cleavage sites in NET formation. A) Representative RP-HPLC chromatograms of acid-extracted histones from NETs, ​​as well as corresponding 1D-SDS-PAGE and immunoblots to identify H3- and H4-containing fractions. Histone-rich supernatants were prepared from neutrophils stimulated for 90 min with PMA. (B) Representative Coomassie-stained blots of pooled H3 fractions, separated by 2D electrophoresis. The inset is a magnification of all spots (1-5) identified as histone H3 by mass spectrometry. [Figure 2B] (C) Schematic representation of the cleavage site in the cleaved H3 product in both the linear sequence of H3 and in a nucleosomal context. H3 is depicted in blue, and the pink tail region and partial alpha helix represent the portion of H3 that is removed. The nucleosome structure is adapted from PDB 2F8N. The structure was assembled by X-ray crystallography and is composed of histones from multiple species. The H3 variant is Xenopus H3.2, but has 100% identity to human H3.2. The protein identifiers for Xenopus and human are P84233 and Q71DI3, respectively. [Figure 3A] Figure 3 shows screening and detection of cleaved H3 and NETs by 3D9. (A) Immunofluorescence microscopy of neutrophils stimulated with PMA and fixed at the indicated times. Samples were stained with Hoechst (DNA-blue) and 3D9 (Alexafluor-568-conjugated secondary antibody-red). NS: unstimulated. Images were taken on an upright fluorescence microscope at 20x. Scale bar-50 μm (full field) and 10 μm (inset). [Figure 3B](B) Immunoblots of lysates prepared from neutrophils stimulated with PMA (50 nM) for the times indicated. H3 C-terminal antibody was used as a control to detect all H3 forms, while a single band (cleaved H3) was detected by 3D9, a newly generated monoclonal antibody. (C) Direct ELISA of cleaved H3 in NETs, ​​chromatin (A549 lung epithelial cells), recombinant histone H3, and DNA. Samples were serially diluted and immobilized on high-affinity ELISA plates according to DNA content (for NETs, ​​chromatin, and DNA) or protein content (for recombinant histone H3) determined by PicoGreen and bicinchoninic acid analysis, respectively. Starting concentrations were 1 μg / ml DNA or protein. Cleaved H3 was detected using 3D9 (2 μg / ml) and HRP-conjugated anti-mouse secondary antibody, and the reaction was developed using TMB (3,3',5,5'-tetramethylbenzidine) as substrate. Data are shown for a dilution of 200 ng / ml. REIRR indicates a non-conjugated branched immune peptide control formed by SEQ ID NO11 and SEQ ID NO26. This peptide was coated at 20 ng / ml. Data represent the mean ± SD of three experiments using independent donors. [Figure 3C] (D) ELISA and subsequent in-house immunoblot and immunofluorescence microscopy screening strategy. [Figure 3D](E) Immunofluorescence microscopy of neutrophils stimulated with PMA for 3 hours in the presence of competitor and control peptides and stained with 3D9 (1 μg / ml). Prior to staining, 3D9 was pre-incubated overnight at 4° C. with the immunizing branched peptides (SEQ ID NOs. 11 and 12) and negative control branched peptides (SEQ ID NOs. 16 and 17) at the indicated molar ratios. (F, G) Neutrophils were seeded in 10 ml Petri dishes and stimulated with PMA for 3 hours. Prior to whole cell lysis and immunoprecipitation, NETs were digested with benzonase to release histones and octamers for antibody binding. Line 1: ISO, Line 2: 3D9, Line 3: beads only; INP: input; M: marker. [Figure 3E] (F) Coomassie stained gel of proteins immunoprecipitated by 3D9 or isotype control and the corresponding silver stained gel. The expected cleaved and intact H3 bands are shown. HC-Ab: antibody heavy chain, LC-Ab: antibody light chain. [Figure 3F] (G) Immunoblots for total H3 (C-terminal antibody) and cleaved H3 on proteins immunoprecipitated with 3D9 or isotype control antibody, and Coomassie stained gels of samples run in parallel. Gels are representative of three independent experiments. [Figure 4A] Figure 4 shows a comparison of NET quantification using anti-chromatin antibody PL2.3 versus 3D9. (A) Confocal immunofluorescence microscopy of neutrophils stimulated with PMA (180 min) and stained with Hoechst and anti-chromatin antibody (PL2.3) or 3D9. Insets represent selected cells examined at high magnification (63x) and displayed as split channels in greyscale or integrated by the whole field (20x magnification). White scale bar - 50 μm, cyan scale bar - 10 μm. Images are representative of three experiments. [Figure 4B](B) Comparison of fluorescence distribution over time (6 h) between PL2.3 and 3D9 staining of PMA-stimulated cells. Staining intensity was normalized across all images for each time course. NS: 360: no stimulation at 360 min. Analysis is performed on one data set, representative of three to four independent time course experiments. [Figure 4C] (C) Comparison of NET quantification using manual or automatic thresholding and segmentation procedures for chromatin antibody (PL2.3) and cleaved H3 antibody (or 3D9). Manual thresholding excludes cells / NETs with weak signal, whereas automatic thresholding includes all subjects regardless of signal. Images for analysis were taken using a fluorescent microscope. Graphs represent the mean ± standard deviation, where n = 3–4. [Diagram 5] Figure 5 shows detection by 3D9 in NETs from various stimuli. Immunofluorescence microscopy of neutrophils left unstimulated (NS), stimulated with PMA (50 nM, 2.5 h), stimulated with nigericin (15 μM, 2.5 h), TNF-sensitized and then stimulated with heme (20 μM, 6 h), and co-cultured with Candida albicans hyphae (MOI 5) for 4 h. Samples were stained with Hoechst, anti-neutrophil elastase (NE), and 3D9. Scale bar - 50 μm. Images were taken at 20× on a confocal microscope and are representative of three experiments with independent donors. Scale bar - 20 μm. [Figure 6] Figure 6 shows detection of NETs in mixed cell fractions. Immunofluorescence microscopy of unpurified peripheral blood mononuclear cell (PBMC) fractions treated with NET stimuli, PMA (50 nM, 2.5 h) or nigericin (15 μM, 2.5 h), and then stained with Hoechst, anti-neutrophil elastase (NE) and 3D9 or PL2.3. Images were taken at 20x magnification on an upright fluorescent microscope and selected images are representative of three independent experiments. Scale bar - 50 μm. [Figure 7A]FIG. 7 shows a comparison of 3D9 detection in response to apoptotic, necroptotic, and necrotic cell death stimuli. A) Confocal immunofluorescence microscopy of neutrophils stimulated with various cell death stimuli and subsequently stained with Hoechst, anti-neutrophil elastase (NE), and 3D9. NETs were induced with PMA (100 nM, 3 h). Apoptosis was induced in resting neutrophils by overnight incubation without stimulation for 24 h. Neutrophils were stimulated with caspase inhibitors Z-VAD-FMK (50 μM) plus SMAC mimetic (100 nM) plus TNF (50 ng / ml) for 6 h to induce necroptosis. Necrosis was induced with the pore-forming toxin α-hemolysin (25 μg / ml). Images were taken at 20× and are representative of three experiments. Scale bar 20 μm. ns: no stimulation. [Figure 7B] B) Comparison was performed with parallel samples stained with the chromatin antibody PL23. ns: no stimulation. [Figure 8A] Figure 8 shows detection of excised histone 3 and NETs in human tissues. Paraffin-embedded sections were stained with Hoechst, anti-NE, and 3D9, or H3 cit antibodies and examined by confocal microscopy. Scale bar - 10 μm. (A) Inflamed human tonsil. (B & C) Inflamed human kidney. [Figure 8B] Figure 8 shows detection of excised histone 3 and NETs in human tissues. Paraffin-embedded sections were stained with Hoechst, anti-NE, and 3D9, or H3 cit antibodies and examined by confocal microscopy. Scale bar - 10 μm. (A) Inflamed human tonsil. (B & C) Inflamed human kidney. [Figure 9] Figure 9 shows a comparison of sectioned H3, H3cit, and H2B staining in the gallbladder. Paraffin-embedded sections were stained with Hoechst, anti-NE and histone antibodies 3D9, H3cit, and H2B and examined by confocal microscopy. Scale bar - 20 μm. [Figure 10]Figure 10 shows a comparison of H3, H3cit, and H2B staining in sectioned appendix. Paraffin-embedded sections were stained with Hoechst, anti-NE, and 3D9 or H3cit antibodies and examined by confocal microscopy. Scale bar - 20 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0158] The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the invention, and therefore can be considered to constitute preferred modes for said practice. However, those of skill in the art should, in light of this disclosure, understand that many changes can be made to the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the invention.

[0159] Further modifications and alternative embodiments of the various aspects of the invention will be apparent to those skilled in the art upon consideration of this specification. Thus, this specification is to be construed as illustrative only and is intended to teach those skilled in the art the general manner of carrying out the invention. It should be understood that the forms of the invention shown and described herein are to be construed as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and steps may be reversed, and certain features of the invention may be utilized independently, all as would become apparent to those skilled in the art after having the benefit of this specification in the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as set forth in the following claims.

[0160] [Example] material and method reagent All reagents were purchased from common suppliers of laboratory reagents, e.g., Sigma Aldrich or VWR Germany, unless otherwise stated.

[0161] Blood collection and ethical approval Venous blood was collected from healthy donors who provided informed consent according to the Declaration of Helsinki. Ethical approval was provided by the Medical Committee of the Charité-Universitatsmedizin Berlin and blood was provided anonymously at the Charité Hospital Berlin.

[0162] Purification and culture of human peripheral blood neutrophils Neutrophils were isolated as described by Amulic et al. (2017). Briefly, venous whole blood was collected in EDTA, layered over an equal volume of Histopaque 1119, and separated by centrifugation at 800 g (20 min). The pinkish neutrophil-rich fraction was collected and washed with 3 volumes of wash buffer (Mg supplemented with 0.5% [w / v] human serum albumin [HSA, Grifols]). 2+ or Ca 2+ The neutrophil fraction was washed once by addition of PBS (Gibco) without HCl and centrifuged at 300g (10 min). The neutrophil fraction was further purified by density gradient centrifugation - Percoll (Pharmacia), 85% to 65% (v / v) gradient. Purified cells were collected from the 80% to 70% fraction and washed once before resuspension in wash buffer. Cells were counted using a CASY cell counter.

[0163] For all experiments, neutrophils were cultured in RPMI (GIBCO 32404014) supplemented with 10 mM HEPES and 0.1% (w / v) HSA and pre-equilibrated for 1 h with CO2 conditions unless otherwise indicated. For some stimuli, the HSA content was reduced to 0.05% or 0% HSA as indicated in the figure legends. Cells were routinely cultured at 37°C and 5% CO2 unless otherwise indicated. For all experiments, stimuli were added to cell reactions as 10x working stock solutions freshly diluted in RPMI. For inhibition experiments, 10x inhibitor stocks and appropriate vehicle controls were added to cells and pre-incubated for the times indicated in the figure legends.

[0164] Preparation of neutrophil and NET lysates To analyze proteins, lysates were prepared from stimulated or resting neutrophils. Cells were diluted to 1 × 10 in medium in a 1.5 ml microcentrifuge tube. 7 5 x 10 cells / ml per time point 6 Cells were seeded in 100 mL of 1000 mM EDTA, 0.1% glycerol, 0.1% EDTA, 0.2% EDTA-treated 100% ethanol. After addition of inhibitors or agonists, cells were gently mixed and incubated at 37°C with gentle rotation. At the indicated time points, protease inhibitors-1 mM AEBSF, 20 μM Cathepsin G Inhibitor I (Calbiochem), 20 μM Neutrophil Elastase Inhibitor GW311616A (Biomol), 2X Halt Protease Inhibitor Cocktail (PIC, Thermofisher Scientific), 10 mM EDTA, 2 mM EGTA-were added directly to the cell suspension. Cells were gently mixed and centrifuged at 1000 g (30 s) and all residual liquid was collected. Boiling 5X sample loading buffer (50 mM Tris-HCl pH 6.8, 2% [w / v] SDS, 10% glycerol, 0.1% [w / v] bromophenol blue, 100 mM DTT) was added to the samples, which were then vortexed briefly, boiled at 98°C for 10 min, flash frozen in liquid nitrogen, and stored at -80°C.

[0165] 1D SDS-PAGE and immunoblotting For routine protein analysis, samples were analyzed by 1D SDS-PAGE and immunoblotted. Samples were thawed on ice, boiled at 98°C (10 min), and sonicated to reduce viscosity (Braun sonicator, 10 sec, cycle 7, 70% power). Proteins were applied to NuPAGE 12% gels (Invitrogen, Thermofisher) and run at 150V in MES buffer (Thermofisher Scientific). Proteins were transferred by Western blot onto PVDF (0.2 μm pore size, Amersham GE Healthcare) using a BioRad wet transfer system (buffer: 25 mM Tris, 192 mM glycine, 20% methanol, protocol: 30 min at 100 mA, 120 min at 400 mA). Blotting efficiency was assessed by Ponceau S staining. Blots were blocked with TBST (TBS pH 7.5, 0.1% [v / v] Tween-20) with 5% [w / v] skim milk for 1 h at RT. Blots were then incubated overnight at 4°C or for 2 h at room temperature with the following primary antibodies: Rabbit anti-histone H3 C-term pAb, 1:15000 (Active motif #61277); rat anti-histone H3 N-term mAb, 1:1000 (Active motif #61647, aa1-19); histone H4 C-term, 1:5000 (Abcam 10158-aa50 to C-term); rabbit anti-histone H4 N-term mAb, 1:30,000 (Upstate, Millipore #05-858, aa17-28); rabbit GAPDH mAb, 1:5000 (Cell Signaling Technology, #2118); mouse 3D9 1ug / ml (produced in this study) were all diluted in TBST with 3% (w / v) skim milk.After washing with TBST (3 x 5 min), the blots were blocked for 15 min as before and then probed with secondary HRP-conjugated antibody (Jackson ImmunoResearch - diluted 1:20000 in 5% skim milk TBST) for 1 h at room temperature. Blots were washed with TBST (3 x 5 min) and developed using SuperSignal™ West Dura Extended Duration Substrate (ThermoFisher Scientific) and an ImageQuant Gel imager (GE Healthcare).

[0166] Immunofluorescence staining of in vitro samples For immunofluorescence imaging of purified cells, neutrophils / PBMCs were plated at 1 × 10 per well in 24-well dishes containing glass coverslips. 5Cells were seeded and incubated at 37°C for 1 h to allow attachment to coverslips. Inhibitors and priming factors were included at this stage, as indicated. Reactions were stopped by the addition of paraformaldehyde (2% [w / v]) for 20 min at room temperature or overnight at 4°C. After fixation, cells were washed and stained as previously described (Brinkmann et al. 2012). Briefly, all steps were performed by floating the coverslip upside down on a drop of buffer on laboratory parafilm. Cells were permeabilized with PBS, pH 7.5, 0.5% (v / v) Triton X-100 for 3 min. For screening and quantitative experiments, this permeabilization step was extended to 10 min. Samples were washed (3x5 min) with PBS, 0.05% (v / v) Tween 20, incubated with blocking buffer - PBS pH 7.5, 0.05% (v / v) Tween 20, 3% (v / v) normal goat serum, 3% (w / v) freshwater fish gelatin, 1% (w / v) BSA - for 20 min at room temperature, then probed with primary antibodies diluted in blocking buffer and incubated overnight at 4°C. Primary antibodies: anti-chromatin (H2A-H2B-DNA complex) mouse mAb, 1 μg / ml (Losman 1992); neutrophil elastase, rabbit pAb 1:500 (Calbiochem); screening mouse serum, 1:100; hybridoma supernatant, neat; 3D9 mouse mAb, 1 μg / ml. Samples were then washed as before. Alexa-labeled secondary antibodies (Invitrogen) were diluted 1 / 500 in blocking buffer and incubated for 2 hours at room temperature. DNA was stained with Hoescht 33342 (Invitrogen, Molecular Probes) at 1 μg / ml and incubated with the secondary antibody step. Samples were washed with PBS followed by water and mounted in Mowiol mounting medium.

[0167] Histone extraction from neutrophils Histone-rich fractions were prepared from resting neutrophils and NETs according to a method modified from Shechter et al. (Shechter, D., Dormann, H.L., Allis, C.D., and Hake, S.B. (2007). Extraction, purification and analysis of histones. Nat Protoc 2, 1445-1457). Neutrophils (4–8 × 10 7 ) were resuspended in 13 ml RPMI (without HSA) in 15 ml polypropylene tubes and incubated with 50 nM PMA at 37°C on a roller for 90 min. After stimulation, 1 mM AEBSF was added to prevent further degradation by NSPs and cells were chilled on ice for 10 min. All subsequent steps were performed on ice or at 4°C when possible. Cells and NETs were pelleted by centrifugation at 1000g for 10 min. Samples were dissolved in 1 ml buffer / 5x10 in ice-cold hypotonic lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM KCL, 1.5 mM MgCl2, 1 mM DTT-1 mM AEBSF, 20 μM NEi, 20 μM CGi, 2XPIC, 10 mM EDTA supplemented with protease inhibitors immediately before use) and diluted with 1 ml buffer / 5x10. 6 The cells were then resuspended using PBS. The cells were then incubated for 30 min at 4°C on a rotator before being passed through a syringe to aid in lysis and shearing of intact cells. Nuclei and NETs were collected by centrifugation at 10,000g for 10 min and the supernatant was discarded. To disrupt nuclei, samples were resuspended in dHO (1ml / 1x10 7The cells were resuspended in 100 mM NaCl (0.2% [v / v]) and incubated on ice for 5 min with intermittent vortexing. NP40 (0.2% [v / v]) was added to aid in lysis and disruption of NETs, ​​and the samples were briefly sonicated (10 s, mode 7, 70% power). To extract histones, H2SO4 (0.4 N) was added to the samples and vortexed briefly. The samples were then incubated for 2–3 h with rotation. The histone-rich fraction was collected by aliquoting the samples into multiple 1.5 ml microcentrifuge tubes and centrifuging at 16,000 g for 10 min, followed by collection of the supernatant. To minimize further processing of the histones, proteins were immediately precipitated overnight by adding trichloroacetic acid dropwise to a final concentration of 33% followed by mixing. The following day, precipitated proteins were pelleted by centrifugation at 16,000 g for 10 min. The supernatant was discarded and the waxy pellet was washed once with an equal volume of ice-cold acetone with 0.2% (v / v) HCl and five times with ice-cold acetone alone. The pellet was air-dried for 5 min and then resuspended in 1 ml (5 × 10 6 The pellets were resuspended with 100% dH2O (per cell). For pellets that were difficult to resuspend, the mixtures were shaken vigorously overnight at 4°C as before, and the samples were centrifuged to remove undissolved protein. Pooled supernatants for each sample were lyophilized and stored at -80°C until histone fractionation.

[0168] Purification of histone H3 Histones were fractionated by RP-HPLC according to a previously described method. After lyophilization, samples were resuspended in 300 μl of buffer A (5% acetonitrile, 0.1% trifluoroacetic acid) and centrifuged at 14,000 g to remove particulate matter. 150 μl of purified sample was mixed with 40 μl of buffer A as described by Schechter et al. (Nat. Protoc. 2007, 2, 1445-1457) before being applied to a C18 column (#218TP53, Grave Vydac) and run on a RP-HPLC (Waters 626 LC system, MA, USA). The flow rate was 1 ml min -1The incubation time was set to 30 min and fractions were collected at 30 sec intervals from 30 min to 55 min. All fractions were lyophilized and stored at -80°C until analysis. To determine which fractions contained H3 and truncated species, each fraction was dissolved in 50 μl dH2O and 5 μl was subjected to SDS-PAGE and stained with Coomassie blue stain or transferred to PVDF and immunoblotted for H3 and H4 as previously described.

[0169] 2D electrophoresis (2-DE) of purified histones To determine the cleavage site, fractions containing H3 were pooled and subjected to a modified 2-DE procedure as described above. Briefly, pooled fractions were denatured with 9 M urea, 70 mM DTT, 2% Servalyte 2-4 and 2% Chaps. Samples (30 μl) were applied to a 1.5 mm thick isoelectric focusing (IEF) gel, using an abbreviated IEF protocol: 20 min 100 V, 20 min 200 V, 20 min 400 V, 15 min 600 V, 5 min 800 V and 3 min 1000 V (total 83 min and 500 V / h) in an 8 cm long IEF tube gel. Separation in the second dimension was performed on a 6.5 cm × 8.5 cm × 1.5 mm SDS-PAGE gel. Duplicate gels were prepared. One was stained with Coomassie Brilliant Blue R250 for removal of spots for mass spectrometry identification. The second was transferred to PVDF as follows: proteins were blotted onto a PVDF blotting membrane (0.2 μm) using a semi-dry blotting procedure in 100 mM borate blotting buffer. Spots were stained with Coomassie Brilliant Blue R250 and analyzed by N-terminal Edman degradation sequencing (Proteome Factory, Berlin, Germany).

[0170] antibody generation The immunizing and screening peptides were as follows: a branched peptide formed by REIRR (SEQ ID NO11) and REIRRKC-NH2 (SEQ ID NO12) bound to KLH for immunization, a branched peptide formed by AARKS (SEQ ID NO13) and AARKS C-NH2 (SEQ ID NO14) for negative screening, a competitive branched peptide formed by SEQ ID NO11 and REIRRKC-NH2 (SEQ ID NO15) for validation, and a branched peptide formed by TGGVK (SEQ ID NO16) and TGGVKK-NH2 (SEQ ID NO17) as a negative control for validation, and SEQ ID NO11 and REIRRKC-NH2 (SEQ ID NO26) not bound to KLH for positive screening and Elisa. In the branched peptides, a first peptide (SEQ ID NO11, SEQ ID NO13, or SEQ ID NO16) is linked via an amide bond between the carboxyl terminus of the amino acid at position 5 and the side chain amine of the lysine at position 6 of a second peptide (SEQ ID NO12, SEQ ID NO14, SEQ ID NO15, SEQ ID NO17, or SEQ ID NO26). These peptides were synthesized by Eurogentec (Belgium). Some were further conjugated to Key Lymphocyte Haemoglutinin (KLH) for immunization. Mice immunization, preliminary ELISA screening, and hybridoma production were performed by Genscript as follows. Six mice (3xBalb / c and 3xC57 / BL6) were immunized with the branched peptides. Mice were bled and effective immunization was evaluated using direct ELISA. The ELISA and subsequent in-house immunoblot and immunofluorescence microscopy screening strategy is outlined in Figure 3D. Following selection of effectively immunized animals, further booster injections of immunogen were administered prior to isolation of spleen cells for hybridoma production. The resulting hybridoma supernatants were similarly screened.Large-scale cultivation of the supernatant and purification of the antibodies were performed by Genscript.

[0171] ELISA To assess 3D9 specificity for NETs, ​​3D9 was used in an indirect ELISA to detect purified NETs, ​​chromatin, recombinant H3, and cleaved H3 in DNA. 6NETs were prepared by seeding 10 neutrophils and incubating with 100 nM PMA for 3–6 h. NETs were gently washed three times with an equal volume of PBS and then collected in 300 μl of PBS. Aggregated NETs were disrupted by brief sonication (3 s, mode 7, 70% power output - Braun sonicator) and then flash frozen and stored at -80 °C. Chromatin was prepared from lung epithelial cells (A549) as previously described by Shechter et al. (Nat. Protoc. 2007, 2, 1445-1457). The final nuclear pellet was resuspended in dH2O, sonicated as before, and stored at -80 °C. DNA content in NETs and chromatin was assessed by PicoGreen analysis according to the manufacturer's instructions (Thermofisher Scientific). Starting at 1 μg / ml (DNA content), serial dilutions in NETs, ​​chromatin, and calf thymus DNA (Invitrogen) were prepared in lo-DNA binding Eppendorf microcentrifuge tubes. A similar dilution series of recombinant histone H3 (New England Biolabs) was prepared starting at 1 μg / ml protein. All dilutions were performed in PBS. One hundred microliters of each sample was aliquoted in duplicate at dilutions from 1 μg / ml to 1 ng / ml into Nunc Maxisorb 96-well dishes and fixed overnight at 4° C. at 250 rpm. The immunizing peptide, REIRR (10 ng / ml), was used as a positive control. The next day, all wells were washed six times with washing buffer (PBS, 0.05% Tween 20) and then blocked for 2 h (room temperature) with 200 μl of blocking solution (1% BSA in washing buffer). Wells were washed once with washing buffer and incubated with 100 μl of 3D9 (2 μg / ml in blocking solution) at room temperature (2 h) with gentle shaking (250 rpm).Wells were washed six times as before and then incubated with 100 μl of secondary HRP-conjugated anti-mouse (Jackson Laboratories) diluted 1:10,0000 in blocking solution and incubated as before. Finally, wells were washed six times as before and HRP activity was detected using TMB (3,3',5,5' tetramethylbenzidine) reagent (BD OptEIA™) according to the manufacturer's instructions (incubate for 15-30 min). The assay was stopped by the addition of 100 μl H2SO4 (0.16 M) and absorbance (450 nm) was measured in a 96-well plate reader (VERSA max, Molecular Devices, CA, USA).

[0172] Quantification of staining characteristics with Image J and R To evaluate the staining properties in antibodies during NET formation, the inventors developed a bundle of Image J and R scripts. These scripts allow an automated workflow starting from two-channel microscopic images of a series of experiments (DNA staining, antibody staining), to the graphical display and classification in individual cells, and finally mapping these classifications back to the original image as a graphic overlay. In a first step, nuclei are fragmented based on an intensity threshold (either programmatic or manual), including options for lower and upper limits of size selection. The same threshold is applied to the entire experimental series, and an upper size limit is used to exclude fused structures that cannot be assigned to individual cells. A quality score is assigned to every image based on the percentage of the total DNA stained area that can be assigned to an individual cell (or NET). This score, together with all other parameters in the analysis, can be exported as a report file and used to automatically exclude images from the analysis. Furthermore, this part of the script generates a result file that contains the area, circumference (as X, Y coordinates), and cumulative intensity of each channel for all detected nuclei, along with information, such as time point or stimulus that can be assigned programmatically. To analyze these data sets, we implemented a series of functions in R, including import of ImageJ result files, classification of cells based on nuclear area and staining intensity, various plotting and data export functions, as well as a mapping function that allows the classification of nuclei as color-coded circles to be displayed overlaid on the original image.

[0173] Quantification of NETs using Image J NETs were quantified by the semi-automated method described by Brinkmann et al. (Brinkmann, et al. (2012). Automatic quantification of in vitro NETformation. Frontiers in immunology 3, 413) and via a second modified method that allows automatic quantification. All microscopy image datasets were processed by both methods to allow comparison. Hoechst was used to stain total DNA, and NETs were further stained with anti-chromatin (PL2.3) or 3D9 antibodies (1 μg / ml) and Alexa-568-conjugated secondary antibodies according to the previous section. Images were acquired using a Leica DMR upright fluorescence microscope equipped with a Jenoptic B / W digital microscope camera and a 10× or 20× objective. For each experiment, the same exposure settings were used for all samples, and a minimum of three random fields of view (FOVs) were collected. Images were analyzed using ImageJ / FIJI software. Each channel was imported as an image sequence and converted to a stack as described by Brinkmann et al., 2012. To count total cells / NETs per FOV, Hoechst channel stacks were imported and fragmented using an autothreshold function (Bernsen method) with radius 15 and parameter 1 set to 35, generating black and white threshold images. Particle analysis was then performed to count all objects with a size equal to or greater than the size of a cell nucleus (10x objective: particle size 25 to infinity; 20x objective: particle size 100 to infinity) and to exclude background staining artifacts. Total NETs were then counted appropriately using anti-chromatin (PL2.3) or 3D9 channels. As for the above method, anti-chromatin staining was fragmented using manually adjusted thresholds such that low intensity staining in stationary cell nuclei was excluded.Particle analysis was then performed, counting all objects larger than a stationary cell nucleus (10x objective: particle size 75-infinity; 20x objective: particle size 250-infinity). This was also performed for 3D9. A second automated NET analysis was performed as the manual semi-automated analysis, except that the workflow was modified, and thus automated Bernsen thresholding and fragmentation was used for both the Hoechst and NET channels, total cells, and NETs, ​​respectively. For each method, the percentage of NETs per FOV was calculated as (total NETs per FOV / total cells per FOV) x 100. The results per FOV were then averaged according to sample.

[0174] Immunofluorescence staining in tissue samples from tissue sections Paraffin sections (2 μm thick) were deparaffinized in two changes of 100% xylene for 5 min each, then rehydrated in two changes of 100% ethanol for 5 min each, followed by two changes of 90% and 70% ethanol for 5 min each. Sections were washed in three changes of deionized water and incubated in TBS (Tris-buffered saline). For antigen retrieval, slides were incubated in a steam cooker (Braun) for 20 min using Target Retrieval Solution (TRS pH 9) (Dako S2367). After cooling to room temperature in antigen retrieval buffer, slides were rinsed three times with deionized water and incubated in TBS until further processing. Slides were blocked with blocking buffer (1% BSA, 5% normal donkey serum, 5% cold water fish gelatin, and 0.05% Tween 20 in TBS, pH 7.4) for 30 min. Blocking buffer was removed and sections were incubated with primary antibodies appropriately diluted in blocking buffer (containing 0.05% Triton-X100) overnight at room temperature. Sections were rinsed with TBS and then incubated with secondary antibodies at appropriate concentrations (1:100) for 45 min at room temperature in the dark, then rinsed three times with TBS for 5 min, followed by rinsing with deionized water. Slides were incubated with DNA stain Hoechst 33342 (1:5000) for 5 min, rinsed with water, and then mounted with Mowiol. Primary antibodies for detection of NETs were: mouse anti-cleaved histone 3 clone 3D9 (2 μg / ml), rat anti-calgranulin A clone 8H6, in-house MPIIB (1:100), rabbit anti-histone H3 antibody (citrulline R2+R8+R17; ab5103, Abcam), rabbit anti-histone citrulline H4 antibody (citrulline 3) Millipore 07-596 (1:100), chicken anti-histone H2B ab134211 Abcam (1:400), rabbit anti-fibrinogen Millipore ABT259 (1:200).Secondary antibodies were: donkey anti-rabbit immunoglobulin G (IgG) heavy and light chain (H&L) Alexa Fluor 488 (Jackson 711-225-152) and donkey anti-mouse IgG H&L Cy3 (Jackson 715-165-151). Donkey anti-chicken IgY H&L Cy3 (Jackson 703-165-155), donkey anti-rat immunoglobulin G (IgG) heavy and light chain (H&L) Cy5 (Jackson). For fluorescence imaging, an upright widefield microscope (Leica DMR) equipped with a JENOPTIK B / W digital microscope camera or a Leica confocal microscope SP8 was used. Z-stack images were collected at a magnification of 63x. Paraffin tissue blocks of human tonsils and kidneys were purchased from AMSbio, and paraffin sections of inflamed tissues - gallbladder and appendix - were provided by Dr. Stefan Florian, Charité Berlin.

[0175] Cytotoxicity analysis To assess inhibitor toxicity, LDH release was assessed using the CytoTox 96® non-radioactive cytotoxicity assay (Promega) according to the manufacturer's instructions. Neutrophils were cultured at 1 × 10 in RPMI in 6-well plates. 5 Cells were seeded at 100 μl / well. Inhibitors were pre-incubated with the cells for 30 min at 37° C. at the concentrations indicated in the figures.

[0176] Reactive oxygen species analysis Reactive oxygen species (ROS) production was measured using the luminol-HRP assay described by Amulic et al. (Amulic, B., Knackstedt, SL, Abu Abed, U., Deigendesch, N., Harbort, CJ, Caffrey, BE, Brinkmann, V., Heppner, FL, Hinds, PW, Zychlinsky, A. (2017). Cell-Cycle Proteins Control Production of Neutrophil Extracellular Traps. Dev Cell 43, 449-462 e445.). Furthermore, since all steps of the assay are performed in atmospheric conditions, normal tissue culture medium was replaced as before with RPMI containing carbonate and without phenol red (Seahorse XF RPMI medium #103336-100, Agilent). Inhibitors were preincubated with cells at the concentrations indicated in the figures for 30 min at 37° C. Immediately prior to stimulation, luminol (50 μM) and HRP (1.2 U / ml) were added, followed by 50 nM PMA. Luminescence measurements were taken every 30 s using a luminescence plate reader (VICTOR Light Luminescence Counter, Perkin Elmer) and expressed as Relative Light Units (RLU).

[0177] mass spectrometry Protein spots were excised and transferred to 0.5 ml Eppendorf tubes. Samples were destained in 500 μl of 200 mM ammonium bicarbonate (ABC) in 50% acetonitrile (ACN) for 30 min at 37° C. and equilibrated in 200 μl of 50 mM ABC, 5% ACN for 30 min at 37° C. Samples were then dried at room temperature for 60 min. Protein digestion was performed overnight at 37° C. with 100 ng trypsin (spots 1, 3, 7 with 200 ng) in 25 μl of 50 mM ABC, 5% ACN. The supernatant was transferred to a new Eppendorf tube and additional peptides were extracted by applying 25 μl of 60% ACN, 0.5% trifluoroacetic acid (TFA) for 10 min, followed by 25 μl of 100% ACN for 10 min. All supernatants were combined and dried at 45°C in an Eppendorf concentrator. After solubilization in 15 μl 0.1% TFA, peptides were desalted using ZipTips and eluted onto a MALDI plate with 1 μl a-cyano-4-hydroxycinnamic acid (5 mg / ml in 60% CAN, 0.3% TFA). Peptide mass fingerprints (PMF) and fragment spectra (MSMS) of the five most intense peaks were measured using a 4700 Proteomics Analyzer (AB Sciex). Database searches were performed applying the Mascot MS / MS ion search function to search Swiss-Prot subsets in human proteins. For fragment mass tolerance, 30 ppm and peptide mass tolerance of ±0.3 Da were allowed. Variable modifications were defined as one missed cleavage, oxidation of methionine, N-terminal acetylation of proteins, propionamide at cysteine ​​residues, and formation of N-terminal pyroglutamic acid. The following identification criteria were used: minimum 30% sequence coverage; or minimum 15% sequence coverage and one MS / MS confirmation; or less than 15% sequence coverage and at least two MS / MS confirmations.

[0178] Immunoprecipitation (IP) of clipped histones Magnetic protein G-coupled beads (Invitrogen) were washed with an equal volume of PBS (x3). For each IP sample, 20 μl of beads were prepared. Since 3D9 is a mouse IgG1, a bridging antibody (anti-mouse, Active motif #53017) was used to improve binding to the protein G-coupled beads. The bridging antibody (50 μg) was incubated with 20 μl of magnetic beads for 1 hour at 4°C with gentle inversion. The beads were washed twice with PBS. The beads were then incubated with 50 μg of 3D9 or isotype control (IgG1, k-Genscript clone 1H7A8) for 3 hours at room temperature (with gentle rotation). The beads were washed three times and resuspended in 2 ml of PBS in a 15 ml Falcon tube. PFA fixation was used to permanently crosslink the antibodies. 2 ml of 1% PFA (in PBS) was added to the beads and immediately vortexed for 1 min. The reaction was then quenched with 125 mM glycine (pH 8.0) and incubated on ice for 5 min. The beads were then washed 5 times with IP lysis buffer (20 mM Tris, 150 mM NaCl, 1% Triton X-100, pH 7.5) and blocked with 1% BSA in IP lysis buffer for 1 h at room temperature. The beads were either used immediately or stored overnight at 4°C. The next day, neutrophils (1 × 10 7) were seeded in Petri dishes with 10 ml of medium and left unstimulated or stimulated with 100 nM PMA for 3 h at 37°C. After stimulation, cells / NETs were washed very gently (x3) with an equal volume of PBS (plus 1 mM AEBSF). After the final wash, NETs were digested in 1 ml of benzonase buffer (20 mM Tris-HCl, pH 7.6, 2 mM MgCl2, 1 mM CaCl2) with 250 U benzonase (Sigma E1014) and 1 mM AEBSF, 20 μM NEi and CGi for 30 min at 37°C with intermittent rocking to disperse the buffer. The reaction was stopped by adding 4 mM EDTA and 4 mM EGTA. Cells were further lysed by adding 300 μl of 5× IP lysis buffer (plus 5× protease inhibitors). Dishes were incubated on ice for 10 min. Cells and NETs were then collected by scraping into Eppendorf tubes. For multiple dishes at the same time point and stimulation, samples were pooled and immediately split for the IP step to be performed. For each IP, 1 × 10 7 Lysates from cells were used with 15 μl of bound beads and incubated overnight at 4° C. with gentle rotation. The next day, beads were washed five times with IP lysis buffer and inhibitors. After the final wash, beads were resuspended in 30 μl of 1× SDS-PAGE sample loading buffer. Samples were analyzed by SDS-PAGE and stained or immunoblotted as indicated. Coomassie Instant Blue staining or Thermofisher silver staining kits were used according to the manufacturer's instructions.

[0179] Peptide competition analysis Neutrophils were seeded (1 × 10) on coverslips in 24-well dishes. 5Cells / well) and stimulated with PMA for 3 h and fixed with 2% PFA. Prior to staining, 3D9 (2 μg / ml = ~12.9 nM) was pre-incubated overnight with rotation in PBS with 0.01-200 fold molar excess of branched REIRR peptide at 4 °C. TGGVK branched peptide was used as a negative control. The next day, competing antibody-peptide solutions were diluted 1:1 with blocking buffer and the immunostaining procedure was performed as before. Peptide competition was visually assessed by fluorescence microscopy.

[0180] Epitope mapping Antibody binding to human H3 was assessed by a combination of linear, conformational, and amino acid substitution analysis epitope mapping using peptide synthesis and ELISA analysis performed by Pepscan Presto BV (Leiden, The Netherlands). A library of peptide-based peptidomimetics was synthesized using Fmoc-based solid-phase peptide synthesis to reconstitute the epitope in the target molecule (H3 amino acid residues 30-70 of PATGGVKKPHRYRPGTVALREIRRYQKSTELIRKLPFQRL SEQ ID NO:25). Amino-functionalized polypropylene supports were obtained by grafting with a proprietary hydrophilic polymer formulation followed by reaction with t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with N-hydroxybenzotriazole (HOBt) followed by cleavage of the Boc group using trifluoroacetic acid (TFA). Peptides were synthesized on amino-functionalized solid supports by custom-modified JANUS liquid handling stations (Perkin Elmer) using standard Fmoc peptide synthesis. Synthesis of structural mimetics (structural analysis) was performed using Pepscan's proprietary Chemically Linked Peptides on Scaffolds (CLIPS) technology. Antibody binding to each of the synthesized peptides was tested by Pepscan-based ELISA. Peptide sequences were incubated (overnight at 4°C) with primary antibodies (3D9 or isotype control) at 0.02 μg / ml for linear and conformational analysis, and with primary antibodies at 0.5 μg / ml for amino acid substitution analysis. After washing, the peptide sequences were incubated with a 1 / 1000 dilution of rabbit anti-mouse IgG (H+L) HRP conjugate (Southern Biotech, Birmingham, Alabama, USA) for 1 h at 25°C.After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μl / ml of 3% H2O2 were added. After 1 h, the color development was quantified using a charge-coupled device (CCD)-camera and an image processing system. Values ​​obtained from the CCD camera ranged from 0 to 3000 mAU, similar to standard 96-well plate ELISA readers. Results were quantified and stored in a Peplab database. To verify the quality of the synthesized peptides, separate sets of positive and negative control peptides were synthesized in parallel. These were screened using the commercially available antibodies 3C9 and 57 (Posthumus, WP, Lenstra, JA, Schaaper, WM, van Nieuwstadt, AP, Enjuanes, L., and Meloen, RH (1990). Analysis and simulation of a neutralizing epitope of transmissible gastroenteritis virus. J Virol 64, 3304-3309).

[0181] Hybridoma antibody gene sequencing Sequencing of the antibody genes of hybridoma 3D9 was performed by Genscript (NJ, USA). Briefly, total RNA was isolated from five clones of 3D9 hybridoma according to the technical manual of TRIzol® reagent. Then, total RNA was reverse transcribed into cDNA using either isotype-specific antisense primers or universal primers according to the technical manual of PrimeScript™ 1st Strand cDNA Synthesis Kit. Heavy and light chain antibody fragments were amplified according to the standard operating procedure of Genscript for rapid amplification of cDNA ends (RACE). The amplified antibody fragments were cloned into the cloning vector pCE2 TA / Blunt-zero vector (CAT#C601-01- Vazyme Biotech Co.,Ltd, China). Colony PCR was performed to screen the clones with the correct size insert. A consensus sequence was provided and the fragments were sequenced using an Applied Biosystems 3730 DNA analyzer. Sequences were then aligned and annotated using NCBI Ig Blast results.

[0182] [Table 1-1]

[0183] [Table 1-2]

[0184] Example 1: Serine protease-dependent cleavage in the N-terminal tail of histone H3 during NET formation Histones are processed in response to phorbol 12 myristate 13 acetate-PMA, and other NET stimuli. In time-course experiments in human neutrophils incubated with PMA, we detected a ∼14 kDa H3 cleavage product as early as 30 min after stimulation (Figure 1), followed by ∼13 kDa and 10 kDa cleavage products. C-terminal histone antibodies detected H3 cleavage products, but N-terminal histone antibodies did not (Figure 1A). Figure 1A shows that PMA-induced histone H3 cleavage occurs at the N-terminal domain and is prevented by serine protease inhibition. These results indicate that in cleaved H3, the N-terminus is truncated.

[0185] Neutrophil azurophil granules are rich in serine proteases that can degrade histones in vitro. Therefore, we tested the contribution of these proteases to H3 cleavage during NET formation. Preincubation with the serine protease inhibitor AEBSF (4-[2-aminoethyl]benzenesulfonyl fluoride) (Figure 1A) inhibited H3 cleavage upon PMA stimulation. AEBSF also inhibited NET formation and nuclear decondensation, as shown by immunofluorescence microscopy (Figure 1B). This data indicates that serine proteases cleave the N-terminus of H3 early in NET formation.

[0186] Example 2: Histone H3 is cleaved at a novel site in the globular domain To identify the exact H3 cleavage site, we prepared histone-rich extracts from primary neutrophils stimulated with PMA for 90 min, and then purified H3 as described in the Methods section. We identified fractions containing H3 and the cleavage products by Western blot using an anti-H3 C-terminal antibody. H3 was the last core histone to elute at 45-46 min (Figure 2A). We separated H3 and the cleavage products by 2D electrophoresis (2D-E) and confirmed their identity by mass spectrometry (Figure 2A). Figure 2B shows a representative Coomassie-stained blot of pooled H3 fractions separated by 2D electrophoresis.

[0187] The sequence coverage did not include residues that would allow the distinction of H3 variants. The N-termini of the isolated H3 fragments shown in Figure 2B were sequenced by Edman degradation from two independent experiments (Table 1). The N-terminal sequence in the highest molecular weight H3 spot matched the N-terminal sequence of intact H3 (spot 1). We did not obtain reliable sequencing in spots 2 and 3, but identified cleavage sites in spots 4 and 5. The most cleaved H3 fragment (spot 5) was cleaved between L48 and R49 in the globular domain of the protein within the nucleosome core structure. This is a previously unidentified cleavage site in H3, and therefore we selected cleavage at H3R49 as a candidate marker for NETs.

[0188] [Table 2]

[0189] Example 3: Generation of histone H3 cleavage site monoclonal antibodies To raise antibodies against the cleaved histone site, we immunized mice with a lysine-branched immunogen containing a 5-amino acid carboxyl group against the H3R49 cleavage site (Figure 3D). First, sera and subsequent hybridoma supernatants were screened by ELISA against the immunogen (branched peptide formed by SEQ ID NO11 and SEQ ID NO12) and control peptides (SEQ ID NO13 and SEQ ID NO14). Screening was then performed for sera or clones that detected cleaved H3 in immunoblots of PMA-stimulated neutrophil cell lysates. Sera and clones that detected full-length histone H3 in addition to cleaved H3 were excluded (Figures 3A and 3B). In parallel, sera were screened and clones that detected NETs by immunofluorescence microscopy but not quiescent chromatin of naive neutrophils were selected. Of the six mice immunized, one stable clone, 3D9, that worked in both Western blots and microscopy was obtained.

[0190] 3D9 recognized a ~10 kDa protein in neutrophils stimulated with PMA for over 120 min, but did not detect the protein in resting or early stimulated cells (Figure 3B). This band matched in size with the smallest H3 fragment detected by the H3 C-terminal antibody. Interestingly, by microscopy, 3D9 only recognized neutrophils undergoing NETosis with chromatin decondensation (Figure 3A). To validate the specificity of the antibody for the de novo N-terminal H3 epitope in NETs, ​​we performed competition experiments with branched immune peptides (SEQ ID NO11 and SEQ ID NO12), and branched negative control peptides (SEQ ID NO16 and SEQ ID NO17), and demonstrated that they were able to block 3D9 binding to NETs, ​​as shown by immunofluorescence microscopy (Figure 3E).

[0191] Indirect ELISA experiments showed that 3D9 specifically binds to cleaved H3, as does 3D9 binding to branched immunopeptides containing the sequence REIRR (SEQ ID NO11 and SEQ ID NO24) and to isolated NETs, ​​but not to equivalent concentrations of chromatin, recombinant H3, or purified calf thymus DNA by direct ELISA (Figure 3C). Furthermore, in immunoprecipitation experiments, 3D9 pulled down H3 in lysates of both naive and activated neutrophils, but only the cleaved fragment of H3 from activated cells, and not the isotype control (Figure 3F). We detected these pulldowns by both Coomassie and silver stained gels. In a similar experiment, immunoprecipitates were immunoblotted with C-terminal antibodies and 3D9 (Figure 3G), showing that only our monoclonal recognizes cleaved H3. Collectively, these data indicate that 3D9 is selective for truncated H3 under the denaturing conditions of SDS-PAGE, but also recognizes full-length H3 under the more native conditions of IP.

[0192] Example 4: Epitope mapping of antibody clone 3D9 against histone H3 We determined the binding site of 3D9 on histone H3 by ELISA using overlapping linear peptide and helical peptidomimetic sequences based on the sequence surrounding the H3R49 cleavage site (PATGGVKKPHRYRPGTVALREIRRYQKSTELIRKLPFQRL = residues 30-70 of SEQ ID NO25). To mimic the potential charge generated at the newly revealed N-terminal R49, the sequences included the sequence of the unmodified peptide. Based on the overlapping peptides, the putative core epitope in the linear sequence was (R)EIRR. Peptides ending in REIRR were in the top two of each peptidomimetic in all cases. Interestingly, N-terminally extended peptides were also recognized. Furthermore, acetylation at the N-terminus of peptides ending in the REIRR sequence did not affect binding, suggesting that the free N-terminus may not be recognized by the antibody. Epitope mapping was refined by amino acid substitution analysis in linear peptides ending in REIRR, and in helical peptidomimetics. Mutations at Glu51, Ile52, and Arg54 negatively affected signaling, indicating that these residues are important for epitope recognition.

[0193] Example 5: Automated quantification of in vitro generated NETs by microscopy We tested by immunofluorescence microscopy how 3D9 stained NETs in samples that had been permeabilized (Triton X-100, 0.5%, 10 min) to facilitate distribution of the antibody throughout the sample. 3D9 detected almost exclusively non-condensed chromatin (Figure 4A). In contrast, an anti-chromatin antibody against the H2A-H2B-DNA epitope (clone PL2.3) stained NETs in addition to condensed nuclei.

[0194] We compared the staining characteristics of 3D9 and PL2.3 during NET formation. Nuclear area and signal intensity were determined at the indicated time points from multiple fields of view (Figure 4B). Both antibodies detected an increase in nuclear area characteristic of NETosis between 15 and 180 min after simulation. At later time points, the intensity of PL2.3 staining decreased and quiescent cell nuclei could not be distinguished from NETosis. In contrast, 3D9 stained nuclei undergoing NETosis more brightly than nuclei of non-activated cells.

[0195] Publicly available software ImageJ can be used to quantify in vitro NETosis. Staining with 3D9 was compared against an anti-chromatin antibody by a previously published manual semi-automated image analysis (Brinkmann et al., 2012) and a modified automated method. Both methods used automatic thresholding of the DNA channel (Hoechst) to count total cells / objects. The historical manual semi-automated method utilizes differential staining with chromatin antibodies in decondensed chromatin (high signal) versus condensed chromatin (weak signal) to count cells with NETosis. This method uses a manual thresholding and fragmentation procedure (denoted as manual in the figure). This manual thresholding step is subject to observer bias. In contrast, the modified method uses automatic thresholding at both stages: total cell and NET counting. Both methods use a size-exclusion particle analysis step and therefore only count structures larger than quiescent nuclei. Both 3D9 and PL2.3 antibodies effectively quantified NETs using previously published methods (Figure 4C - manual). However, PL2.3 was unable to accurately quantify the number of NETs using a fully automated method, especially at early time points (15 min) after stimulation, where weakly stained, lobulated nuclei may spread over a larger surface area during cell activation and adhesion. Collectively, these data indicate that a modified automated method using 3D9 staining reduces experimental bias.

[0196] Example 5: 3D9 detects NETs induced by multiple stimuli Histone H3 cleavage occurs in NETs induced by multiple stimuli. 3D9 detected NETs induced by the bacterial toxin nigericin, which induces NETs independently of NADPH oxidase activation, by heme in TNF-sensitized neutrophils, and by the fungal pathogen Candida albicans (Figure 5). Interestingly, we detected both 3D9-positive and 3D9-negative NETs in C. albicans infections.

[0197] Example 6: 3D9 identifies NETs in mixed cell samples Histone H3 clipping was observed in mast cells and in unstimulated PBMC fractions, although at other sites in the N-terminal tail. Of note, PBMC fractions often contain contaminating neutrophils. To test whether 3D9 specifically stained neutrophils treated with NET stimuli, PBMCs were incubated with PMA or nigericin (Figure 6). Importantly, 3D9 only detected nuclei that appeared decondensed in cells that were positive for NE, a specific neutrophil marker. In contrast, chromatin antibodies stained both neutrophils and other cell nuclei in NETosis. This indicates that 3D9 specifically detects NETs, ​​even in the presence of other blood cells.

[0198] Example 7: 3D9 distinguishes between NETosis and other forms of cell death in neutrophils It is well known that neutrophils can engage in different cell death pathways. We induced apoptosis by overnight incubation in the presence of SMAC (second mitochondria-derived caspase activator) mimics and caspase inhibition, and necroptosis with TNFα stimulation. Interestingly, the anti-chromatin antibody PL2.3 (Figure 7B) stained the condensed nuclei of cells undergoing apoptosis, whereas 3D9 (Figure 7A) did not, and neither antibody stained cells undergoing necrosis induced by the staphylococcal toxin α-hemolysin.

[0199] Example 8: 3D9 labels NETs in human tissue sections NETs are found in inflamed tissues based on the juxtaposition of chromatin with granule makers and the detection of citrullinated H3. 3D9 stains regions of decondensed DNA that colocalize with NE (Hoechst) in both inflamed human tonsils (Figure 8A) and human kidneys (Figure 8B). This indicates that 3D9 labels NETs in histological samples. Indeed, in kidneys (Figure 8C), inflamed appendix (Figure 9), and gallbladder (Figure 10), 3D9 labeled DNA in the same clusters as anti-H3cit or anti-H2B antibodies. Interestingly, 3D9 stained decondensed, more NET-like structures, whereas anti-H3cit or anti-H2B antibodies stained relatively condensed chromatin.

[0200] Example 9: Identification of the cleavage site L48R49 in other histone H3 variants By performing sequence alignment analysis, it was observed that the cleavage site L48R49 in SEQ ID NO25, which corresponds to the histone H3 sequence Q71DI3 lacking the first Met, also exists at the same position in other histone H3 variants P0DPK2, Q6NXT2, Q16695, P84243, and P68431. Thus, the cleavage site L48R49 in SEQ ID NO25 corresponds to the site L49R50 in Q71DI3, P0DPK2, Q6NXT2, Q16695, P84243, and P68431.

Claims

1. 1. An isolated antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of a truncated histone H3, wherein the antibody comprises: a) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having greater than 90% sequence identity with the amino acid sequence of SEQ ID NO:3; b) an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having greater than 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; c) an H-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having greater than 90% sequence identity with the amino acid sequence of SEQ ID NO:5; d) an L-CDR1 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having greater than 90% sequence identity with the amino acid sequence of SEQ ID NO:6; e) an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having greater than 90% sequence identity with the amino acid sequence of SEQ ID NO: 7; f) A L-CDR3 comprising the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having greater than 90% sequence identity with the amino acid sequence of SEQ ID NO:

8.

2. 2. An isolated antibody, or an antigen-binding portion thereof, that specifically binds to a C-terminal fragment of a truncated histone H3 according to claim 1, wherein the antibody comprises: I) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO:1; and II) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having greater than 90% sequence identity with the amino acid sequence of SEQ ID NO:

2.

3. 3. An isolated antibody, or an antigen-binding portion thereof, that specifically binds to the C-terminal fragment of a truncated histone H3 of claim 1 or claim 2, wherein the antigen-binding portion is selected from the group comprising a single chain variable fragment (scFv), a variable domain (Fv) fragment, a fragment antigen-binding (Fab) fragment, an F(ab)2 fragment, a peptide, or a proteolytic fragment comprising an epitope-binding region of an antibody.

4. A nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody or an antigen-binding portion thereof that specifically binds to the C-terminal fragment of a truncated histone H3 described in any one of claims 1 to 3.

5. A nucleic acid composition comprising one or more nucleic acid molecules encoding an antibody, or an antigen-binding portion thereof, that specifically binds to the C-terminal fragment of truncated histone H3 of any one of claims 1 to 3, wherein the nucleic acid composition comprises a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:9 and a nucleic acid sequence having greater than 90% sequence identity to SEQ ID NO:

10.

6. A host cell comprising the nucleic acid composition of claim 4 or claim 5.

7. 1. A method for in vitro detection of neutrophil extracellular traps in an isolated biological sample, said method comprising: 1) contacting the isolated biological sample with an antibody or antigen-binding portion thereof that specifically binds to the C-terminal fragment of truncated histone H3 according to any one of claims 1 to 3; 2) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, that specifically binds to the C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; 3) determining whether the antibody, or an antigen-binding portion thereof, that specifically binds to the C-terminal fragment of cleaved histone H3 binds to the isolated biological sample.

8. 8. The method of claim 7, wherein the antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of cleaved histone H3 is linked to a detectable label.

9. 1. A method for in vitro assessment of a disease state in an individual, said method comprising: i) contacting an isolated biological sample obtained from said individual with an antibody, or an antigen-binding portion thereof, that specifically binds to the C-terminal fragment of truncated histone H3 according to any one of claims 1 to 3; ii) providing conditions sufficient to allow the antibody, or an antigen-binding portion thereof, that specifically binds to the C-terminal fragment of cleaved histone H3 to bind to the isolated biological sample; iii) determining whether the antibody, or an antigen-binding portion thereof, that specifically binds to the C-terminal fragment of cleaved histone H3 binds to the isolated biological sample; wherein the disease is associated with NET formation; wherein the disease is selected from the group including thrombotic diseases, neurological diseases, bacterial infections, viral infections, fungal infections, inflammatory diseases, autoimmune diseases, cancer diseases, and cancer metastasis diseases.

10. 10. The method of claim 9, wherein the autoimmune disease is selected from the group including psoriasis, vasculitis, systemic lupus erythematosus (SLE), rheumatoid arthritis, ulcerative colitis, Crohn's disease, immune-mediated or type 1 diabetes, immune-mediated glomerulonephritis, inflammatory bowel disease (IBD), antiphospholipid syndrome, and immune thrombocytopenia.

11. 11. The method of claim 9 or claim 10, wherein the antibody, or antigen-binding portion thereof, that specifically binds to a C-terminal fragment of cleaved histone H3 is linked to a detectable label.

12. An isolated fragment of human histone H3 cleaved at site L48R49 of SEQ ID NO25, wherein said fragment of human histone H3 cleaved at site L48R49 consists of the amino acid sequence contained between residues R49 and A135 of SEQ ID NO25, or a fragment thereof, or consists of the amino acid sequence contained between residues A1 and L48 of SEQ ID NO25, or a fragment thereof. Isolated fragment.

13. A kit for evaluating a disease comprising an antibody, or an antigen-binding portion thereof, that specifically binds to the C-terminal fragment described in any one of claims 1 to 3, and / or a fragment of human histone H3 described in claim 12, wherein the disease is selected from the group including thrombotic diseases, neurological diseases, bacterial infections, viral infections, fungal infections, inflammatory diseases, autoimmune diseases, cancer diseases, and cancer metastasis diseases, and wherein the disease is associated with NET formation.

14. 14. The kit for assessing a disease according to claim 13, wherein the autoimmune disease is selected from the group including psoriasis, vasculitis, systemic lupus erythematosus (SLE), rheumatoid arthritis, ulcerative colitis, Crohn's disease, immune-mediated or type 1 diabetes, immune-mediated glomerulonephritis, inflammatory bowel disease (IBD), antiphospholipid syndrome, immune thrombocytopenia.

15. Use of the histone H3 cleavage site located at L48R49 of SEQ ID NO25 for the specific detection of neutrophil extracellular traps.

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