Antibodies that bind to citrullinated histone 2A and / or 4

Abstract

The present invention provides antibodies or binding fragments thereof directed against citrullinated epitopes. The antibodies or binding fragments thereof described in the present invention can be used for treatment, for example, for treating or preventing neutrophil extracellular trap (NET)-related pathologies.

Description

Technical Field

[0001] The present invention provides antibodies or binding fragments thereof directed against citrullinated epitopes. The antibodies or binding fragments thereof of the present invention can be used for treatment, for example, for treating or preventing pathologies associated with neutrophil extracellular traps (NETs). The antibodies or binding fragments thereof of the invention can be used to treat or prevent NET-related pathologies, such as systemic lupus erythematosus (SLE), lupus, sepsis, vasculitis, inflammatory arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, Sjogren's disease, antiphospholipid syndrome, Behcet's disease, spondylitis, spondyloarthropathies, multiple system atrophy, Parkinson's disease, Lewy body dementia asthma, allergic rhinovirus-exacerbated asthma, allergic asthma, cystic fibrosis, fibrosis and idiopathic pulmonary fibrosis, dry eye disease, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, bronchitis or other NET-related pathologies, such as in vivo or in vitro wound healing in diabetes, cancer, cancer metastasis, and transplant organ health. The present invention also provides pharmaceutical compositions and methods for treating or preventing pathologies associated with NETs, ​​such as SLE, lupus, sepsis, vasculitis, inflammatory arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, Sjögren's disease, antiphospholipid syndrome, Behcet's disease, spondylitis, spondyloarthropathies, multiple system atrophy, Parkinson's disease, Lewy body dementia asthma, allergic rhinovirus-exacerbated asthma, allergic asthma, cystic fibrosis, fibrosis and idiopathic pulmonary fibrosis, dry eye disease, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, bronchitis or other NET-related pathologies, such as wound healing in diabetes, cancer, cancer metastasis, and transplanted organ health in vivo or in vitro. Background Art

[0002] Inflammation symptoms, whether chronic or acute, are a significant problem in the healthcare industry. In short, chronic inflammation is considered to be inflammation of long duration (weeks or months) in which active inflammation, tissue destruction, and healing attempts occur simultaneously. Although chronic inflammation can be secondary to an acute inflammatory episode, it can also begin as an insidious process that develops over time, such as due to persistent infection (e.g., tuberculosis, syphilis, fungal infections) leading to delayed hypersensitivity reactions, prolonged exposure to endogenous (e.g., elevated blood lipids) or exogenous (e.g., silica, asbestos, cigarette tar, surgical sutures) toxins, or autoimmune reactions against the body's own tissues (e.g., rheumatoid arthritis, systemic lupus erythematosus, vasculitis, multiple sclerosis, psoriasis).

[0003] One of the results of inflammation is the formation of neutrophil extracellular traps (NETs). NETs are also known to cause inflammation. NETs are structures containing DNA and histones, which are produced by neutrophils and are part of the host's defense mechanism against pathogens. They can capture and kill a variety of bacterial, fungal, viral and protozoan pathogens, and their release is one of the first lines of defense against pathogens. Upon activation by microorganisms or cytokines, histones become hypercitrullinated and the nucleus of neutrophils undergoes a process of chromatin decondensation that leads to the formation of NETs through NETosis (neutrophil net capture death, a form of neutrophil death).

[0004] NETs play a pathological role in a variety of diseases, for example by causing abnormal inflammation. Thus, NETs are involved in the pathology of various inflammatory conditions, such as systemic lupus erythematosus (SLE), lupus, sepsis, vasculitis, inflammatory arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, Behcet's disease, spondylitis, spondyloarthropathies, multiple system atrophy, Parkinson's disease, Lewy body dementia asthma, allergic rhinovirus-induced asthma, cystic fibrosis, and idiopathic pulmonary fibrosis.

[0005] For example, NETs can cause exposure of autoantigens to the extracellular space and subsequent production of pathological autoantibodies by the subject. In addition, NETs and NET remnants contain toxic histones, which can cause vascular damage and subsequent organ damage and failure. Therefore, in such diseases, interfering with the formation of NETs and inducing the clearance of NETs and NET remnants from the circulation and tissues would have therapeutic benefits.

[0006] Neutrophils are also increasingly being recognized as important factors in tumor progression. They play an important role in almost every stage of tumor progression, and a large number of studies have shown that their presence is essential for the development of tumors. Research has also implicated NETs as promoters of tumor progression and metastasis. It has also been shown that neutrophils provide scaffolds and stimulation for platelet adhesion, thrombosis, and coagulation in tumors by producing NETs.

[0007] Furthermore, NETs have been implicated in reducing organ health after transplantation. NETs cause primary graft dysfunction, leading to early mortality after lung transplantation. NETs have been shown to play a pathological role in solid organ transplantation.

[0008] Therefore, the identification of therapeutic agents that can prevent NET formation, clear NETs and / or prevent NETosis will have important implications for inflammatory diseases such as inflammatory arthritis, rheumatoid arthritis and osteoarthritis, as well as other NET-related pathologies such as systemic lupus erythematosus (SLE), lupus, sepsis, vasculitis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, Sjögren's disease, antiphospholipid syndrome, Behcet's disease, spondylitis, spondyloarthropathies, multiple system atrophy, Parkinson's disease, asthma with Lewy bodies, allergic rhinovirus-exacerbated asthma, allergic asthma, cystic fibrosis, fibrosis and idiopathic pulmonary fibrosis, dry eye disease, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, bronchitis, wound healing in diabetes, cancer, cancer metastasis, and transplanted organ health in vivo or in vitro.

[0009] There remains a need for compounds useful for treating or preventing pathologies associated with NETs.

[0010] Antibodies that bind to citrullinated epitopes on deiminated human histone 2A and histone 4 are described in WO2009147201, WO2011070172 and WO2016092082. Summary of the invention

[0011] The present inventors have created improved antibodies that bind to citrullinated epitopes on the amino terminus of histone 2A and / or histone 4. These antibodies can be used to treat diseases or pathologies associated with citrullination, such as NET-related pathologies and inflammatory disorders.

[0012] The inventors have created antibodies that show improved properties over the therapeutic antibodies disclosed in WO2009147201, WO2011070172 and WO2016092082. The inventors found through accelerated stability testing and mass spectrometry that the isomerization of certain amino acid residues in the complementarity-determining region 1 (CDR1) of the light chain of the antibodies disclosed in WO2009147201, WO2011070172 and WO2016092082 caused the antibody to decrease in binding affinity to the tested histone-derived peptide over time. The inventors then conducted a thorough analysis of the CDR1 light chain mutants to address the isomerization problem while attempting to retain the binding properties of the antibodies. Multiple attempts have resulted in antibodies with reduced binding affinity to the target peptide.

[0013] Finally, the inventors succeeded in identifying a set of mutations in the CDR1 of the light chain that eliminated the isomerization problem while maintaining the binding properties of the original antibody. Surprisingly, the mutant antibodies showed improved properties over the original antibody both in vitro and in vivo.

[0014] Therefore, the present invention provides:

[0015] -An antibody or a binding fragment thereof that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, wherein the antibody or the binding fragment thereof comprises:

[0016] a) CDR1 of the light chain variable domain (VL), wherein the CDR comprises or consists of the amino acid sequence QSL-X1-D-X2-D-X3-KTY, wherein X1 is V or L, X2 is T, S, A or N, and X3 is G or A, with the proviso that the amino acid sequence is not QSLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37); and

[0017] b) at least one CDR selected from SEQ ID NOs: 1 to 5.

[0018] The present invention also provides:

[0019] -An antibody or binding fragment thereof that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, wherein the antibody or binding fragment thereof comprises the CDRs of:

[0020] a) CDR1 of SEQ ID NO: 13, 14, 15, 16 or 17; and

[0021] b) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12.

[0022] The present invention also provides:

[0023] - A polynucleotide encoding an antibody or a binding fragment thereof as defined herein, a cloning or expression vector comprising said polynucleotide, or a host cell comprising said cloning or expression vector.

[0024] The present invention also provides:

[0025] - A method for producing an antibody or a binding fragment thereof which specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, comprising culturing a host cell as defined herein and isolating said antibody or binding fragment thereof from said cell.

[0026] The present invention also provides:

[0027] A pharmaceutical composition comprising an antibody or binding fragment thereof as defined herein and at least one pharmaceutically acceptable diluent or carrier.

[0028] The present invention also provides:

[0029] - An antibody or binding fragment thereof as defined herein or a pharmaceutical composition as defined herein, for use in therapy.

[0030] The present invention also provides:

[0031] - Use of an antibody or binding fragment thereof as defined herein, or a pharmaceutical composition as defined herein, in a method for treating or preventing a pathology associated with NETs.

[0032] The present invention also provides:

[0033] - A method of treating a patient comprising administering to said patient a therapeutically effective amount of an antibody or binding fragment thereof as defined herein or a pharmaceutical composition as defined herein.

[0034] Brief Description of Sequence Listing

[0035] Antibody Name

[0036] CDR = complementarity determining region.

[0037] VH = heavy chain variable domain.

[0038] VL = light chain variable domain.

[0039] CH = heavy chain constant domain.

[0040] CL = light chain constant domain.

[0041] msVH22.101 = mouse VH of therapeutic antibody.

[0042] msVL22.101 = mouse VL of therapeutic antibody.

[0043] hVH22.101x = humanized VH of therapeutic antibody, "x" refers to the heavy chain.

[0044] hVL22.101y = humanized VL of therapeutic antibody, "y" refers to the light chain.

[0045] hVH22.101(HC)x = optimized humanized VH of therapeutic antibody, "(HC)x" refers to the heavy chain.

[0046] hVL22.101(LC)y = optimized humanized VL of therapeutic antibody, "(LC)y" refers to the light chain.

[0047] hMQ22.101x / y = humanized therapeutic antibody, "x" refers to the heavy chain and "y" refers to the light chain.

[0048] hMQ22.101 (HC)x / (LC)y = optimized humanized therapeutic antibody of the present invention, "(HC)x" refers to the heavy chain, and "(LC)y" refers to the light chain.

[0049]

[0050] BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 - Accelerated stability test of hMQ22.101j / e and hMQ22.101f / g

[0052] 0.75 ml aliquots (glass tubes) containing hMQ22.101j / e (12.5 mg / ml) or hMQ22.101f / g (3.31 mg / ml) in 25 mM Tris-HCl (trishydroxymethylaminomethane hydrochloride) and pH 8.0 were stored at 37° C. for 8 weeks. Several 10 μl and 20 μl samples were taken from each glass tube under sterile conditions every week and stored at -80° C. until further analysis (ELISA and mass spectrometry).

[0053] hMQ22.101j / e samples from weeks 0, 2, 4, 6, and 8 and hMQ22.101f / g samples from weeks 0, 3, and 6 were subjected to a house-validated CMC ELISA in which binding to a histone-derived peptide (SEQ ID NO: 18) was assessed.

[0054] The antibody binding affinity from the accelerated stability sample at week 0 was set to 100%, and all other binding affinity values ​​from the accelerated stability samples (weeks 2, 3, 4, 6, and 8) were recalculated as percentages of week 0 (100%) and plotted as a bar graph.

[0055] Figure 2-Mass spectrometry analysis of hMQ22.101x / y antibodies

[0056] a) Mass spectrometry (MS) analysis of accelerated stability samples from antibody hMQ22.101j / e.

[0057] 0.75 ml aliquots (glass tubes) containing hMQ22.101j / e (12.5 mg / ml) were each stored at 37° C. for 8 weeks. Samples were removed from each glass tube under sterile conditions every week and stored at −80° C. until MS analysis.

[0058] MS analysis was performed as described in Example 2. The table shows the relative aspartate (D) isomerization levels within CDR1 and near CDR2 of hVL22.101e.

[0059] b) Antigen binding assay was performed using a humanized antibody comprising the aspartic acid mutated CDR1 of hVL22.101y.

[0060] The generated CDR1 aspartate-mutated antibodies hMQ22.101j / h, hMQ22.101j / i and hMQ22.101j / j were compared with the aspartate-containing antibody hMQ22.101j / e using an internally validated CMC ELISA as described in Example 1. The figure shows the optical density results of the three hVL22.101y CDR1 mutants (hVL22.101h with CDR1=DS site mutated to AS; hVL22.101i with CDR1=DS site mutated to ES; and hVL22.101j with CDR1=DS site mutated to SS).

[0061] c) MS analysis of accelerated stability samples from antibody hMQ22.101j / i. MS analysis was performed as described in Example 2. The table shows the relative aspartate (D) isomerization levels within CDR1 and near CDR2 of hVL22.101i.

[0062] Figure 3 - Generation and affinity analysis of hMQ22.101 isomerization mutants

[0063] a) Table showing the 17 CDR1 mutation domains of hVL22.101(LCy) that were created, and the unmutated CDR1 of hVL22.101e and hVL22.101g.

[0064] b) The graph shows the dissociation rate (kdis×E-07 (1 / s)) of the isomerization mutant into the citrullinated H2A-derived peptide (SEQ ID NO: 18) and H4-derived peptide (SEQ ID NO: 20) measured using an Octet RED96 instrument. A lower dissociation rate indicates a higher affinity of the antibody for the antigen.

[0065] Figure 4 - Accelerated stability testing of hMQ22.101 isomerization mutants

[0066] 0.4 ml aliquots (glass tubes) containing the indicated mutant antibodies (range 2.06-4.29 mg / ml) were each stored at 37°C for 6 weeks. Samples were removed from each glass tube under sterile conditions each week and stored at -80°C until further analysis. Samples from weeks 0, 3, and 6 were subjected to an internal validation CMC ELISA in which binding to a citrullinated H2A-derived peptide (SEQ ID NO: 18) was assessed.

[0067] The recalculated antibody binding affinity from the Week 0 accelerated stability sample was set to 100% and all other binding affinity values ​​for the accelerated stability samples were recalculated as percentages of Week 0 (100%) and plotted as a bar graph.

[0068] The preferred heavy chains for accelerated stability testing were hVH22.101f and hVH22.101HC9. Nine combinations of heavy chain and CDR1 mutated light chains were tested. hMQ22.101f / LC41, hMQ22.101f / LC42, hMQ22.101HC9 / LC21, hMQ22.101HC9 / LC27 and hMQ22.101HC9 / LC42 showed the greatest stability after 6 weeks.

[0069] Figure 5 -Mass spectrometry analysis of isomerization mutants of hMQ22.101

[0070] 0.4 ml aliquots (glass tubes) containing the specified mutant antibodies (range 2.06-4.29 mg / ml) were each stored at 37°C for 6 weeks. Samples were removed from each glass tube under sterile conditions every week and stored at -80°C until further analysis. Mass spectrometry (MS) analysis of the hMQ22.101 antibodies (isomerization mutants) with VL CDR1 mutations was performed as described in Example 2, except that accelerated stability samples from weeks 0 and 6 were used and compared with the isomerization levels of hMQ22.101j / e. The table shows the relative aspartic acid (D) isomerization levels in CDR1 of hVL22.101 (LC) y. MS analysis of hMQ22.101 isomerization mutants showed that hMQ22.101f / LC41 showed the least isomerization (0.5%) over time and was therefore the most preferred candidate. Other preferred candidates are hMQ22.101f / LC42 and hMQ22.101HC9 / LC42.

[0071] Figure 6 - Aggregation and degradation assays of selected hMQ22.101 isomerization mutants

[0072] 0.4 ml aliquots (glass tubes) containing the specified mutant antibodies (range 2.06-4.29 mg / ml) were each stored at 37°C for 6 weeks. Samples were taken out from each glass tube under sterile conditions every week and stored at -80°C until further analysis. Aggregation and degradation analysis as described in Example 10 was performed using stability samples of hMQ22.101f / LC41, hMQ22.101f / LC42 and hMQ22.101HC9 / LC42 isomerization mutants at week 0 and week 6. Measured on an Agilent 1200 system combined with an Agilent ZorbaxGF-250 chromatographic column. Protein was detected using 240nm ultraviolet light. The main antibody peak was detected at approximately 4.25 minutes. The shoulder peaks before and after the main peak were quantified and were the percentages of aggregation and degradation levels, respectively. hMQ22.101f / LC41, hMQ22.101f / LC42, and hMQ22.101HC9 / LC42 showed acceptable aggregation and degradation profiles, indicating that they can be used for further development.

[0073] Figure 7 - NETosis inhibition experiments using the preferred isomerization mutants hMQ22.101f / LC41 and hMQ22.101f / LC42

[0074] Neutrophils from healthy volunteers (donors 154 and 155) were stimulated with the calcium ionophore A23187 for 4 hours. The effect of antibodies that reduce neutrophil extracellular traps (NETs) was tested by adding antibodies or assay buffer at a concentration of 25 μg / ml 15 minutes before adding A23187 to the cells. After incubation for 4 hours at 37°C and 5% CO2, the cells were washed and the extracellular DNA was subsequently digested with S7 nuclease. NET fragments were harvested from the wells and quantified by measuring MPO activity in the samples by adding 50 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate to 50 μl of harvested NETs. After incubation for 10 minutes at room temperature, 50 μl of H2SO4 was added and the optical density was measured at 450 nm. The background signal from neutrophils not treated with A23187 was subtracted, and the signal from neutrophils treated with A23187+non-relevant antibodies was set to 100%. Signals from all other treatment groups were set as a percentage of the irrelevant antibody treatment.

[0075] Figure 8 - Dose response of hMQ22.101f / LC41, hMQ22.101f / LC42, and hMQ22.101f / g in the mouse CAIA model

[0076] Lead optimized candidate antibodies can prevent the onset of inflammation. The collagen antibody-induced arthritis (CAIA) model was used to test the dose-response efficacy of hMQ22.101f / LC41, hMQ22.101f / LC42 or hMQ22.101f / g. Groups of 5 mice were treated with 2.8 mg anti-collagen II antibody by intraperitoneal injection on day 0. On day 3, LPS (25 μg / mouse) was injected intraperitoneally, and at the same time, hMQ22.101f / LC41, hMQ22.101f / LC42 or hMQ22.101f / g were injected respectively; each at 6.25, 12.5 and 25 mg / kg of a non-related isotype-matched control antibody (25 mg / kg of MQR2.201) or no antibody (placebo). The degree of swelling of the paws was scored for 2 weeks and is depicted in the figure as "average arthritis score / mouse".

[0077] Figure 9 - In vitro NET inhibition and NET binding by hMQ22.101f / LC41

[0078] Bone marrow-derived mouse neutrophils were stimulated with A23187 to induce in vitro release of NETs. NET release was inhibited by hMQ22.101f / LC41, but not by MQR2.201 (Fig. A; left bar graph, quantification of colocalization of Hoechst (DNA) and citrullinated histone 3 (citH3); right bar graph, quantification of Hoechst alone). In addition, hMQ22.101f / LC41 bound to expelled NETs (yellow arrows) and pre-NETs (white arrows), which may be the first step in macrophage clearance of NETs (Fig. B). Sytox Green was used to detect DNA including NETs and pre-NETs, ​​while anti-hIgG was used to detect hMQ22.101f / LC4 bound to NETs and pre-NETs. Scale bar: 25 μm.

[0079] FIG10 - In vivo NET inhibition and NET binding using hMQ22.101f / LC41

[0080] A pristane-induced peritoneal cell influx mouse model was used to induce NET formation in vivo. 50 mg / kg of MQR2.201 or hMQ22.101f / LC41 was administered immediately after injection of 500 μl of pristane oil, and then 50 mg / kg of MQR2.201 or hMQ22.101f / LC41 was injected again 12 hours later. After 24 hours, the cells were harvested. When mice were treated with hMQ22.101f / LC41 instead of MQR2.201, inhibition of NET release in vivo was observed.

[0081] (Panel A) Representative images. (Panel B) Quantification of colocalization of NETs by nicotinate (DNA) and citrullinated histone 3 (citH3). (Panel C) hMQ22.101f / LC41j binds to NETs and pre-NETs, ​​which may be the first step in NET clearance by macrophages. Glycogreen was used to detect DNA including NETs and pre-NETs, ​​while anti-hIgG was used to detect hMQ22.101f / LC41j bound to NETs and pre-NETs.

[0082] Scale bar: 50 μm (panel A) or 25 μm (panel C).

[0083] Fig.11 - NETs enriched with hMQ22.101f / LC41 are engulfed by mouse macrophages in vivo

[0084] A pristane-induced peritoneal cell influx mouse model was used to induce NET formation in vivo. 50 mg / kg of MQR2.201 or hMQ22.101f / LC41 was administered immediately after injection of 500 μl of pristane oil, and then 50 mg / kg of MQR2.201 or hMQ22.101f / LC41 was injected again 12 hours later. 24 hours later, cells were harvested and stained with nicotinate hexadecanine (DNA: blue), macrophage marker anti-F4 / 80 (magenta), anti-NE (green), anti-citH3 (yellow), and anti-hIgG (cyan). NET particles containing NE (blue arrow), citH3 (red arrow), and hMQ22.101f / LC41 (white arrow) were present in macrophages (F4 / 80). Scale bar: 10 μm.

[0085] Figure 12 - hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice

[0086] (Panel A) Schematic diagram of the CIA mouse model of RA. To induce chronic arthritis, mice were injected with CII twice (day 0 and day 21). Therapeutic treatment was initiated after disease onset (between days 21-28) when MAS ≥ 0.75. Treatment consisted of 4 injections (4 days apart) with a tapered dosing schedule: MQR2.201 (50 / 50 / 50 / 50 mg / kg) or hMQ22.101j / e (30 / 30 / 30 / 10, 50 / 50 / 50 / 15 or 50 / 10 / 10 / 10 mg / kg). Treatment of mice was terminated 14 days after initiation. (Panel B) Mean arthritis scores (MAS) of CIA mice were evaluated for 14 days (n = 10 mice per group; MQR2.201 was used to calculate statistical differences). (Panel C) Bone lesions in the left and right hind knee and ankle joints were analyzed by X-ray on day 14 after the first injection of the antibody (n = 10). Histological analysis of left and right ankle joints using H&E and SO staining determined inflammatory cell influx (D), bone erosion (E), cartilage erosion (F), cartilage proteoglycan depletion (G), and chondrocyte death (H) at day 14 after the first antibody injection (n=16-20 mouse ankles). (Figure I) Representative immunofluorescence and H&E images of NET release in the right hind paw joint showing citrullinated histone 3 (citH3; red), DAPI (blue), neutrophil marker Ly6G (green), and myeloperoxidase (MPO; yellow). DAPI was used as a nuclear and extracellular DNA stain. Scale bar: 100 μm. Quantification of Ly6G (J) and NET (colocalization of citH3 and MPO) in the tibiotarsal joint, proximal intertarsal joint, distal intertarsal joint, and tarsometatarsal joint of the right hind paw of mice (n=10). (Figure L) Significant correlation between macroscopic score (paw swelling) and NET per joint. (Figure M) Significant correlation between macroscopic score (paw swelling) and neutrophils (Ly6G) per joint. Results are expressed as mean ± SEM. Two-way ANOVA with Dunnett's multiple comparison test (B), unpaired two-tailed Student's t test (C), two-tailed Mann-Whitney statistical test (D to H, J and K), or Spearman's r test (L and M), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0087] Fig.13 -hMQ22.101j / e does not bind to healthy leukocytes

[0088] PBMCs and neutrophils were isolated from the blood of healthy volunteers. CD45 was used to distinguish leukocytes from erythrocytes and platelets, while CD3, CD11c, CD14, CD20, CD56 and CD66b were used to label T cells, DCs, monocytes, B cells, NK cells and neutrophils, respectively. No binding of hMQ22.101j / e coupled to HiLyteTMFluor488 was detected to healthy inactive T cells, B cells, monocytes, NK cells, DCs and neutrophils. Activated neutrophils (5μM A23187, 45 minutes) were used as a positive control and showed increased binding of hMQ22.101j / e coupled to HiLyteTMFluor488. ****P<0.001 by ordinary one-way ANOVA with Dunnett's multiple comparison test. DETAILED DESCRIPTION

[0089] It will be appreciated that different applications of the disclosed invention can be adapted to the specific needs of the art.It will also be appreciated that the terminology used herein is for the purpose of describing particular embodiments of the present invention only and is not intended to be limiting.

[0090] In addition, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to the singular form "an antibody" includes the plural form "antibodies" and so forth.

[0091] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0092] The present invention relates to antibodies or binding fragments thereof that specifically bind to citrullinated epitopes on deiminated human histone 2A and / or histone 4. Deimination of human histones 2A and 4 can be performed by enzymes such as peptidyl arginine deiminases (PADs), such as PAD2 and PAD4. The antibodies of the present invention can also specifically bind to citrullinated epitopes on human histone 3. The antibodies of the present invention can specifically bind to citrullinated epitopes on human histone 2A and / or histone 4 and / or histone 3. The present invention also relates to the use of such antibodies or binding fragments thereof, such as therapeutic uses.

[0093] The present invention relates to an antibody or a binding fragment thereof that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, for use in treating or preventing pathologies associated with NETs. The antibodies or binding fragments thereof of the invention can be used to treat or prevent pathologies associated with NETs, ​​such as SLE, lupus, sepsis, vasculitis, inflammatory arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, Sjogren's disease, antiphospholipid syndrome, Behcet's disease, spondylitis, spondyloarthropathies, multiple system atrophy, Parkinson's disease, Lewy body dementia asthma, allergic rhinovirus-exacerbated asthma, allergic asthma, cystic fibrosis, fibrosis and idiopathic pulmonary fibrosis, dry eye disease, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, bronchitis, bronchitis or other pathologies associated with NETs, ​​such as wound healing in diabetes, cancer, cancer metastasis, and transplanted organ health in vivo or in vitro.

[0094] Targets of the antibodies or binding fragments thereof of the present invention

[0095] Citrulline is an amino acid that is not incorporated into proteins during normal translation, however, it may be produced by post-translational modification of arginine residues by enzymes such as PAD (EC 3.5.3.15). To date, in mammals (human, mouse, and rat), five PAD isoforms have been identified (PAD1-PAD6; "PAD4" and "PAD5" are used for the same isoform), each encoded by a different gene.

[0096] Citrullination of histone 2A and / or histone 4 is associated with the formation of NETs. The downstream pathological effects of NET formation may be numerous. For example, subjects may expose autoantigens to the extracellular space and subsequently produce pathological autoantibodies. NET-derived histones may be toxic to vascular walls and organs, leading to vascular damage and organ failure. NETs can lead to the formation of autoantigen / autoantibody immune complexes, which further exacerbate inflammation, such as in the kidneys of SLE patients. NETs are also involved in metastasis in cancer progression.

[0097] The antibody or its binding fragment according to the present invention specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4. The antibody of the present invention may also specifically bind to a citrullinated epitope on deiminated human histone H3. In a specific embodiment, the antibody or its binding fragment according to the present invention specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, wherein the epitope comprises a peptide selected from SEQ ID NO: 18, 19, 20, 21 and 22. The antibody or its binding fragment may also bind to an epitope comprising a peptide of SEQ ID NO: 53 or 54.

[0098] Antibodies or binding fragments thereof

[0099] As used herein, the terms "antibodies (plural)", "antibody (singular)" or "binding fragments" thereof refer to structures, preferably protein or polypeptide structures, that are capable of specifically binding to a target molecule, generally referred to as an "antigen".

[0100] The antibody molecule used herein refers to an antibody or its binding fragment. The term "antibody" used herein generally refers to a complete (whole) antibody, i.e., an element comprising 2 heavy chains and 2 light chains. The antibody may include other additional binding domains, such as DVD-Ig molecules disclosed in WO 2007 / 024715, or so-called (FabFv)2Fc according to WO2011 / 030107. Therefore, the "antibody" used herein includes monovalent, bivalent, trivalent or tetravalent full-length antibodies.

[0101] Binding fragments of antibodies include single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv, monovalent, bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabodies, tribodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see, e.g., Holliger P and Hudson PJ, 2005, Nat. Biotechnol., 23,: 1126-1136; Adair JR and Lawson ADG, 2005, Drug Design Reviews—Online, 2, 209-217). Methods for producing and manufacturing these antibody fragments are well known in the art (see, e.g., Verma R et al., 1998, J. Immunol. Methods, 216, 165-181). The Fab-Fv format was first disclosed in WO2009 / 040562, and its disulfide-stabilized variant, Fab-dsFv, was first disclosed in WO2010 / 035012. Other antibody fragments for use in the present invention include Fab and Fab' fragments. Multivalent antibodies may contain multiple specificities, e.g., bispecific or may be monospecific.

[0102] The antibody or its binding fragment can be selected from: single-chain antibodies, single-chain variable fragments (scFv), variable fragments (Fv), fragment antigen binding regions (Fab), recombinant antibodies, monoclonal antibodies, fusion proteins including antigenic domains of natural antibodies or aptamers, single-domain antibodies (sdAb), also known as VHH antibodies, nanobodies (camelid-derived single-domain antibodies), shark-derived IgNAR-derived single-domain antibody fragments (called VNARs), diabodies, triabodies, anti-lipids (Anticalins), aptamers (DNA or RNA) and active ingredients or fragments thereof.

[0103] IgG1 (e.g., IgG1 / κ) antibodies with IgG1 heavy and light chains can be advantageously used in the present invention. However, the present invention also encompasses other human antibody isotypes, including IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE in combination with κ or λ light chains. Likewise, all animal-derived antibodies of various isotypes can be used in the present invention. The antibody can be a full-size antibody, or an antigen-binding fragment of an antibody, including Fab, F(ab')2, a single-chain Fv fragment, or a single-domain VHH, VH or VL single domain.

[0104] In the present context, the term "specifically binds to citrulline" or "specifically binds to a citrullinated epitope" means that the antibody or its binding fragment binds to a structure such as a peptide containing citrulline residues, whereas the antibody or its binding fragment does not bind very strongly or preferably does not bind at all to the same structure containing arginine residues instead of citrulline residues. The term "peptide" should be interpreted as a structure that is capable of presenting citrulline residues under the right circumstances so as to immunoreact with the antibody or its binding fragment as described herein, preferably under the same circumstances as occurs in the human or animal body, preferably in the case of a native polypeptide.

[0105] The antibody or binding fragment thereof of the present invention specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4. The binding of the antibody or binding fragment thereof to a citrullinated epitope on deiminated human histone 2A and / or histone 4 prevents the formation of NETs. Citrullination of histones is associated with the formation of NETs.

[0106] The blocking of NET formation can be all or part. For example, the antibody or its binding fragment of the present invention can reduce NET formation by 10 to 50%, at least 50% or at least 70%, 80%, 90%, 95% or 99%. NET blocking can be measured by any suitable means, such as by measuring NETosis in vitro (Kraaij T et.al, 2016, Autoimmun. Rev. 15, 577-584).

[0107] The terms "binding activity" and "binding affinity" are intended to refer to the tendency of an antibody molecule to bind or not bind to a target. Binding affinity can be quantified by determining the dissociation constant (Kd) of the antibody and its target. Similarly, the binding specificity of an antibody relative to its target can be defined by a comparative dissociation constant (Kd) comparing the dissociation constant of the antibody to its target with the dissociation constant for the antibody and another non-target molecule.

[0108] Typically, the Kd of an antibody relative to a target is 2 times smaller, preferably 5 times smaller, more preferably 10 times smaller than the Kd of other non-target molecules (e.g., unrelated substances or accompanying substances in the environment). More preferably, the Kd will be 50 times smaller, even more preferably 100 times smaller, and still more preferably 200 times smaller.

[0109] The value of the dissociation constant can be determined directly by well-known methods and can be calculated, even for complex mixtures, by methods such as those described in Catechims and Cacheris WP (1984, Byte, 9, 340-362). For example, Kd can be established using a double filtration nitrocellulose filter binding assay, such as that disclosed by Wong I and Lohman TM (1993, Proc. Natl. Acad. Sci. USA, 90, 5428-5432), or, for example, by using Octet surface plasmon resonance.

[0110] One method of assessing binding affinity to deiminated human histone 2A and / or histone 4 is by ELISA. Other standard assays for assessing the binding ability of ligands, such as antibodies, to targets are known in the art, including, for example, Western blots, RIA, and flow cytometry analysis. The binding kinetics (e.g., binding affinity) of antibodies can also be assessed by standard assays known in the art, such as surface plasmon resonance, such as by Biacore. TM System analysis to evaluate.

[0111] Preferably, the antibodies of the present invention have a binding affinity of 1 nM or less for deiminated human histone 2A and / or histone 4. Preferably, the antibodies of the present invention have a binding affinity of 0.5 nM or less, 0.1 nM or less, 50 pM or less, 10 pM or less, 5 pM or less, 2 pM or less or 1 pM or less for deiminated human histone 2A and / or histone 4 and / or deiminated human histone H3.

[0112] The antibody or binding fragment thereof may also be a fusion protein comprising the antigen binding domain of a natural antibody or an aptamer (eg, an aptamer in the form of DNA or RNA).

[0113] Preferably, the antibody of the present invention or its binding fragment is a monoclonal antibody.Monoclonal antibodies are immunoglobulin molecules that are identical to each other and have a single binding specificity and avidity for a specific epitope.Monoclonal antibodies (mAbs) of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as those disclosed in "Monoclonal Antibodies: Technical Manual" (Zola H, 1987, CRC Press) and "Monoclonal Hybridoma Antibodies: Technology and Application" (Hurrell JGR, 1982 CRC Press).

[0114] The antibodies or binding fragments thereof of the present invention comprise a binding domain. The binding domain will generally include 6 CDRs (3 in the case of VHH), 3 of which are from the heavy chain and 3 from the light chain. In one embodiment, each CDR is in the framework and together forms a variable region or domain. Therefore, in one embodiment, the antibody or binding fragment comprises a specific binding domain for an antigen, which comprises a light chain variable region or domain and a heavy chain variable region or domain.

[0115] The residues in the variable domain of antibodies are usually numbered according to IMGT (http: / / www.imgt.org). This system was proposed by Lefranc MP (1997, J, Immunol. Today, 18, 509). Unless otherwise indicated, this numbering system is used in this specification.

[0116] IMGT residue names do not always correspond directly to the linear numbering of amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict IMGT numbering, corresponding to shortening of, or additions to, the resulting components of the basic variable domain structure (structural components of the framework or CDRs). The correct IMGT numbering of residues for a given antibody can be determined by aligning homologous residues in the antibody sequence with the sequence of the "standard" IMGT numbering.

[0117] According to the IMGT numbering system, the CDRs of the heavy chain variable domain are located at residues 27-38 (CDR1 of VH), residues 56-65 (CDR2 of VH), and residues 105-117 (CDR3 of VH).

[0118] According to the IMGT numbering system, the CDRs of the light chain variable domain are located at residues 27-38 (CDR1 of VL), residues 56-65 (CDR2 of VL), and residues 105-117 (CDR3 of VL).

[0119] The antibodies or binding fragments thereof of the present invention are disclosed by the primary amino acid sequences of their CDR regions. The antibodies or binding fragments thereof of the present invention are disclosed by the primary amino acid sequences of their heavy and light chains.

[0120] The present invention is based on the discovery that a modified CDR1 of the VL of an antibody or binding fragment thereof that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4 provides improved properties to the antibody or binding fragment thereof relative to an antibody or binding fragment thereof comprising an unmodified form of the CDR1 of the VL. The unmodified CDR1 of the VL from which the antibody of the invention is derived comprises or consists of the amino acid sequence QSLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37).

[0121] The modified CDR1 of the VL chain of the antibody or binding fragment thereof of the present invention comprises or consists of the amino acid sequence QSL-X1-D-X2-D-X3-KTY, wherein X1 is V or L, X2 is T, S, A or N, and X3 is G or A, provided that the amino acid sequence is not QSLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37). Compared to the unmodified CDR1 of SEQ ID NO: 36 or 37, the modified CDR1 of the VL chain of the antibody or binding fragment thereof of the present invention shows reduced isomerization, but maintains the binding properties of the unmodified CDR1.

[0122] The amino acid sequences of the CDRs of VH of the specific antibody or binding fragment thereof of the present invention are shown in SEQ ID NOs: 1, 2 and 3. The CDRs 2 and 3 of VL are shown in SEQ ID NOs: 4 and 5.

[0123] The amino acid sequences of VH and VL of the specific antibodies or binding fragments thereof of the present invention are shown in SEQ ID NOs: 11 and 13. The CDRs of VH are shown in SEQ ID NOs: 1, 2 and 3. The CDRs of VL are shown in SEQ ID NOs: 6, 4 and 5.

[0124] The amino acid sequences of VH and VL of another antibody or binding fragment thereof of the present invention are shown in SEQ ID NOs: 11 and 14. The CDRs of VH are shown in SEQ ID NOs: 1, 2 and 3. The CDRs of VL are shown in SEQ ID NOs: 7, 4 and 5.

[0125] The amino acid sequences of VH and VL of another antibody or binding fragment thereof of the present invention are shown in SEQ ID NOs: 11 and 15. The CDRs of VH are shown in SEQ ID NOs: 1, 2 and 3. The CDRs of VL are shown in SEQ ID NOs: 8, 4 and 5.

[0126] The amino acid sequences of VH and VL of another antibody or binding fragment thereof of the present invention are shown in SEQ ID NOs: 11 and 16. The CDRs of VH are shown in SEQ ID NOs: 1, 2 and 3. The CDRs of VL are shown in SEQ ID NOs: 9, 4 and 5.

[0127] The amino acid sequences of VH and VL of another antibody or binding fragment thereof of the present invention are shown in SEQ ID NOs: 11 and 17. The CDRs of VH are shown in SEQ ID NOs: 1, 2 and 3. The CDRs of VL are shown in SEQ ID NOs: 10, 4 and 5.

[0128] The amino acid sequences of VH and VL of another antibody or binding fragment thereof of the present invention are shown in SEQ ID NOs: 12 and 13. The CDRs of VH are shown in SEQ ID NOs: 1, 2 and 3. The CDRs of VL are shown in SEQ ID NOs: 6, 4 and 5.

[0129] The amino acid sequences of VH and VL of another antibody or binding fragment thereof of the present invention are shown in SEQ ID NOs: 12 and 14. The CDRs of VH are shown in SEQ ID NOs: 1, 2 and 3. The CDRs of VL are shown in SEQ ID NOs: 7, 4 and 5.

[0130] The amino acid sequences of VH and VL of another antibody or binding fragment thereof of the present invention are shown in SEQ ID NOs: 12 and 15. The CDRs of VH are shown in SEQ ID NOs: 1, 2 and 3. The CDRs of the VL chain are shown in SEQ ID NOs: 8, 4 and 5.

[0131] The amino acid sequences of VH and VL of another antibody or binding fragment thereof of the present invention are shown in SEQ ID NOs: 12 and 16. The CDRs of VH are shown in SEQ ID NOs: 1, 2 and 3. The CDRs of VL are shown in SEQ ID NOs: 9, 4 and 5.

[0132] The amino acid sequences of VH and VL of another antibody or binding fragment thereof of the present invention are shown in SEQ ID NOs: 12 and 17. The CDRs of VH are shown in SEQ ID NOs: 1, 2 and 3. The CDRs of VL are shown in SEQ ID NOs: 10, 4 and 5.

[0133] In one embodiment of the present invention, the antibody of the present invention comprises a heavy chain variable domain amino acid sequence of SEQ ID NO: 11, a light chain variable domain amino acid sequence of SEQ ID NO: 16, a heavy chain constant region amino acid sequence of SEQ ID NO: 23 or 56, and a light chain constant region amino acid sequence of SEQ ID NO: 24.

[0134] In one embodiment of the present invention, the antibody of the present invention comprises a heavy chain variable domain amino acid sequence of SEQ ID NO: 11, a light chain variable domain amino acid sequence of SEQ ID NO: 16, a heavy chain constant region amino acid sequence of SEQ ID NO: 23 or 56, and a light chain constant region amino acid sequence of SEQ ID NO: 24.

[0135] The antibodies or binding fragments thereof of the present invention may include one or more of the CDR sequences of any specific antibody described above, except that the CDR1 of VL is always present in a form comprising, or consisting of, the amino acid sequence QSL-X1-D-X2-D-X3-KTY, wherein X1 is V or L, X2 is T, S, A or N, and X3 is G or A, provided that the amino acid sequence is not QSLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37), or does not include or consist of SEQ ID NO: 6, 7, 8, 9 or 10.

[0136] The antibodies or binding fragments thereof of the present invention may include one or more VL CDR sequences of the specific antibody in addition to VL CDR1, and alternatively or additionally, include one or more VH CDR sequences. The antibodies or binding fragments thereof of the present invention may include 1, 2 or all 3 of the VH CDR sequences of the specific antibody or binding fragments thereof as described above, and alternatively or additionally, 1, 2 or all 3 of the VH chain CDR sequences of the specific antibody or binding fragments thereof, including VL CDR1. The antibodies or binding fragments thereof of the present invention may include all 6 CDR sequences of the specific antibody or binding fragments thereof as described above. For example, the antibodies of the present invention may include one of SEQ ID NO: 6, 7, 8, 9 or 10 and one or more of SEQ ID NO: 1, 2, 3, 4 and 5.

[0137] In one embodiment of the present invention, the modified CDR1 of the VL chain of the antibody or binding fragment thereof of the present invention comprises or consists of the amino acid sequence QSL-Z1-Z2-Z3-Z4-Z5-KTY, wherein Z1 is V or L, Z2 is D or E, Z3 is T, S, A or N, Z4 is D, E, S or A, and Z5 is G or A, provided that the amino acid sequence is not QSLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37). Compared to the unmodified CDR1 of SEQ ID NO: 36 or 37, the modified CDR1 of the VL chain of the antibody or binding fragment thereof of the present invention shows reduced isomerization, but retains the binding properties of the unmodified CDR1. The modified CDR1 of the VL chain of the antibody or binding fragment thereof of the present invention may include or consist of SEQ ID NO: 6, 7, 8, 9, 10, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52. In one embodiment of the present invention, the antibody of the present invention may include one of SEQ ID NO: 6, 7, 8, 9, 10, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52, and one or more of SEQ ID NO: 1, 2, 3, 4 and 5. In one embodiment of the present invention, the antibody of the present invention includes one of SEQ ID NO: 6, 7, 8, 9, 10, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52, and all of SEQ ID NO: 1, 2, 3, 4 and 5.

[0138] The antibodies or binding fragments thereof of the present invention may alternatively include a variant of one of these heavy chain variable domain or CDR sequences in CDR2 or 3 of VL. For example, the variant may be a substitution, deletion or addition variant of any of the above amino acid sequences.

[0139] Variant antibodies may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions from the above-mentioned specific sequences and fragments while maintaining the activity of the antibodies described herein. "Deletion" variants may include, for example, the deletion of 1, 2, 3, 4 or 5 single amino acids, or one or more small amino acid groups, such as the deletion of 2, 3, 4 or 5 amino acids. "Small amino acid groups" may be defined as being continuous with each other, or close but not continuous. "Replacement" variants preferably involve replacing one or more amino acids with the same number of amino acids and performing conservative amino acid substitutions. For example, amino acids may be substituted with alternative amino acids having similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, another aliphatic amino acid, another tiny amino acid, another small amino acid or another large amino acid. Some of the characteristics of the 20 main amino acids that can be used to select suitable substitutions are as follows:

[0140] Ala Aliphatic, hydrophobic, neutral Met Hydrophobic, neutral Cys Polar, hydrophobic, neutral Asn Polar, hydrophilic, neutral Asp Polar, hydrophilic, charged (-) Pro Hydrophobic, neutral Glu Polar, hydrophilic, charged (-) Gln Polar, hydrophilic, neutral Phe Aromatic, hydrophobic, neutral Arg Polar, hydrophilic, charged (+) Gly Aliphatic, neutral Ser Polar, hydrophilic, neutral His Aromatic, polar, hydrophilic, charged (+) Thr Polar, hydrophilic, neutral Ile Aliphatic, hydrophobic, neutral Val Aliphatic, hydrophobic, neutral Lys Polar, hydrophilic, charged (+) Trp Aromatic, hydrophobic, neutral Leu Aliphatic, hydrophobic, neutral Tyr Aromatic, polar, hydrophobic

[0141] Preferred "derivatives" or "variants" include those in which the amino acids appearing in the sequence are structural analogs of naturally occurring amino acids rather than the naturally occurring amino acids. The amino acids used in the sequence may also be derivatized or modified, such as labeled, provided that the function of the antibody is not significantly adversely affected.

[0142] Derivatives and variants as described above can be prepared during antibody synthesis or by post-production modification, or when the antibody is in recombinant form, using known techniques for site-directed mutagenesis, random mutagenesis or enzymatic cleavage and / or nucleic acid ligation.

[0143] Preferably, the amino acid sequence of the variant antibody according to the present invention has greater than 60% or greater than 70% amino acid identity with the VL and / or VH of the disclosed antibody described herein or its fragment, for example, 75% or 80%, preferably greater than 85%, for example greater than 90%, 95%, 96%, 97%, 98% or 99% amino acid identity. This amino acid identity level can be found in the full length of the relevant SEQ ID NO sequence or a portion of the sequence, for example in 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

[0144] Preferably, the variant antibody comprises one or more of the CDR sequences described herein.

[0145] With respect to amino acid sequences, "sequence identity" means that the sequences have the stated values ​​when assessed using ClustalW (Thompson JD et al., 1994, Nucleic Acid Res., 22, 4673-4680) with the following parameters:

[0146] Pairwise alignment parameters-Method: slow / accurate, matrix: PAM, gap open penalty: 10.00, gap extension penalty: 0.10;

[0147] Multiple Alignment Parameters - Matrix: PAM, Gap Open Penalty: 10.00, Delayed Percent Identity: 30, Penalize end gaps: On, Gap Interval Distance: 0, Negative Matrix: No, Gap Extension Penalty: 0.20, Residue-specific gap penalties: On, Hydrophilic gap penalties: On, Hydrophilic residues: G, P, S, N, D, Q, E, K, R. Sequence identity at a particular residue is intended to include identical residues which have been simply derivatized.

[0148] Thus, the present invention provides antibodies having specific VH and VL amino acid sequences and variants and fragments thereof which retain the function or activity of these VH and VL.

[0149] Accordingly, the present invention encompasses antibodies or binding fragments thereof comprising variants of VH that retain the ability to specifically bind to citrullinated epitopes on deiminated human histone 2A and / or histone 4. The heavy chain variant may have at least 70% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid identity with the unmodified VH. Variants of VH may include a fragment of at least 7 amino acids of hVH22.101f or hVH22.101HC9 (SEQ ID NOs: 11 and 12, respectively), wherein the antibody or its binding fragment retains the ability to specifically react with citrullinated epitopes on deiminated human histone 2A and / or histone 4; or a variant of hVH22.101f or hVH22.101HC9 (SEQ ID NOs: 11 and 12, respectively), which has at least 70% amino acid sequence identity with the sequence of hVH22.101f or hVH22.101HC9 (SEQ ID NOs: 11 and 12, respectively), wherein the antibody or its binding fragment retains the ability to specifically react with citrullinated epitopes on deiminated human histone 2A and / or histone 4.

[0150] Polynucleotides, vectors and host cells

[0151] The present invention also encompasses polynucleotides, vectors, and expression vectors encoding the antibodies or binding fragments thereof described herein.

[0152] The present invention also relates to polynucleotides encoding antibodies of the present invention. Therefore, the polynucleotides of the present invention can encode any antibody or fragment described herein. The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, which are deoxyribonucleotides or ribonucleotides, or their analogs. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, genomic DNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. The polynucleotides of the present invention can be provided in an isolated or purified form.

[0153] A nucleic acid sequence "encoding" a selected polypeptide is a nucleic acid molecule that is transcribed (for DNA) and translated (for mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. For the purposes of the present invention, such nucleic acid sequences may include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. The transcription termination sequence may be located 3' to the coding sequence. In one embodiment, the polynucleotide of the present invention comprises a sequence encoding a VH or VL amino acid sequence as described above. As disclosed herein, the polynucleotide may encode a VH or VL sequence of a specific antibody or binding fragment thereof.

[0154] Therefore, the antibody of the present invention or its binding fragment can be produced or delivered in the form of a polynucleotide that encodes it and can express it. When the antibody includes 2 or more chains, the polynucleotide of the present invention can encode one or more antibody chains. For example, the polynucleotide of the present invention can encode an antibody light chain, an antibody heavy chain or both. Two polynucleotides can be provided, one of which encodes an antibody light chain, and the other encodes the corresponding antibody heavy chain. Such polynucleotides or polynucleotide pairs can be expressed together to produce antibodies of the present invention.

[0155] The polynucleotides of the present invention can be synthesized according to methods well known in the art, for example as described in Sambrook J et al. (1989, Molecular cloning: a laboratory manual; Cold Spring Harbor: New York: Cold Spring Harbor Laboratory Press).

[0156] Nucleic acid molecules of the present invention can be provided in the form of expression cassettes, and expression cassettes include control sequences operably connected to insertion sequences, thereby allowing in vivo expression of antibodies of the present invention. These expression cassettes are usually provided in vectors (e.g., plasmids or recombinant viral vectors). This expression cassette can be directly applied to a host subject. Alternatively, a vector comprising a polynucleotide of the present invention can be applied to a host subject. Preferably, genetic vectors are used to prepare and / or administer polynucleotides. Suitable vectors can be any vector that can carry a sufficient amount of genetic information and allow expression of a polypeptide of the present invention.

[0157] Therefore, the present invention includes expression vectors comprising such polynucleotide sequences. Such expression vectors are conventionally constructed in the field of molecular biology, and for example may relate to the use of plasmid DNA and suitable initiators, promoters, enhancers and other elements, such as polyadenylation signals that may be necessary and positioned in the correct direction to allow expression of the peptide of the present invention. Other suitable vectors will be apparent to those skilled in the art. In this regard, as a further example, we relate to Sambrook J et al. (1989, Molecular cloning: a laboratory manual; Cold Spring Harbor: New York: Cold Spring Harbor Laboratory Press).

[0158] One skilled in the art can use the sequences described herein to clone or generate cDNA or genomic sequences, for example, as described in the examples below. These sequences are cloned into a suitable eukaryotic expression vector, such as pcDNA3 (expression vector (Invitrogen)) or a derivative thereof, and then transfected into mammalian cells (such as CHO cells) in combination with a suitable vector containing light and heavy chains, resulting in expression and secretion of antibodies as described herein.

[0159] A skilled person can also prepare analogs of the antibodies or binding fragments thereof described herein by using the specific binding domains of the antibody sequences and expressing them in different contexts, such as polypeptides, such as fusion proteins. This is well known in the art.

[0160] The present invention also includes cells that have been modified to express the antibodies of the present invention. Such cells include transient or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells, such as yeast or prokaryotic cells, such as bacterial cells. Specific exemplary cells that can be modified by inserting a vector or expression cassette encoding an antibody of the present invention include mammalian HEK293, CHO, HeLa, NS0 and COS cells. Preferably, the cell line selected will not only be stable, but also allow mature glycosylation.

[0161] Such cell lines of the present invention can be cultured using conventional methods to produce antibodies or binding fragments thereof of the present invention, or can be used for delivering antibodies or binding fragments thereof of the present invention to a subject in treatment or prevention. Alternatively, the polynucleotides, expression cassettes or vectors of the present invention can be administered to cells from a subject ex vivo, and then the cells are returned to the subject.

[0162] The present invention also includes a method for producing an antibody or a binding fragment thereof that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, the method comprising culturing the host cell described herein and isolating the antibody or the binding fragment thereof from the cell.

[0163] Pharmaceutical composition

[0164] The present invention encompasses a pharmaceutical composition comprising an antibody or binding fragment thereof of the present invention. The present invention encompasses a pharmaceutical composition comprising an antibody or binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.

[0165] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for parenteral, such as intravenous, intraocular, intramuscular, subcutaneous, intradermal or intraperitoneal administration (e.g., by injection or infusion). In certain embodiments, the pharmaceutically acceptable carrier includes at least one selected from the group consisting of a cosolvent solution, a liposome, a micelle, a liquid crystal, a nanocrystal, a nanoparticle, an emulsion, a microparticle, a microsphere, a nanosphere, a nanocapsule, a polymer or a polymer carrier, a surfactant, a suspending agent, a complexing agent such as a cyclodextrin or an adsorbent molecule such as albumin, a surface active particle, and a chelating agent. In a further embodiment, the polysaccharide comprises hyaluronic acid and its derivatives, dextran and its derivatives, cellulose and its derivatives (e.g., methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate butyrate, hydroxypropyl methylcellulose phthalate), chitosan and its derivatives, β-glucan, arabinoxylan, carrageenan, pectin, glycogen, fucoidan, chondroitin, dermatan, heparanoids, heparin, pentosan, keratan, alginate, cyclodextrin and their salts and derivatives, including their esters and sulfates.

[0166] Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in the pharmaceutical composition of the present invention include water, buffered water, and normal saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. For example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersion, and by using a surfactant, suitable fluidity can be maintained. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols, such as mannitol, sorbitol, or sodium chloride.

[0167] The pharmaceutical composition of the present invention may also include a pharmaceutically acceptable antioxidant. These compositions may also include adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. The presence of microorganisms can be ensured by the above-mentioned sterilization procedures and by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. It may also be necessary to include isotonic agents in the composition, such as sugars, sodium chloride, etc. In addition, the absorption of injectable drug forms can be extended by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0168] The therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage.The pharmaceutical composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0169] Sterile injection solutions can be prepared by incorporating the desired amount of an active agent (e.g., an antibody) into an appropriate solvent (having one or a combination of the above-listed components as required) followed by sterilization microfiltration. Typically, dispersions are prepared by incorporating the active agent into a sterile carrier comprising an alkaline dispersion medium and the desired other components listed above. For sterile powders for the preparation of sterile injection solutions, preferred methods of preparation are vacuum drying and freeze drying (lyophilization), producing a powder of the active agent plus any other desired ingredients from a previously sterile filtered solution thereof.

[0170] The pharmaceutical compositions of the present invention may comprise additional active ingredients as well as the antibodies of the present invention. As described above, the compositions of the present invention may comprise one or more antibodies of the present invention. They may also comprise other therapeutic or prophylactic active agents.

[0171] Depending on the route of administration, the antibody or binding fragment thereof may be coated in a material to protect the antibody from acids and other natural conditions that might inactivate or denature the antibody.

[0172] In a preferred embodiment, the pharmaceutical composition according to the present invention is in a form selected from aqueous solution, gel, hydrogel, film, paste, cream, spray, ointment or wrap.

[0173] In a further embodiment, pharmaceutical compositions as described herein can be administered, for example, by intravenous, subcutaneous, intraocular, intramuscular, intraarticular, intradermal, intraperitoneal, spinal or other parenteral routes of administration, for example, by injection or infusion. Administration can be rectal, oral, ocular, topical, epidermal or by mucosal routes. Administration can be by intracavitary infusion, intracapsular administration or by suction at the resection margin of the tumor, in the lesion, locally around the lesion (including around the tumor, beside the tumor (juxtatumoral), in the tumor) to carry out. In a preferred embodiment, pharmaceutical compositions are administered intravenously or subcutaneously.

[0174] Kits comprising the antibodies or other compositions of the invention and instructions for use are also within the scope of the invention.The kit may further comprise one or more additional agents, such as other therapeutic or prophylactic agents described herein.

[0175] Therapeutic uses of the antibodies and binding fragments thereof of the present invention

[0176] Antibodies or binding fragments thereof according to the present invention can be used for treatment. In therapeutic applications, the antibody or composition is administered in sufficient amounts to a subject already suffering from a disease or condition to cure, alleviate or partially prevent one or more of the condition or its symptoms. This therapeutic treatment may result in a reduction in the severity of disease symptoms, or an increase in the frequency or duration of symptom-free periods. An amount sufficient to achieve this effect is defined as a "therapeutically effective amount". The effective amount for a given purpose will depend on the severity of the disease or injury and the subject's weight and overall condition. As used herein, the term "subject" includes any person.

[0177] In certain embodiments, the antibodies or binding fragments thereof of the invention may be linked (directly or indirectly) to another moiety. The other moiety may be a therapeutic agent, such as a drug. The other moiety may be a detectable label. The other moiety may be a binding moiety, such as an antibody or polypeptide binding domain specific for a therapeutic target. The antibodies or binding fragments thereof of the invention may be bispecific antibodies.

[0178] Therapeutic agents or detectable labels can be directly attached to the antibodies of the present invention or their binding fragments, for example, by chemical coupling (conjugation). Methods for coupling coupling agents or labels to antibodies are known in the art. For example, carbodiimide coupling (Bauminger S and Wilchek M, 1980, Methods Enzymol, 70, 151-159) can be used to couple a variety of reagents including doxorubicin to antibodies or peptides. Water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is particularly useful for coupling the functional moiety to the binding moiety.

[0179] Other methods may also be used to couple a moiety to the antibody. For example, sodium periodate oxidation may be used followed by reductive alkylation with a suitable reactant, or glutaraldehyde cross-linking may be used. However, it is recognized that no matter which method is chosen to produce the coupling of the present invention, it must be ensured that the antibody retains its targeting ability and that the functional moiety retains its associated function.

[0180] The therapeutic agent linked to the antibody may comprise a polypeptide or a polynucleotide encoding a polypeptide having therapeutic benefit. Examples of such polypeptides include anti-proliferative or anti-inflammatory cytokines.

[0181] The antibody can be linked to a detectable label. "Detectable label" means that the antibody is linked to a moiety that can be detected in a non-invasive manner, usually from outside the body and at the site where the target is located, when the moiety is located at the target site after the antibody is administered to the patient. Therefore, the antibody can be used for imaging and diagnosis.

[0182] Typically, the label is or includes a radioactive atom that can be used for imaging. Suitable radioactive atoms include 99mTc and 123I for scintigraphic studies. Other labels include, for example, spin labels for magnetic resonance imaging (MRI), such as 123I, 131I, 111In, 19F, 13C, 15N, 17O, gadolinium, manganese or iron, for example. Obviously, a sufficient number of appropriate atomic isotopes must be attached to the antibody to make the molecule easy to detect.

[0183] Radio tags or other labels can be incorporated in a known manner. For example, antibodies or fragments thereof can be biosynthesized, or can be synthesized by chemical amino acid synthesis using suitable amino acid precursors (e.g., replacing hydrogen with F19). For example, labels such as 99Tc, 123I, 186Rh, 188Rh and 111In can be connected, for example, by cysteine ​​residues in polypeptides. 90Y can be connected by lysine residues. Preferably, detectable labels include radioactive atoms, such as technetium-99m or iodine-123. Alternatively, detectable labels can be selected from: iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, iron.

[0184] In one embodiment, the antibodies of the invention are capable of selectively binding directly or indirectly to a cytotoxic moiety or a detectable marker. Thus, in this embodiment, the antibody is linked to a moiety that selectively binds to a cytotoxic or other compound or component that is easily detected.

[0185] The antibody or binding fragment of the present invention, or a composition comprising the antibody or fragment, can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As recognized by those skilled in the art, the route of administration and / or mode will vary according to the desired result. The preferred route of administration of the antibody or composition of the present invention includes intravenous, subcutaneous, intraocular, intramuscular, intradermal, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral administration" as used herein refers to a mode of administration other than enteral and topical administration, usually by injection. Administration can be rectal, oral, ocular, topical, epidermal or through a mucosal route. Administration can be by intracavitary infusion, intracapsular administration or by inhalation at the resection margin of the tumor, in the lesion, locally around the lesion (including around the tumor, beside the tumor, in the tumor). In a preferred embodiment, the pharmaceutical composition is administered intravenously or subcutaneously.

[0186] The suitable dosage of antibody or its binding fragment of the present invention can be determined by skilled medical practitioners. The actual dosage level of active ingredient in the pharmaceutical composition of the present invention can be changed to obtain the amount of active ingredient that realizes the effective therapeutic response of expectation for specific patients, compositions and administration modes, and is nontoxic to patients. The dosage level selected will depend on a variety of pharmacokinetic factors, which include the activity of the specific antibody used, the route of administration, the time of administration, the antibody excretion rate, the duration of treatment, other drugs, compounds and / or the materials used in combination with the specific composition used, the age, sex, weight, condition, general health and previous medical history of the treated patient, and similar factors well-known in the medical field.

[0187] The suitable dosage of the antibody or its binding fragment of the present invention can be, for example, in the range of about 0.1 μg / kg to about 100 mg / kg of the body weight of the patient to be treated. For example, a suitable dosage can be about 1 μg / kg to about 50 mg / kg body weight per week, about 100 μg / kg to about 25 mg / kg body weight per week, or about 10 μg / kg to about 12.5 mg / kg body weight per week.

[0188] Suitable dosages may be about 1 μg / kg to about 50 mg / kg body weight per day, about 100 μg / kg to about 25 mg / kg body weight per day, or about 10 μg / kg to about 12.5 mg / kg body weight per day.

[0189] The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single suppository can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased according to the urgency of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for subjects to be treated; each unit contains a predetermined amount of active compound, which is calculated to produce the desired therapeutic effect together with the desired pharmaceutical carrier.

[0190] The antibody can be administered in a single dose or multiple doses. Multiple doses can be administered by the same or different routes and at the same or different locations. Alternatively, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required. The dosage and frequency can vary depending on the half-life of the antibody in the patient and the desired duration of treatment. The dosage and frequency of administration can also vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses can be administered at relatively infrequent intervals over a long period of time. In therapeutic applications, relatively high doses can be administered, for example, until the patient shows partial or complete improvement of disease symptoms.

[0191] The combined administration of two or more agents can be achieved in a variety of different ways. In one embodiment, the antibody or its binding fragment and the other agent can be administered together in a single composition. In another embodiment, the antibody and the other agent can be administered as part of a combination therapy in a separate composition. For example, the antibody or its binding fragment can be administered before, after, or simultaneously with the other agent.

[0192] Disease to be treated

[0193] The antibodies or binding fragments thereof of the invention, or the pharmaceutical compositions defined herein, are particularly suitable for treating or preventing pathologies associated with citrullination, such as NET-related pathologies and inflammatory disorders.

[0194] The present invention also includes a method of treating a patient, the method comprising administering to the patient a therapeutically effective amount of an antibody or binding fragment thereof as defined herein or a pharmaceutical composition as defined herein, optionally to treat or prevent pathologies associated with citrullination, such as NET-related pathologies and inflammatory disorders.

[0195] The invention also encompasses an antibody or binding fragment thereof as defined herein or a pharmaceutical composition as defined herein for use in the manufacture of a medicament for the prevention or treatment of pathologies associated with citrullination, such as NET-related pathologies and inflammatory disorders.

[0196] The present invention also encompasses a pharmaceutical composition comprising an antibody or binding fragment thereof of the present invention for use in treating or preventing pathologies associated with citrullination, such as NET-related pathologies and inflammatory disorders.

[0197] A pathology associated with citrullination can be defined as any disease or condition in which citrullination is associated with the pathological state of the disease or condition. Whether citrullination plays a role in the pathogenesis of the disease can be easily determined by a skilled person using routine tests available in the art. For example, these diseases can be characterized by the presence of abnormal levels of citrullinated proteins in tissues affected or associated with the disease. This can be accomplished by immunological tests such as Western blotting or ELISA, in which the affected tissue is used as an antigen and the citrullination of the antigen can be detected with the aid of anti-citrulline antibodies described herein. Alternatively, one skilled in the art can use proteomics applications, such as mass spectrometry, to compare the level and type of citrullination in diseased tissue and healthy tissue from affected patients.

[0198] Pathologies associated with NETs can be considered to be pathologies associated with citrullination. Pathologies associated with NETs can be defined as a disease or condition in which the formation of NETs and NETosis is associated with the pathological state of the disease or condition. Whether the formation of NETs and NETosis plays a role in the pathogenesis of the disease can be readily determined by a skilled artisan using routine tests available in the art. For example, these diseases can be characterized by the presence of NETs in the relevant tissues.

[0199] Therefore, the present invention relates to antibodies or binding fragments thereof for use in treating or preventing pathologies associated with NETs.

[0200] Thus, the invention relates to a method of treating a patient in need thereof with a therapeutically effective amount of an antibody or binding fragment thereof of the invention, wherein the patient suffers from a pathology associated with NETs.

[0201] Examples of NET-related pathologies include inflammatory conditions or diseases, ocular inflammatory diseases, autoimmune diseases, cancer, and post-transplant organ health.

[0202] "Inflammatory disorder" or "inflammatory disease" refers to any of a variety of disorders or diseases characterized by vascular changes: edema and infiltration of neutrophils (e.g., acute inflammatory reactions); infiltration of tissues by monocytes; destruction of tissues by inflammatory cells, connective tissue cells, and their cell products; and attempts to repair by connective tissue replacement (e.g., chronic inflammatory reactions). For example, these diseases are inflammatory arthritis, including rheumatoid arthritis and osteoarthritis, SLE, lupus, sepsis, vasculitis, multiple sclerosis, psoriatic arthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, spondyloarthropathies, multiple system atrophy, Parkinson's disease, Lewy body dementia, idiopathic pulmonary fibrosis, dry eye disease, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, and lung diseases such as COPD and bronchitis. Nongranulomatous uveitis may be associated with neutrophil-dominant inflammation, and granulomatous uveitis may be associated with macrophage-dominant inflammation.

[0203] NETs play a role in the pathology of autoimmune diseases including RA, SLE, and vasculitis. The pathway by which therapeutic antibodies or binding fragments thereof improve disease may be through inhibition of NETosis, through clearance of NET remnants (including toxic histones) and other autoantigens from tissues and circulation, and through clearance of NET remnants and toxic histones from tissues and circulation. For many autoimmune diseases, it has been shown that pathology is improved in PAD knock-out models or in wild-type animals treated with PAD inhibitors, implying a close correlation with the number of NETs and disease severity.

[0204] Thus, inflammatory conditions or diseases as well as autoimmune diseases can be treated by the antibodies and binding fragments thereof of the present invention.

[0205] In a preferred embodiment, the disease to be treated is a NET-associated pathology, such as SLE, lupus, sepsis, vasculitis, inflammatory arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, Sjögren's disease, antiphospholipid syndrome, Behcet's disease, spondylitis, spondyloarthropathies, multiple system atrophy, Parkinson's disease, dementia with Lewy bodies asthma, allergic rhinovirus-exacerbated asthma, allergic asthma, cystic fibrosis, fibrosis and idiopathic pulmonary fibrosis, dry eye disease, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, bronchitis or other NET-associated pathologies, such as wound healing in diabetes, cancer, cancer metastasis, and transplanted organ health in vivo or in vitro.

[0206] In a preferred embodiment, the disease to be treated is an inflammatory disorder, such as SLE, lupus, sepsis, vasculitis, inflammatory arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, Sjögren's disease, antiphospholipid syndrome, Behcet's disease, spondylitis, spondyloarthropathies, multiple system atrophy, Parkinson's disease, dementia with Lewy bodies, asthma, allergic rhinovirus-exacerbated asthma, allergic asthma, cystic fibrosis, fibrosis, idiopathic pulmonary fibrosis, dry eye disease, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, bronchitis.

[0207] Further embodiments

[0208] The present invention is further described by the following examples:

[0209] 1. An antibody or a binding fragment thereof that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, wherein the antibody or the binding fragment thereof comprises:

[0210] a) CDR1 of VL, wherein the CDR comprises or consists of the amino acid sequence QSL-X1-D-X2-D-X3-KTY, wherein X1 is V or L, X2 is T, S, A or N, and X3 is G or A, with the proviso that the amino acid sequence is not QSLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37); and

[0211] b) at least one CDR selected from SEQ ID NOs: 1 to 5.

[0212] 2. The antibody or binding fragment thereof according to 1, wherein the antibody or binding fragment thereof comprises:

[0213] a) CDR1 of VL, wherein the CDR comprises or consists of the amino acid sequence QSL-X1-D-X2-D-X3-KTY, wherein X1 is V or L, X2 is T, S, A or N, and X3 is G or A, with the proviso that the amino acid sequence is not QSLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37); and

[0214] b) CDRs of SEQ ID NO:3 and SEQ ID NO:5.

[0215] 3. The antibody or binding fragment thereof according to 2, wherein the antibody or binding fragment thereof comprises:

[0216] a) one of the CDRs of SEQ ID NOs: 6, 7, 8, 9 and 10; and

[0217] b) CDRs of SEQ ID NO:3 and SEQ ID NO:5.

[0218] 4. The antibody or binding fragment thereof according to 2, wherein the antibody or binding fragment thereof comprises:

[0219] a) CDR1 of VL, wherein the CDR comprises or consists of the amino acid sequence QSL-X1-D-X2-D-X3-KTY, wherein X1 is V or L, X2 is T, S, A or N, and X3 is G or A, with the proviso that the amino acid sequence is not QSLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37); and

[0220] b) CDRs of SEQ ID NOs: 1 to 5.

[0221] 5. The antibody or binding fragment thereof according to any one of the preceding examples, wherein the antibody or binding fragment thereof comprises:

[0222] a) one of the CDRs of SEQ ID NOs: 6, 7, 8, 9 and 10;

[0223] b) CDRs of SEQ ID NOs: 1 to 5.

[0224] 6. The antibody or binding fragment thereof according to 1 or 2, wherein the antibody or binding fragment thereof comprises:

[0225] a) CDR1 of VL, wherein the CDR comprises or consists of the amino acid sequence QSL-X1-D-X2-D-X3-KTY, wherein X1 is V or L, X2 is T, S, A or N, and X3 is G or A, with the proviso that the amino acid sequence is not QSLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37);

[0226] b) at least one of the CDRs of SEQ ID NOs: 4 and 5; and

[0227] c)

[0228] i) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12; or

[0229] ii) a fragment of at least 7 amino acids of (i), wherein the antibody or binding fragment thereof retains the ability to specifically react with a citrullinated epitope on deiminated human histone 2A and / or histone 4; or

[0230] iii) A variant of (i) having at least 70% amino acid sequence identity with the sequence of (i), wherein the antibody or binding fragment thereof retains the ability to specifically react with citrullinated epitopes on deiminated human histone 2A and / or histone 4.

[0231] 7. The antibody or binding fragment thereof according to 6, wherein the antibody or binding fragment thereof comprises:

[0232] a) CDR1 of VL, wherein the CDR comprises or consists of the amino acid sequence QSL-X1-D-X2-D-X3-KTY, wherein X1 is V or L, X2 is T, S, A or N, and X3 is G or A, with the proviso that the amino acid sequence is not QSLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37);

[0233] b) at least one of the CDRs of SEQ ID NOs: 4 and 5; and

[0234] c) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12.

[0235] 8. The antibody or binding fragment thereof according to 7, wherein the antibody or binding fragment thereof comprises:

[0236] a) CDR1 of VL, wherein the CDR comprises or consists of the amino acid sequence QSL-X1-D-X2-D-X3-KTY, wherein X1 is V or L, X2 is T, S, A or N, and X3 is G or A, with the proviso that the amino acid sequence is not QSLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37);

[0237] b) the CDRs of SEQ ID NOs: 4 and 5; and

[0238] c) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12.

[0239] 9. The antibody or binding fragment thereof according to 8, wherein the antibody or binding fragment thereof comprises:

[0240] a) one of the CDRs of SEQ ID NOs: 6, 7, 8, 9 and 10;

[0241] b) the CDRs of SEQ ID NOs: 4 and 5; and

[0242] c) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12.

[0243] 10. The antibody or binding fragment thereof according to any one of the preceding examples, wherein the antibody or binding fragment thereof comprises:

[0244] a) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 and the light chain variable domain amino acid sequence of SEQ ID NO: 13;

[0245] b) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 and the light chain variable domain amino acid sequence of SEQ ID NO: 14;

[0246] c) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 and the light chain variable domain amino acid sequence of SEQ ID NO: 15;

[0247] d) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 and the light chain variable domain amino acid sequence of SEQ ID NO: 16;

[0248] e) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 and the light chain variable domain amino acid sequence of SEQ ID NO: 17;

[0249] f) the heavy chain variable domain amino acid sequence of SEQ ID NO: 12 and the light chain variable domain amino acid sequence of SEQ ID NO: 13;

[0250] g) the heavy chain variable domain amino acid sequence of SEQ ID NO: 12 and the light chain variable domain amino acid sequence of SEQ ID NO: 14;

[0251] h) the heavy chain variable domain amino acid sequence of SEQ ID NO: 12 and the light chain variable domain amino acid sequence of SEQ ID NO: 15;

[0252] i) the heavy chain variable domain amino acid sequence of SEQ ID NO: 12 and the light chain variable domain amino acid sequence of SEQ ID NO: 16; or

[0253] j) The heavy chain variable domain amino acid sequence of SEQ ID NO: 12 and the light chain variable domain amino acid sequence of SEQ ID NO: 17.

[0254] 11. An antibody or binding fragment thereof that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, wherein the antibody or binding fragment thereof comprises the following CDRs:

[0255] a) CDR1 of SEQ ID NO: 13, 14, 15, 16 or 17; and

[0256] b) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12.

[0257] 12. The antibody or binding fragment thereof according to any of the preceding examples, which specifically binds to a peptide selected from SEQ ID NO: 18, 19, 20, 21 and 22, and binds to deiminated human histone 2A and / or histone 4.

[0258] 13. The antibody or binding fragment thereof according to any of the preceding examples, which specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4 with an affinity of at least 1 nM or less.

[0259] 14. The antibody or binding fragment thereof according to any one of the preceding examples is selected from recombinant antibodies, single-chain antibodies, single-chain variable fragments (scFv), variable fragments (Fv), fragment antigen binding regions (Fab), single domain antibodies (sdAb), VHH antibodies, nanobodies, single domain antibodies derived from camelids, single domain antibody fragments (VNAR) derived from shark IgNAR, diabodies, triabodies, anti-lipids and aptamers.

[0260] 15. The antibody or binding fragment thereof according to any one of 1 to 13, wherein the antibody is preferably a full-length antibody.

[0261] 16. The antibody or binding fragment thereof according to 15, comprising an Fc region, such as an IgG1, IgG2, IgG3 or IgG4 region.

[0262] 17. The antibody or binding fragment thereof according to 16, wherein the heavy chain constant region comprises SEQ ID NO: 23, and / or the light chain constant region comprises SEQ ID NO: 24.

[0263] 18. An antibody or binding fragment thereof according to any of the preceding examples, coupled to another moiety.

[0264] 19. A polynucleotide encoding the antibody or binding fragment thereof according to any one of 1 to 17, a cloning or expression vector comprising the polynucleotide, or a host cell comprising the cloning or expression vector.

[0265] 20. A method for producing an antibody or a binding fragment thereof that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, comprising culturing the host cell of 19 and isolating the antibody or the binding fragment thereof from the cell.

[0266] 21. A pharmaceutical composition comprising the antibody or binding fragment thereof according to any one of 1 to 18 and at least one pharmaceutically acceptable diluent or carrier.

[0267] 22. The pharmaceutical composition according to 21, further comprising other active ingredients.

[0268] 23. Use of the antibody or binding fragment thereof according to any one of 1 to 18, or the pharmaceutical composition according to 21 or 22 in therapy.

[0269] 24. Use of the antibody or binding fragment thereof according to any one of 1 to 18, or the pharmaceutical composition according to 21 or 22, in a method for treating or preventing a pathology associated with NET.

[0270] 25. The use of an antibody, a binding fragment thereof or a pharmaceutical composition according to 24, wherein the pathology associated with NET is systemic lupus erythematosus (SLE), lupus, sepsis, vasculitis, inflammatory arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, Sjogren's disease, antiphospholipid syndrome, Behcet's disease, spondylitis, spondyloarthropathies, multiple system atrophy, Parkinson's disease, Lewy body dementia asthma, allergic rhinovirus-exacerbated asthma, allergic asthma, cystic fibrosis, fibrosis and idiopathic pulmonary fibrosis, dry eye disease, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, bronchitis or other pathologies associated with NET, such as wound healing in diabetes, cancer, cancer metastasis, and transplanted organ health in vivo or in vitro.

[0271] 26. The use of an antibody, a binding fragment thereof or a pharmaceutical composition for use according to any one of 23 to 25, wherein the antibody, a binding fragment thereof or a pharmaceutical composition is administered by a parenteral route, such as intravenous, subcutaneous, intraocular, intramuscular, intradermal, intraperitoneal, spinal route or by injection or infusion; or by other routes of administration, such as rectal, oral, ocular, topical, epidermal, mucosal, local, peritumoral, paratumoral, intratumoral administration to the resection margin of the tumor, within the lesion, around the lesion, by intracavitary infusion, intracapsular administration, or by inhalation.

[0272] 27. A method for treating a patient, comprising administering to the patient a therapeutically effective amount of the antibody or binding fragment thereof as defined in any one of 1 to 18, or the pharmaceutical composition according to 21 or 22.

[0273] 28. The method of 27, wherein the treatment is a NET-related pathology.

[0274] The present invention is further illustrated by the following examples, which should not be construed as further limiting.The contents of all figures and all references, patents and published patent applications cited in this application are expressly incorporated herein by reference.

[0275] Example

[0276] Example 1: Accelerated stability test of hMQ22.101j / e and hMQ22.101f / g.

[0277] 0.75 ml aliquots (glass test tubes) containing hMQ22.101j / e (12.5 mg / ml) or hMQ22.101f / g (3.31 mg / ml) in 25 mM Tris-HCl were stored at pH 8.0 at 37° C. for 8 weeks each. Several 10 μl and 20 μl samples were removed from each glass tube under sterile conditions each week and stored at -80° C. until further analysis (ELISA and mass spectrometry).

[0278] hMQ22.101j / e samples from weeks 0, 2, 4, 6, and 8 and hMQ22.101f / g samples from weeks 0, 3, and 6 were subjected to an internally validated CMC ELISA. A 96-well ELISA plate was coated with neutravidin (0.1 μg / well) by overnight incubation at 4°C. The wells were washed 5 times with PBS-Tween 20 (PBS-T) and blocked by incubation with PBS-T + 1% bovine serum albumin (BSA) for 2 hours at room temperature (RT). After washing more than 5 times with PBS-T, the wells were incubated in PBS-T + 0.2% BSA with a histone-derived peptide (SEQ ID NO 18: SGXGKQGGKARA) containing citrulline (X) at position 3 and C-terminal biotin (40 ng / well) for 1 hour at room temperature. After washing 5 times with PBS-T, a calibration curve was prepared from a reference batch of hMQ22.101j / e or hMQ22.101f / g by adding hMQ22.101j / e or hMQ22.101f / g to the wells starting at 1350ng / well and then diluting 1:1 to a concentration of 0.66ng / ml in PBS-T+0.2%BSA. Spiked quality control (QC) samples were prepared from the same reference batch of hMQ22.101j / e or hMQ22.101f / g at higher (HQC, 250ng / ml), mid (MQC, 50ng / ml), lower (LQC 3, 75ng / ml) and lower limit quality control (LLQC, 1.25ng / ml), which were diluted in PBS-T+0.2%BSA and then also added to the plate. Use these QC samples to validate the ELISA results.

[0279] Finally, the accelerated stability samples incubated at 37°C for 0, 2, 3, 4, 6 and 8 weeks were added to the same plate at a concentration of 40 ng / ml in PBS-T+0.2% BSA and incubated for 2 hours at room temperature. The wells were washed 5 times with PBS-T and incubated with rabbit anti-human HRP antibody (1:12.000 in PBS-T+0.2% BSA) for 1 hour at room temperature, then washed 3 times with PBS-T and 3 times with PBS. Before the reaction was stopped with 2M H2SO4, the wells were incubated with TMB substrate for 10 minutes, and the optical density was measured at a wavelength of 450nm. An S-shaped calibration curve was drawn and fitted using the values ​​from the serial dilution reference antibody. The concentrations of the QC samples and accelerated stability samples were recalculated using the S-shaped fitting curve equation. The antibody concentration recalculated from the Week 0 accelerated stability sample was set to 100%, and all other accelerated stability recalculated concentrations (Weeks 2, 3, 4, 6, and 8) were calculated as a percentage of Week 0 (100%) and plotted as a bar graph ( Figure 1 The top group is for hMQ22.101j / e, Figure 1 The bottom group is for hMQ22.101f / g).

[0280] Accelerated stability testing showed that the binding affinity of hMQ22.101j / e and hMQ22.101f / g to histone-derived citrulline-containing peptides decreased over time.

[0281] Example 2: Mass spectrometry analysis of hMQ22.101j / e accelerated stability samples.

[0282] The reasons why the binding affinity of hMQ22.101j / e antibodies decreases over time were investigated. hMQ22.101j / e has multiple potential aspartate isomerization sites in or near the VLCDR region (CDR1 and CDR2). The purpose of this example is to determine the sensitivity of aspartate residues to isomerization by peptide mapping based on liquid chromatography (LC)-mass spectrometry (MS).

[0283] Before digestion, 50 μg of each accelerated stability sample (weeks 0, 4, and 8) was desalted, reduced with dithiothreitol, and alkylated with iodoacetamide. After reduction and alkylation, the samples were digested at 37°C for 18 hours using sequencing-grade modified trypsin (Promega) at an enzyme / protein ratio of 1 / 50 (w / w). The digests were stored at -20°C until LC-MS analysis. Trypsin is a serine protease that specifically cleaves at the C-terminus of arginine or lysine. Analysis of tryptic digests was performed using reverse phase liquid chromatography (RPLC) combined with UV and mass spectrometry detection (RPLC-UV-MS). Data were acquired using an Agilent Technologies 1290UHPLC system hyphenated with an Agilent Technologies 6540Q-TOF equipped with a jetstream electrospray ionization (ESI) source. Before UV 214nm and MS ( / MS) detection, water, trifluoroacetic acid and acetonitrile were used as mobile phase components to separate samples on RPLC columns (AdvanceBio peptide map C18, 250mm L, 2.1mm ID, 2.7μm dp, Agilent Technologies). Approximately 4.5μg of the amount was loaded onto the chromatographic column. The MS system was operated in extended dynamic range mode (2GHz), and the operation was 20,000 at a resolution of 922.009798 for mass, and without using a reference mass, with a higher mass accuracy (usually <10ppm). 2 spectra were acquired per second, and the acquisition range was 100-3000amu in MS and MS / MS modes. MS / MS data were acquired in data-dependent mode. Collision energy was optimized for peptide fragments. All MS measurements were performed in positive ionization mode.

[0284] The measured signal was matched to the sequence using the Agilent MassHunter software containing the BioConfirm algorithm. The mass tolerance for matching the experimental data to the sequence was set to 20ppm. The specified enzyme was trypsin (C-terminal cleavage of lysine or arginine), allowing 0-2 missing cleavages. The peak area of ​​the extracted ion chromatogram obtained at 20ppm mass accuracy was used to quantify the modification. Considering the nearly complete sequence coverage, all candidate aspartate isomerization sites in the hCDR region were covered. These peptides were manually integrated. When present, the peptide containing isoaspartate was eluted, followed by the peptide containing aspartate. The relative isomerization level was then calculated in each case.

[0285] The relative aspartate (D) isomerization level of VL CDR1 of hMQ22.101j / e increases over time ( Figure 2A The isomerization sites tested in CDR1 and CDR2 of VL were Figure 2A Isomerization of CDR1 of VL is believed to be responsible for the loss of binding affinity of antibodies over time.

[0286] Example 3: Generation of hMQ22.101 antibody with three VL CDR1 aspartate mutations.

[0287] It was then investigated whether the absence of a non-germline isomerization site in the CDR1 of VL prevented isomerization. DNA comprising three hVL22.101y domains containing a single aspartate mutation at each amino acid position 31 (L: Asp31) was synthesized by GeneArt. Based on amino acid similarity, such as 1) size, 2) polarity and 3) charge, L: Asp31 was mutated to alanine, glutamine or serine. These aspartate-mutated VL domains were cloned into a mammalian expression vector encoding a full-length human light chain. Subsequently, these light chain constructs (hVL22.101h, hVL22.101i, and hVL22.101j) were used, all in combination with a full-length human heavy chain construct (hVH22.101j), to transiently transfect HEK293 cells to produce hMQ22.101j / h, hMQ22.101j / i, and hMQ22.101j / j, respectively. Full-size antibodies were purified from culture supernatants using a MabSelect SuRe affinity column, followed by buffer exchange to 25 mM Tris-HCl, pH 8.0 using a desalting column, both performed on an Akta-FPLC system. Next, the antibodies were polished with an ion exchange spin column to remove host cell proteins and residual protein A-derivatized resin, followed by an endotoxin removal step using a high-capacity endotoxin removal resin. Finally, antibodies were concentrated using a MicroSep Advance Centrifugal Device (10K MWCO).

[0288] Example 4: Antigen binding assay using hMQ22.101 antibody with VL CDR1 aspartic acid mutation.

[0289] As described in Example 1, the generated VL CDR1 aspartate-mutated antibodies hMQ22.101j / h, hMQ22.101j / i and hMQ22.101j / j were compared with the aspartate-containing antibody hMQ22.101j / e using an internally validated CMC ELISA. Here, the hMQ22.101j / e reference batch was used for calibration curves at 5, 10, 20, 30, 40, 60, 80 and 100 ng / ml, and spiked QC samples at 10, 20, 60 and 80 ng / ml were separated. hMQ22.101j / h, hMQ22.101j / i, hMQ22.101j / j and hMQ22.101j / e were tested at 10, 20, 30, 40, 80 and 100 ng / ml, and dose response curves were plotted in the figure ( Figure 2B ).

[0290] Figure 2B The optical density results of three VL CDR1 mutants (hVL22.101h with CDR1=DS site mutated to AS, hVL22.101i with CDR1=DS site mutated to ES, and hVL22.101jj with CDR1=DS site mutated to SS) are shown. The hMQ22.101j / i antibody obtained the highest improved optical density result.

[0291] Example 5: Mass spectrometry analysis of hMQ22.101j / i with VL CDR1 aspartate mutations after accelerated stability testing.

[0292] 0.75 ml aliquots (glass tubes) containing hMQ22.101j / i (12.5 mg / ml) in 25 mM Tris-HCl (pH 8.0) were stored for 4 weeks at 37° C. Several 10 μl and 20 μl samples were removed from each glass tube under sterile conditions each week and stored at −80° C. until further analysis (mass spectrometry).

[0293] Mass spectrometry analysis was performed identically to that described in Example 2, except that only accelerated stability samples from week 0 and week 4 were used. The isomerization percentages of hMQ22.101j / i were compared to those of antibodies comprising hMQ22.101j / e ( Figure 2C ).

[0294] Mass spectrometry data of hMQ22.101j / i antibody ( Figure 2C ) showed that isomerization in CDR1 of VL still increased over time, however, deletion of the non-germline DS isomerization site in CDR1 of VL did greatly resolve the isomerization problem.

[0295] However, hMQ22.101j / i has lower affinity for the target (SEQ ID NO: 18) than hMQ22.101j / e, and therefore is not a suitable therapeutic antibody candidate.

[0296] Example 6: Generation of additional hMQ22.101 isomerization mutants.

[0297] A comprehensive mutational analysis of the isomerization sites in the CDR1 of the VL was then performed to investigate whether it was possible to remove isomerization of the CDR1 while maintaining the affinity of the mutant antibody for its target. 17 mutant CDR1 domains of hVL22.101 were created. The sequences of these 17 VL CDR1 mutants and the sequences of the unmutated CDR1 of hVL22.101e and hVL22.101g were compared in Figure 3A Listed in.

[0298] DNA of 17 mutant VL CDR1 domains of hVL22.101 and 4 VH domain variants of hVH22.101 were synthesized by GeneArt. All mutant VL and VH domains were cloned into mammalian expression vectors encoding full-length human light and heavy chains, respectively. 17 mutant light chains (hVL22.101LC16, hVL22.101LC17, hVL22.101LC19, hVL22.101LC20, hVL22.101LC21, hVL22.101LC22, hVL22.101LC23, hVL22.101LC24, hVL22.101LC25, hVL22.101LC26, hVL22.101LC27, hVL22.101LC28, hVL22.101LC29, hVL22.101LC30, hVL22.101LC31, hVL22.101LC32, hVL22.101LC33, hVL22.101LC34, hVL22.101LC35, hVL22.101LC36, hVL22.101LC37, hVL22.101LC38 The 4 variant heavy chains (hVH22.101HC7, hVH22.101HC8, hVH22.101HC9 and hVH22.101HC10) were combined with the non-variant heavy chain hVH22.101j or the 4 variant heavy chains (hVH22.101HC7, hVH22.101HC8, hVH22.101HC9 and hVH22.101HC10). Thus, the light chains (hVL22.101e, hVL22.101LC16, hVL22.101LC17, hVL22.101LC19, hVL22.101LC20, hVL22.101LC21, hVL22.101LC22, hVL22.101LC23, hVL22.101LC24, hVL22.101LC25, hVL22.101LC26, hVL22.101LC27, hVL22.101LC All possible combinations of heavy chain (hVH22.101, hVH22.101HC7, hVH22.101HC8, hVH22.101HC9 and hVH22.101HC10) constructs were used to transiently transfect HEK293 cells to produce full-size antibodies (isomeric mutants). Antibodies were purified, desalted, polished and concentrated as described in Example 3.

[0299] Example 7: Dissociation rate analysis of hMQ22.101 isomerization mutants.

[0300] Off-rate screening of isomerization mutant antibodies was performed on an Octet RED96 instrument (Pall ForteBio). All measurements were performed at 30°C. The streptavidin (SA) biosensor was first washed with PBS for 50 seconds. 1 ug / ml N-terminal histone 2A (SEQ ID NO: 18) and histone 4 (SEQ ID NO: 20) peptides containing citrulline at position 3 and biotin at the C-terminus were immobilized on the SA biosensor for 200 seconds, washed with PBS for 50 seconds, and excess reactive streptavidin molecules were blocked with EZ-link biocytin for 200 seconds. After two additional 50-second washing steps in PBS, antibodies diluted in PBS at a concentration of 72 nM were bound to the biosensor for 200 seconds. The sensor was then placed in PBS for 4000 seconds to measure its dissociation rate.

[0301] Before plotting the dissociation curves for each antibody, the background signals generated by the uncoated biosensors exposed to each antibody and the signals generated by the coated biosensors not exposed to each antibody were subtracted. The dissociation rate constants (kdis×E-07 (1 / s)) for histone 2A and histone 4 of each antibody were calculated using ForteBio data analysis software 8.1, applying a 1:1 interaction model (local fit, complete fit).

[0302] The results are shown in Figure 3B Medium. Lower numbers indicate slower off-rates, which means slower release of the antigen. 1×E-07 1 / s is the minimum value, detected by Octet, which means there is almost no measurable off-rate.

[0303] Several hMQ22.101 isomerization mutants showed off-rates of 1×E-07 (1 / s). Preferred heavy chains: hVH22.101j and hVH22.101HC9. Preferred light chains: hVL22.101LC17, hVL22.101LC21, hVL22.101LC27, hVL22.101LC41 and hVL22.101LC42.

[0304] Example 8: Accelerated stability testing of 9 best hMQ22.101 isomerization mutants.

[0305] 0.4 ml aliquots (glass tubes) of the mutant antibodies selected from the following (range 2.06-4.29 mg / ml) in 25 mM Tris-HCl (pH 8.0) were each stored at 37°C for 6 weeks.

[0306] hMQ22.101f / LC17 hMQ22.101f / LC27 hMQ22.101f / LC41 hMQ22.101f / LC42 hMQ22.101HC9 / LC17 hMQ22.101HC9 / LC21 hMQ22.101HC9 / LC27 hMQ22.101HC9 / LC41 hMQ22.101HC9 / LC42

[0307] Several 10 μl and 20 μl samples were aseptically removed from each glass tube every week and stored at -80°C until further analysis (ELISA and MS analysis).

[0308] An internally validated CMC ELISA was performed on week 0, 3, and 6 antibody samples as described in Example 1, except that only the hMQ22.101f / g reference batch was used for the calibration curve and HQC, MQC, LQC, and LLQC spike QC samples.

[0309] The results are shown in Figure 4 The five best performing isomerization mutants (hMQ22.101f / LC41, hMQ22.101f / LC42, hMQ22.101HC9 / LC21, hMQ22.101HC9 / LC27, hMQ22.101HC9 / LC42, boxed) were used to assess isomerization at week 0 and week 6 by MS analysis.

[0310] Example 9: Mass spectrometric analysis of the five best hMQ22.101 isomerization mutants.

[0311] The 37°C accelerated stability samples from these five antibodies performed best in the accelerated stability test (Example 5) and were further analyzed for isomerization levels in CDR1 of VL by MS analysis.

[0312] hMQ22.101f / LC41 hMQ22.101f / LC42 hMQ22.101HC9 / LC21 hMQ22.101HC9 / LC27 hMQ22.101HC9 / LC42

[0313] MS analysis was performed in the same manner as described in Example 2, except that only the accelerated stability samples from week 0 and week 6 were used. The percentage of isomerization was compared with that of antibody hMQ22.101j / e ( Figure 5 ). hMQ22.101f / LC41 showed little isomerization over time (0.5%) and was considered a preferred candidate. The second best antibodies were hMQ22.101f / LC42 and hMQ22.101HC9 / LC42. The preferred second best antibody was hMQ22.101f / LC42 because HC chain f is more humanized than HC9 and the difference in isomerization between week 0 and week 6 was smaller (1.9% vs. 2.6%).

[0314] Example 10: Aggregation and degradation analysis of the three best performing hMQ22.101 isomerization mutants.

[0315] The 37°C accelerated stability samples from three antibodies showed less isomerization in CDR1 of their VL (Example 6) and were further analyzed for aggregation and degradation levels.

[0316] hMQ22.101f / LC41 hMQ22.101f / LC42 hMQ22.101HC9 / LC42

[0317] The measurements were performed on an Agilent 1200 system equipped with a G1311A quaternary pump, G1322A degasser, G1329A autosampler, G1330B thermostat, G1316A column oven, and G1314B VWD detector (Agilent Technologies) in conjunction with an Agilent Zorbax GF-250, 4 μm, 9.4×250 mm column. 10 μl of antibody was injected and the mobile phase consisted of a solution of 200 mM NaH2PO4 in water (pH 7.0) and was run at a flow rate of 2 ml / min for 10 minutes. Proteins were detected using 240 nm UV light. The main antibody peak was detected at approximately 4.25 minutes. The shoulders before and after the main peak were quantified, and the brightness of the shoulders were respectively the aggregation and degradation levels. The results are shown in Figure 6 shown.

[0318] hMQ22.101f / LC41, hMQ22.101f / LC42, and hMQ22.101HC9 / LC42 showed acceptable aggregation and degradation profiles, indicating that they were acceptable for further development.

[0319] Example 11: Fragmentation analysis of the best performing hMQ22.101 isomerization mutants.

[0320] The analysis of complete mAb samples was carried out using reverse phase liquid chromatography (RP-HPLC) in combination with UV and mass spectrometry (MS) detection (RP-HPLC-UV-MS). Data were obtained using an Agilent Technologies 1290UHPLC system connected to an Agilent Technologies 6540 Q-TOF character equipped with a jet electrospray ionization (ESI) source. Samples were separated on a RPLC column (Zorbax 300SB-C8, 100mm L, 2.1mm ID, 1.8 μm dp, Agilent Technologies), wherein 0.1% TFA in water was used as mobile phase A, and 0.1% TFA in acetonitrile was used as mobile phase B. A concentration gradient of 15% to 80% B was applied in 65 minutes. Approximately 5 μg was loaded onto the column. The MS system was operated in high resolution mode (4 GHz), wherein fragmentor voltage was 350 V, and Quad AMU was set to 300. In positive MS mode, 1 spectrum was acquired per second with an acquisition range of 300-3200 amu. The raw spectra were deconvoluted using the maximum entropy algorithm, which was incorporated into Agilent Technologies MassHunter software with the BioConfirm accessory. The measured MW was compared with the theoretical MW determined by the full sequence, taking into account potential C-terminal lysine truncations and N-glycosylation.

[0321] Using RP-HPLC-UV-MS analysis, increased fragmentation was observed for both hMQ22.101f / LC41 and hMQ22.101j / e samples incubated at 37°C for 6 weeks compared to unstressed samples. The amount of fragmentation was similar to the fragmentation distribution observed for other therapeutic antibodies used in clinical studies and was acceptable.

[0322] Example 12: Human neutrophil extracellular trap assay.

[0323] Whole blood from 2 different healthy donors was collected in sodium heparin tubes (Beckton Dickinson). 30 ml of blood from each donor was mixed with 15 ml of 6% dextran in 0.9% NaCl and incubated at room temperature for 60 minutes. After incubation, 2 clear layers were visible, with the bottom layer containing most of the red blood cells and the top layer containing neutrophils. The top layer was collected and spun down at 300 g for 10 minutes at room temperature. The pellet was resuspended in 25 ml PBS and subjected to density gradient centrifugation with Ficoll-Paque Plus (GE Healthcare), followed by a 10-minute red blood cell lysis step at room temperature, thereby isolating the neutrophils. Guava EasyCyte flow cytometer was used to count the cells. 900.000 neutrophils per well were seeded into 24-well tissue culture plates (Greinerbio-one) in neutrophil extracellular trap (NET) assay buffer (RPMI 1640 medium containing glutamine (Life Technologies)) supplemented with 1% heat-inactivated fetal bovine serum and 1mM CaCl2. Neutrophils were stimulated for 4 hours with calcium ionophore A23187 (Molecular Probes). The effect of reducing NET antibodies was tested by adding one of the following antibodies (hMQ22.101f / g, hMQ22.101f / LC41, hMQ22.101f / LC42 and isotype control antibody MQR2.201) or assay buffer at a concentration of 25 μg / ml 15 minutes before adding A23187 to the cells. After incubation for 4 hours at 37°C and 5% CO2, the cells were washed very finely twice using NET assay buffer. Extracellular DNA was then digested with S7 nuclease (7.5U / 0.5ml) at 37°C for 15 minutes, and 10μl 500mM EDTA was then added to stop further digestion. NETs were collected from the wells and spun down at 20g for 5 minutes to remove intact cells. MPO activity in the sample was measured by adding 50μl 3,3′,5,5′-tetramethylbenzidine (TMB) substrate to 50μl of harvested NETs to quantify the number of NETs. After incubation at room temperature for 10 minutes, 50μl H2SO4 was added and the optical density was measured at 450nm. The background signal from neutrophils not treated with A23187 was subtracted, and the signal from neutrophils treated with A23187+MQR2.201 was set to 100%. The signals from all other antibody-treated groups were compared with the A23187+MQR2.201-treated group ( Figure 7 ).

[0324] Surprisingly, the development candidate hMQ22.101ffLC41 outperformed hMQ22.101f / LC42 and hMQ22.101f / g at a concentration of 25 μg / ml (n=2). The isomerized mutant antibody not only maintained the properties of the non-mutated antibody, but also improved upon them.

[0325] Example 13: Experimental mouse model for inflammation.

[0326] The goal of this study was to test a dose response range using the development candidates designated hMQ22.101f / LC41 or hMQ22.101f / LC42 (in which isomerization issues are eliminated) compared to an earlier candidate hMQ22.101f / g and an isotype-matched control antibody MQR2.201 in a mouse model of collagen antibody-induced arthritis (CAIA). Paw and ankle swelling were quantified.

[0327] According to the manufacturer's instructions, a commercially available CAIA mouse model from ModiQuest Research BV (Catalog No.: MQ18.101) was used to induce arthritis in mice. For this purpose, 2.8 mg of an anti-collagen II antibody mixture was injected intraperitoneally in DBA / J1 mice. Three days later, the mice received another intraperitoneal injection containing 25 μg of LPS to synchronize inflammation between mice. Simultaneously with LPS, mice received tACPAs hMQ22.101f / g, hMQ22.101f / LC41 or hMQ22.101f / LC42 (6.25, 12.5 and 25 mg / kg), a non-related isotype-matched control antibody MQR2.201 (25 mg / kg) or a placebo (0.9% NaCl in saline). Typically, inflammation of the front and hind paws can be seen starting 2 days after LPS injection (i.e., day 5). The degree of swelling of the paws was macroscopically scored starting from day 0 for a period of 13 days. The maximum swelling score is 8 (divided among the 4 paws). See the table below for the scoring system.

[0328] 1-2 swollen toes 0.25 3-4 swollen toes 0.50 Slight swelling in the pads or ankles 0.50-0.75 Swelling of the pads or ankles + / - toes 1.00 Swollen toes + slightly swollen pads 1.25 Swollen toes + swollen pads 1.5 Swollen foot pads + swollen ankles 2.00

[0329] The results are shown in Figure 8 The paws of mice treated with the therapeutic antibodies showed significantly reduced inflammation in a dose-dependent manner compared to mice treated with control antibodies or saline solution. Both the optimized lead antibodies hMQ22.101f / LC41 and hMQ22.101f / LC42 (in which the isomerization problem is removed) were more effective in preventing inflammation than the previous lead candidate hMQ22.101f / g, as clearly shown at a dose of 25 mg / kg ( Figure 8 , top group). No adverse reactions were observed. At the lowest dose of 6.25 mg / kg ( Figure 8 , bottom panel), hMQ22.101f / LC41 was superior to all other antibodies, with Student t-test p values ​​of p<0.001, p<0.05, and p=0.46 for hMQ22.101f / LC41, hMQ22.101f / LC42, and hMQ22.101f / g, respectively, compared with the placebo-treated group on day 13.

[0330] Example 14: Further characterization of development candidate hMQ22.101f / LC41 in mouse in vitro NET assay.

[0331] To further strengthen the notion that hMQ22.101f / LC41 is a potent inhibitor of NET formation, the binding of hMQ22.101f / LC41 to mouse NETs and pre-NTTs (pre-NETs) and the inhibition of mouse NET formation have been studied as follows. Pre-NETs are defined as neutrophils with an amorphous decondensed nuclear structure that includes citrullinated chromatin that remains present within the cell, while the nuclear membrane is collapsed.

[0332] The aim of this study was to test whether the development candidate designated hMQ22.101f / LC41 could inhibit NET formation in mice. TM Neutrophils were isolated from the bone marrow of C57BL / 6J mice by negative selection using the Mouse Neutrophil Enrichment Kit (Stemcell Technologies). The purity of the isolated neutrophils was checked by flow cytometry using Ly6G (Biolegend) antibody and was above 90%. The concentration of isolated bone marrow neutrophils was adjusted to 2 × 10 6Cells / ml. A total of 100 μl of cell suspension was added to each well of an 8-well chamber slide (Thermo Fisher Scientific). 25 μg / ml hMQ22.101f / LC41, MQR2.201, or no antibody was incubated with cells for 15 minutes before adding 150 μl of HBSS containing 1 μg / ml A23187 or vehicle control to the cells. The chamber slides were incubated at 37°C and 5% CO2 for 3 hours. Subsequently, 2% (v / v) paraformaldehyde (Merck) was added to each well, and the preparations were incubated at 4°C for 12h. The samples were blocked with 10% fetal calf serum (FCS; Biochrome) in PBS for 1 hour at room temperature. Rabbit anti-citH3 primary antibody (Abcam, ab5103; 1:200) or TRITC-conjugated goat anti-human IgG (Jackson Immunoresearch, 109-025-003; 1:100) was added in 10% FCS in PBS at 4°C for 12 hours. The slides were washed three times with PBS, and then Cy5-conjugated goat anti-rabbit IgG secondary antibody (Jackson ImmunoResearch, 111-175-144; 1:400) was added for 1.5 hours in the dark at room temperature. The slides were washed again with PBS three times. PBS staining solution containing 2.5 μM nicotinate hexadecanine (Hoechst) was added for 15 minutes at room temperature. After washing with PBS, the samples were embedded in mounting medium (BIOZOL). The slides were analyzed on a BZ-X710 microscope (Keyence), and NETs and pre-NETs were quantified using Fiji imaging software ( Fig. 9A Representative images showing hMQ22.101f / LC41 binding (hIgG; red) to NETs (yellow arrows) and pre-NETs (white arrows) are shown in Fig. 9B Displayed in.

[0333] In vitro treatment of mouse bone marrow (BM)-derived neutrophils with hMQ22.101f / LC41 resulted in reduced A23187-induced NET extrusion compared with MQR2.201-treated mouse BM-derived neutrophils ( Fig. 9A In addition, hMQ22.101f / LC41 was expressed in the mouse NETs ( Fig. 9B ; yellow arrow) and anterior NET ( Fig. 9B ; white arrows), which may be the first step in the clearance of NETs by macrophages.

[0334] Example 15: Further characterization of the development candidate hMQ22.101f / LC41 in an in vivo NET / macrophage assay in mice using a pristane-induced peritonitis mouse model

[0335] Used previously by The ability of the development candidate hMQ22.101f / LC41 to inhibit NET formation in vivo was tested in a mouse model of pristane-induced peritoneal cell influx described by et al. (JCI Insight 2017;2(1):e92920).

[0336] Briefly, 50 mg / kg MQR2.201 or hMQ22.101f / LC41 was administered immediately after the injection of 500 μl pristane (Sigma-Aldrich), and then 50 mg / kg MQR2.201 or hMQ22.101f / LC41 LC41 was injected again 12 hours later. After a total of 24 hours, inflammatory cells were isolated from the peritoneum and adjusted to 1×10 6 Cells / ml and transferred to flow chamber slides or cytospin slides for analysis by immunofluorescence microscopy. The slides were then blocked with PBS + 10% FCS and incubated with rabbit anti-citH3 (Abcam, ab5103; 1:200), rabbit anti-NE (Abcam, ab21595; 1:200), AF488-conjugated rat anti-mouse F4 / 80 (Biolegend, 123120; 1:200) or TRITC-conjugated goat anti-human IgG (Jackson Immunoresearch, 109-025-003; 1:100). The slides were washed 3 times with PBS and Cy5-conjugated goat anti-rabbit IgG secondary antibody (Jackson ImmunoResearch, 111-175-144; 1:400) was added in the dark at room temperature for 1.5 hours. The slides were washed again with PBS 3 times. A staining solution containing 2.5 μM nicotinate in PBS was added for 15 min at room temperature. After washing with PBS, the samples were embedded in mounting medium (BIOZOL). The slides were analyzed on a BZ-X710 microscope (Keyence) ( Fig. 10A and C), and NET and pre-NET were quantitatively analyzed using Fiji imaging software ( Fig. 10B ). Fig. 10C Binding of hMQ22.101f / LC41 to NETs and pre-NETs is shown. Fig.11 The uptake of hMQ22.101f / LC41-enriched NETs by macrophages is shown.

[0337] When compared to peritoneal cells from MQR2.201-treated mice, a decrease in NET filaments containing DNA and citrullinated histone 3 (citH3) was observed in peritoneal cells from hMQ22.101f / LC41-treated mice ( Fig. 10AQuantification of NETs (colocalization of citH3 and DNA (nicotinate)) confirmed this observation ( Fig. 10B ). Colocalization of DNA and citH3 is a hallmark of NET formation. In addition, hMQ22.101f / LC41 binds to expelled mouse NETs and mouse pro-NETs ( Fig. 10C ), which may be the first step in the clearance of NETs by macrophages. Indeed, F4 / 80-positive macrophages were observed in cell infiltrates that contained engulfed hMQ22.101f / LC41 in combination with citH3 or neutrophil elastase ( Fig.11 ).

[0338] Example 16: CIA Mouse Model of RA

[0339] To investigate the efficacy of tACPA against NET-induced tissue damage, different tapered tACPA strategies were used in the chronic collagen-induced arthritis (CIA) mouse model of RA.

[0340] To induce chronic arthritis, bovine collagen II was diluted to a concentration of 2 mg / ml in 0.05 M acetic acid and emulsified in an equal volume of Freund's complete adjuvant. On day 0, 10-12 week old male DBA / J1 mice were immunized intradermally at the base of the tail with 100 μg bovine CII. On day 21, mice received an intraperitoneal booster injection of 50 μg bovine CII dissolved in PBS, and arthritis developed a few days later ( Fig. 12A ). Mice were considered to have arthritis when significant changes in redness and / or swelling were noted in the toes or other areas of the paw. Joint inflammation in each paw was scored as described above (CAIA mouse model of RA). Therapeutic treatment was initiated after disease onset (between 21-28 days) when the mean arthritis score (MAS) was ≥ 0.75 (0-2 per paw) on an arbitrary scale of 0-8. Therapeutic dosing was performed with 4 repeated intravenous injections spaced 4 days apart, with the specified doses of hMQ22.101j / e (50 / 10 / 10 / 10, 30 / 30 / 30 / 10 and 50 / 50 / 50 / 15 mg / kg) reducing MAS by 38%, 52% and 81% respectively on day 14 compared to 50 / 50 / 50 / 50 mg / kg MQR2.201. Fig. 12B ). Mice were sacrificed on day 14 after the start of treatment. Ankle and knee joints were collected and stored in formalin for histological analysis.

[0341] It is worth mentioning that all hMQ22.101j / e treatments prevented disease progression within the first 8 days, after which MAS began to rise, likely due to the development of anti-drug antibodies in these mice. Only treatment with 50 / 50 / 50 / 15 mg / kg hMQ22.101j / e completely stabilized disease for 14 days, with MAS not exceeding 0.75.

[0342] To further investigate the effect of tACPA on bone damage, X-rays of the knee and ankle of all hind paws were analyzed following hMQ22.101j / e and MQR2.201 treatment regimens. Consistent with the observed MAS, all hMQ22.101j / e treatments suppressed bone damage in the ankle and knee ( Fig. 12C To further understand the protective effect of tACPA, histological analysis of ankle joints was performed using H&E and safranin O (SO) staining. hMQ22.101j / e inhibited the influx of inflammatory cells compared with mice treated with MQR2.201 ( Fig.12D In addition, hMQ22.101j / e significantly reduced bone and cartilage erosion as well as cartilage proteoglycan depletion and chondrocyte death compared with MQR2.201-treated mice ( Fig.12E to H). These data suggest that tACPA can strongly alleviate arthritis symptoms including joint damage.

[0343] We then investigated the presence of neutrophils and NETs in the paws of CIA mice treated with 50 / 50 / 50 / 15 mg / kg hMQ22.101j / e or 50 / 50 / 50 / 50 mg / kg MQR2.201. Mouse neutrophil markers Ly6G, citrullinated histone 3 (citH3), and myeloperoxidase (MPO) were demonstrated in MQR2.201-treated animals, whereas these markers were almost absent in hMQ22.101j / e-treated mice ( Fig.12I ). DAPI was used as a nuclear and extracellular DNA stain ( Fig.12I Quantification of neutrophils (Ly6G) and NETs (colocalization of citH3 and MPO) was performed by analyzing multiple joints of the right hind paw of each animal, including the tibiotarsal, proximal intertarsal, distal intertarsal, and metatarsotarsal joints. Neutrophils ( Fig.12J ) and NET( Figure 12K We found that the number of NETs in the joints was significantly correlated with macroscopic paw swelling ( Figure 12L ; r = 0.6120, P = 0.0041). Similarly, a significant correlation was observed between paw swelling and the presence of neutrophils in the joints ( Figure 12M ; r = 0.8729, P < 0.0001). Taken together, these data suggest that tACPA treatment eliminates NETs in inflamed tissues in vivo, thereby preventing severe bone and tissue destruction.

[0344] Example 17: hMQ22.101j / e does not bind to healthy leukocytes

[0345] Blood from healthy volunteers (HV) was obtained from the Sanquin blood bank in Nijmegen, the Netherlands, and collected in lithium heparin tubes. All blood donors expressed informed consent. Ficoll density gradient centrifugation was performed to separate peripheral blood mononuclear cells (PBMC) and neutrophils. PBMC was collected and washed 3 times with RPMI 1640 (hereinafter referred to as RPMI 10%) supplemented with 10% (v / v) heat-inactivated fetal calf serum (FCS) and 50U / ml penicillin-streptomycin to remove platelets. Neutrophil / erythrocyte suspension was mixed with 6% (w / v) of dextran in 0.9% NaCl and incubated at room temperature for 25 minutes. Subsequently, neutrophils were collected, exposed to ammonium chloride (ammonium-chloride-potassium, ACK) buffer for 10 minutes at room temperature to dissolve the remaining red blood cells, and washed 2 times with RPMI 10%.

[0346] PBMCs and neutrophils were cultured at 2 × 10 in FACS buffer. 5Cells were seeded at a density of 10 cells / well in 96-well V-bottom plates. Cells were incubated with human Trustain FcX (diluted 1:50 in FACS buffer) for 20 minutes at room temperature to block Fc receptors. Subsequently, PBMCs were incubated for 45 minutes at room temperature with an antibody mixture containing 6.25 μg / ml HiLyte™ Fluor 488-conjugated hMQ22.101j / e, 0.17 μg / ml anti-CD3, 1 μg / ml anti-CD11c, 0.33 μg / ml anti-CD14, 0.17 μg / ml anti-CD20, 83 ng / ml anti-CD45 and 0.17 μg / ml anti-CD56, while neutrophils were incubated with an antibody mixture containing 6.25 μg / ml HiLyte™ Fluor 488-conjugated hMQ22.101j / e, 83 ng / ml anti-CD45 and 83 ng / ml anti-CD66b. As a positive control for HiLyte™ Fluor 488-conjugated hMQ22.101j / e binding, neutrophils were stimulated with 5 μM A23187 for 45 minutes before blocking Fc receptors. After antibody incubation, PBMCs and neutrophils were fixed with 4% formaldehyde for 15 minutes at room temperature, washed with FACS buffer, and analyzed using a CytoFLEX flow cytometer.

[0347] HiLyteTM Fluor 488-conjugated hMQ22.101j / e does not bind to healthy inactive T cells, B cells, monocytes, natural killer (NK) cells, dendritic cells (DCs), or neutrophils, but does bind to activated neutrophils ( Fig.13 ). The hMQ22.101f / LC41 antibody is expected to have comparable results.

[0348] Sequence Listing

[0349] SEQ ID NO: 1—CDR1 of msVH22.10 and hVH22.101(HC)x

[0350] GYTFTNYG

[0351] SEQ ID NO:2—CDR2 of msVH22.101 and hVH22.101(HC)x

[0352] INTYSGEA

[0353] SEQ ID NO: 3—CDR3 of msVH22.101 and hVH22.101(HC)x

[0354] LRGYTYQSFDEGGDY

[0355] SEQ ID NO:4 - CDR2 of msVL22.101 and hVL22.101(LC)y

[0356] LVS

[0357] SEQ ID NO: 5 - CDR3 of msVL22.101 and hVL22.101(LC)y

[0358] WQH

[0359] SEQ ID NO: 6—CDR1 of hVL22.101LC17

[0360] QSLDTDGKTY

[0361] SEQ ID NO: 7—CDR1 of hVL22.101LC21

[0362] QSLDSDAKTY

[0363] SEQ ID NO: 8—CDR1 of hVL22.101LC27

[0364] QSLDTDAKTY

[0365] SEQ ID NO:9—CDR1 of hVL22.101LC41

[0366] QSLLDADGKTY

[0367] SEQ ID NO: 10—CDR1 of hVL22.101LC42

[0368] QSLLDNDGKTY

[0369] SEQ ID NO: 11-hVH22.101f

[0370] RIQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS

[0371] SEQ ID NO: 12-hVH22.101HC9

[0372] RIQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYVDDFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS

[0373] SEQ ID NO:13-hVL22.101LC17

[0374] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDTDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0375] SEQ ID NO:14-hVL22.101LC21

[0376] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDAKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0377] SEQ ID NO:15-hVL22.101LC27

[0378] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDTDAKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0379] SEQ ID NO:16-hVL22.101LC41

[0380] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDADGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0381] SEQ ID NO:17-hVL22.101LC42

[0382] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDNDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0383] SEQ ID NO: 18—SEQ ID NO 1 of histone 2A from WO2016092082 (used in Example 1 / 7)

[0384] SGXGKQGGKARA

[0385] Where X is citrulline

[0386] SEQ ID NO: 19—SEQ ID NO 2 of histone 4 from WO2016092082 (used in Example 7)

[0387] SGXGKGGKGLGKGGAKRHRKVLR

[0388] Where X is citrulline

[0389] SEQ ID NO: 20 - shortened SEQ ID NO 2SGXGKGGKGLGK of histone 4 from WO2016092082 (used in Example 7)

[0390] Where X is citrulline

[0391] SEQ ID NO: Peptide No. 21-4 (human histone 2A) (SEQ ID NO 24 from WO2011070172)

[0392] QFPVGXVHRLLR

[0393] Where X is citrulline

[0394] SEQ ID NO: Peptide No. 22-6: (human histone 2A) (SEQ ID NO 26 from WO2011070172)

[0395] VHRLLXKGNYSE

[0396] Where X is citrulline

[0397] SEQ ID NO: 23—Human heavy chain constant domain of IgG1

[0398] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0399] SEQ ID NO: 24 - Human κ-chain constant region

[0400] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0401] SEQ ID NO: 25 - msVH22.101

[0402] RIQLVQSGPELKKPGEAVKISCKASGYTFTNYGMHWMKQTPGKDFRWMGWINTYSGEATYVDDFKGRFAFSLGTSASTAYLQINNLKNDDTATYFCLRGYTYQSFDEGGDYWGQGTALTVSS

[0403] SEQ ID NO: 26 - hVH22.101j

[0404] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS

[0405] SEQ ID NO: 27 - hVH22.101HC7

[0406] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS

[0407] SEQ ID NO:28-hVH22.101HC8

[0408] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYVDDFQGRVTITADESTSTAYMELSSLRSEDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS

[0409] SEQ ID NO:29-hVH22.101HC10

[0410] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYVDDFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS

[0411] SEQ ID NO:30-msVL22.101

[0412] DVVMTQTPLTLSVTTGQPASISCKSSQSLLDSDGKTYLNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPYTFGGGTNLEIK

[0413] SEQ ID NO:31-hVL22.101e

[0414] DVVMTQSPLSLPVTLGQPASISCRSSQSLVDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0415] SEQ ID NO:32-hVL22.101g

[0416] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0417] SEQ ID NO: 33 - hVL22.101h

[0418] DVVMTQSPLSLPVTLGQPASISCRSSQSLVASDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0419] SEQ ID NO: 34 - hVL22.101i

[0420] DVVMTQSPLSLPVTLGQPASISCRSSQSLVESDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0421] SEQ ID NO: 35 - hVL22.101j

[0422] DVVMTQSPLSLPVTLGQPASISCRSSQSLVSSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0423] SEQ ID NO: 36 - CDR1 of msVL22.101 and hVL22.101g

[0424] QSLLDSDGKTY

[0425] SEQ ID NO: 37 - CDR1 of hVL22.101e

[0426] QSLVDSDGKTY

[0427] SEQ ID NO: 38 - CDR1 of hVL22.101h

[0428] QSLVASDGKTY

[0429] SEQ ID NO: 39—CDR1 of hVL22.101i

[0430] QSLVESDGKTY

[0431] SEQ ID NO:40—CDR1 of hVL22.101j

[0432] QSLVSSDGKTY

[0433] SEQ ID NO:41 - CDR1 of hVL22.101LC16

[0434] QSLLESDGKTY

[0435] SEQ ID NO: 42—CDR1 of hVL22.101LC19

[0436] QSLLDSEGKTY

[0437] SEQ ID NO: 43—CDR1 of hVL22.101LC20

[0438] QSLLDSSGKTY

[0439] SEQ ID NO: 44 - CDR1 of hVL22.101LC22

[0440] QSLLESEGKTY

[0441] SEQ ID NO: 45—CDR1 of hVL22.101LC23

[0442] QSLLESSGKTY

[0443] SEQ ID NO: 46—CDR1 of hVL22.101LC24

[0444] QSLLESDAKTY

[0445] SEQ ID NO: 47 - CDR1 of hVL22.101LC25

[0446] QSLLDTEGKTY

[0447] SEQ ID NO: 48—CDR1 of hVL22.101LC26

[0448] QSLLDTSGKTY

[0449] SEQ ID NO: 49—CDR1 of hVL22.101LC37

[0450] QSLLDSAGKTY

[0451] SEQ ID NO: 50—CDR1 of hVL22.101LC38

[0452] QSLLESAGKTY

[0453] SEQ ID NO: 51 - CDR1 of hVL22.101LC39

[0454] QSLLDAEGKTY

[0455] SEQ ID NO: 52—CDR1 of hVL22.101LC40

[0456] QSLLDNEGKTY

[0457] SEQ ID NO: 53-msFibβXG (SEQ ID NO 37 from WO2011070172)

[0458] EPTDSLDAXGHRPVDRR

[0459] Where X is citrulline

[0460] SEQ ID NO: 54 - msVim XS / XL (SEQ ID NO 38 from WO2011070172)

[0461] YVTXSSAVXLXSSVP

[0462] Where X is citrulline

[0463] SEQ ID NO: 55 - CDR2 vicinity of msVL22.101 and hVL22.101(LC)y

[0464] LVSKLDS

[0465] SEQ ID NO: 56—Heavy chain constant domain of hCH22.101f

[0466] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence Listing <110> MotiXster Pte Ltd <120> Antibody <130> N413524WO <150> GB 1813597.0 <151> 2018-08-21 <150> GB 1900983.6 <151> 2019-01-24 <160> 56 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR1 of msVH22.101 and hVH22.101(HC)x <400> 1 Gly Tyr Thr Phe Thr Asn Tyr Gly 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR2 of msVH22.101 and hVH22.101(HC)x <400> 2 Ile Asn Thr Tyr Ser Gly Glu Ala 1 5 <210> 3 <211> 15 <212> PRT <213> Artificial sequence <220> <223> CDR3 of msVH22.101 and hVH22.101(HC)x <400> 3 Leu Arg Gly Tyr Thr Tyr Gln Ser Phe Asp Glu Gly Gly Asp Tyr 1 5 10 15 <210> 4 <400> 4 000 <210> 5 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR3 of msVL22.101 and hVL22.101(LC)y <400> 5 Trp Gln Gly Thr His Phe Pro Tyr Thr 1 5 <210> 6 <211> 11 <212> PRT <213> Homo sapiens <400> 6 Gln Ser Leu Leu Asp Thr Asp Gly Lys Thr Tyr 1 5 10 <210> 7 <211> 11 <212> PRT <213> Homo sapiens <400> 7 Gln Ser Leu Leu Asp Ser Asp Ala Lys Thr Tyr 1 5 10 <210> 8 <211> 11 <212> PRT <213> Homo sapiens <400> 8 Gln Ser Leu Leu Asp Thr Asp Ala Lys Thr Tyr 1 5 10 <210> 9 <211> 11 <212> PRT <213> Homo sapiens <400> 9 Gln Ser Leu Leu Asp Ala Asp Gly Lys Thr Tyr 1 5 10 <210> 10 <211> 11 <212> PRT <213> Homo sapiens <400> 10 Gln Ser Leu Leu Asp Asn Asp Gly Lys Thr Tyr 1 5 10 <210> 11 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> hVH22.101f <400> 11 Arg Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Ser Gly Glu Ala Thr Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Leu Arg Gly Tyr Thr Tyr Gln Ser Phe Asp Glu Gly Gly Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 12 <211> 122 <212> PRT <213> Homo sapiens <400> 12 Arg Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Ser Gly Glu Ala Thr Tyr Val Asp Asp Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Leu Arg Gly Tyr Thr Tyr Gln Ser Phe Asp Glu Gly Gly Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 13 <211> 112 <212> PRT <213> Homo sapiens <400> 13 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Thr 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 14 <211> 112 <212> PRT <213> Homo sapiens <400> 14 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Ala Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 15 <211> 112 <212> PRT <213> Homo sapiens <400> 15 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Thr 20 25 30 Asp Ala Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 16 <211> 112 <212> PRT <213> Homo sapiens <400> 16 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ala 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 17 <211> 112 <212> PRT <213> Homo sapiens <400> 17 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Asn 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 18 <211> 12 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa = citrulline <400> 18 Ser Gly Xaa Gly Lys Gln Gly Gly Lys Ala Arg Ala 1 5 10 <210> 19 <211> 23 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa = citrulline <400> 19 Ser Gly Xaa Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly Ala Lys 1 5 10 15 Arg His Arg Lys Val Leu Arg 20 <210> 20 <211> 12 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (3) <223> Xaa = Citrulline <400> 20 Ser Gly Xaa Gly Lys Gly Gly Lys Gly Leu Gly Lys 1 5 10 <210> twenty one <211> 12 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (6) <223> Xaa = Citrulline <400> twenty one Gln Phe Pro Val Gly Xaa Val His Arg Leu Leu Arg 1 5 10 <210> twenty two <211> 12 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (6) <223> Xaa = Citrulline <400> twenty two Val His Arg Leu Leu Xaa Lys Gly Asn Tyr Ser Glu 1 5 10 <210> twenty three <211> 329 <212> PRT <213> Homo sapiens <400> 23 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 24 <211> 107 <212> PRT <213> Homo sapiens <400> 24 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 25 <211> 122 <212> PRT <213> Mus musculus <400> 25 Arg Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Ala Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met His Trp Met Lys Gln Thr Pro Gly Lys Asp Phe Arg Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Ser Gly Glu Ala Thr Tyr Val Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Gly Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Asp Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Leu Arg Gly Tyr Thr Tyr Gln Ser Phe Asp Glu Gly Gly Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ala Leu Thr Val Ser Ser 115 120 <210> 26 <211> 122 <212> PRT <213> Homo sapiens <400> 26 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Ser Gly Glu Ala Thr Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Leu Arg Gly Tyr Thr Tyr Gln Ser Phe Asp Glu Gly Gly Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 27 <211> 122 <212> PRT <213> Homo sapiens <400> 27 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Ser Gly Glu Ala Thr Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Leu Arg Gly Tyr Thr Tyr Gln Ser Phe Asp Glu Gly Gly Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 28 <211> 122 <212> PRT <213> Homo sapiens <400> 28 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Ser Gly Glu Ala Thr Tyr Val Asp Asp Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Leu Arg Gly Tyr Thr Tyr Gln Ser Phe Asp Glu Gly Gly Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 29 <211> 122 <212> PRT <213> Homo sapiens <400> 29 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Ser Gly Glu Ala Thr Tyr Val Asp Asp Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Leu Arg Gly Tyr Thr Tyr Gln Ser Phe Asp Glu Gly Gly Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 30 <211> 112 <212> PRT <213> Mus musculus <400> 30 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Thr Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Phe Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys 100 105 110 <210> 31 <211> 112 <212> PRT <213> Homo sapiens <400> 31 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 32 <211> 112 <212> PRT <213> Homo sapiens <400> 32 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 33 <211> 112 <212> PRT <213> Homo sapiens <400> 33 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ala Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 34 <211> 112 <212> PRT <213> Homo sapiens <400> 34 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Glu Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 35 <211> 112 <212> PRT <213> Homo sapiens <400> 35 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ser Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 36 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CDR1 of msVL22.101 and hVL22.101g <400> 36 Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr 1 5 10 <210> 37 <211> 11 <212> PRT <213> Homo sapiens <400> 37 Gln Ser Leu Val Asp Ser Asp Gly Lys Thr Tyr 1 5 10 <210> 38 <211> 11 <212> PRT <213> Homo sapiens <400> 38 Gln Ser Leu Val Ala Ser Asp Gly Lys Thr Tyr 1 5 10 <210> 39 <211> 11 <212> PRT <213> Homo sapiens <400> 39 Gln Ser Leu Val Glu Ser Asp Gly Lys Thr Tyr 1 5 10 <210> 40 <211> 11 <212> PRT <213> Homo sapiens <400> 40 Gln Ser Leu Val Ser Ser Asp Gly Lys Thr Tyr 1 5 10 <210> 41 <211> 11 <212> PRT <213> Homo sapiens <400> 41 Gln Ser Leu Leu Glu Ser Asp Gly Lys Thr Tyr 1 5 10 <210> 42 <211> 11 <212> PRT <213> Homo sapiens <400> 42 Gln Ser Leu Leu Asp Ser Glu Gly Lys Thr Tyr 1 5 10 <210> 43 <211> 11 <212> PRT <213> Homo sapiens <400> 43 Gln Ser Leu Leu Asp Ser Ser Gly Lys Thr Tyr 1 5 10 <210> 44 <211> 11 <212> PRT <213> Homo sapiens <400> 44 Gln Ser Leu Leu Glu Ser Glu Gly Lys Thr Tyr 1 5 10 <210> 45 <211> 11 <212> PRT <213> Homo sapiens <400> 45 Gln Ser Leu Leu Glu Ser Ser Gly Lys Thr Tyr 1 5 10 <210> 46 <211> 11 <212> PRT <213> Homo sapiens <400> 46 Gln Ser Leu Leu Glu Ser Asp Ala Lys Thr Tyr 1 5 10 <210> 47 <211> 11 <212> PRT <213> Homo sapiens <400> 47 Gln Ser Leu Leu Asp Thr Glu Gly Lys Thr Tyr 1 5 10 <210> 48 <211> 11 <212> PRT <213> Homo sapiens <400> 48 Gln Ser Leu Leu Asp Thr Ser Gly Lys Thr Tyr 1 5 10 <210> 49 <211> 11 <212> PRT <213> Homo sapiens <400> 49 Gln Ser Leu Leu Asp Ser Ala Gly Lys Thr Tyr 1 5 10 <210> 50 <211> 11 <212> PRT <213> Homo sapiens <400> 50 Gln Ser Leu Leu Glu Ser Ala Gly Lys Thr Tyr 1 5 10 <210> 51 <211> 11 <212> PRT <213> Homo sapiens <400> 51 Gln Ser Leu Leu Asp Ala Glu Gly Lys Thr Tyr 1 5 10 <210> 52 <211> 11 <212> PRT <213> Homo sapiens <400> 52 Gln Ser Leu Leu Asp Asn Glu Gly Lys Thr Tyr 1 5 10 <210> 53 <211> 17 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (9)..(9) <223> Xaa = Citrulline <400> 53 Glu Pro Thr Asp Ser Leu Asp Ala Xaa Gly His Arg Pro Val Asp Arg 1 5 10 15 Arg <210> 54 <211> 15 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (4) <223> Xaa = Citrulline <220> <221> MISC_FEATURE <222> (9)..(9) <223> Xaa = Citrulline <220> <221> MISC_FEATURE <222> (11)..(11) <223> Xaa = Citrulline <400> 54 Tyr Val Thr Xaa Ser Ser Ala Val Xaa Leu Xaa Ser Ser Val Pro 1 5 10 15 <210> 55 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDR2 vicinity of msVL22.101 and hVL22.101(LC)y <400> 55 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 56 <211> 330 <212> PRT <213> Homo sapiens <400> 56 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, wherein the antibody or antigen-binding fragment thereof comprises: a) VL CDR1 of SEQ ID NO: 9 (QSLLDADGKTY); and b) VH CDR1 of SEQ ID NO: 1 (GYTFTNYG), VH CDR2 of SEQ ID NO: 2 (INTYSGEA), VH CDR3 of SEQ ID NO: 3 (LRGYTYQSFDEGGDY), VL CDR2 of SEQ ID NO: 4 (LVS), and VL CDR3 of SEQ ID NO: 5 (WQGTHFPYT).

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: a) VL CDR1 of SEQ ID NO: 9; b) VL CDR2 of SEQ ID NO: 4 and VL CDR3 of SEQ ID NO: 5; and c) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises: a) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 and the light chain variable domain amino acid sequence of SEQ ID NO: 16; or b) the heavy chain variable domain amino acid sequence of SEQ ID NO: 12 and the light chain variable domain amino acid sequence of SEQ ID NO:

16.

4. The antibody or antigen-binding fragment thereof that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4 according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises the following CDRs: a) CDR1 present in SEQ ID NO: 16; and b) The heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12. 5 . The antibody or antigen-binding fragment thereof according to claim 1 , which specifically binds to a peptide selected from the group consisting of SEQ ID NOs: 18 and 20, and binds to deiminated human histone 2A and / or histone 4. 6 . The antibody or antigen-binding fragment thereof according to claim 1 , which specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4 with an affinity of at least 1 nM or less. 7 . The antibody or antigen-binding fragment thereof according to claim 1 , which is selected from the group consisting of a recombinant antibody, a single-chain antibody, a single-chain variable fragment (scFv), a variable fragment (Fv) and a fragment antigen-binding region (Fab). The antibody or antigen-binding fragment thereof according to claim 1 , which is a full-length antibody.

9. The antibody or antigen-binding fragment thereof according to claim 8, which comprises an Fc region.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the Fc region is an IgG1 region. The antibody or antigen-binding fragment thereof according to claim 9 , wherein the Fc region is an IgG2 region.

12. The antibody or antigen-binding fragment thereof according to claim 9, wherein the Fc region is an IgG3 region.

13. The antibody or antigen-binding fragment thereof according to claim 9, wherein the Fc region is an IgG4 region.

14. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region comprises SEQ ID NO: 23 or 56, and the light chain constant region comprises SEQ ID NO:

24.

15. The antibody or antigen-binding fragment thereof according to claim 14, wherein the antibody comprises a heavy chain variable domain amino acid sequence of SEQ ID NO: 11, a light chain variable domain amino acid sequence of SEQ ID NO: 16, a heavy chain constant region amino acid sequence of SEQ ID NO: 23 or 56, and a light chain constant region amino acid sequence of SEQ ID NO:

24.

16. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, a cloning or expression vector comprising the polynucleotide, or a host cell comprising the cloning or expression vector.

17. A method for producing an antibody or an antigen-binding fragment thereof that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, comprising culturing a host cell expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, and isolating the antibody or antigen-binding fragment thereof from the cell.

18. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 and at least one pharmaceutically acceptable diluent or carrier.

19. The pharmaceutical composition according to claim 18, further comprising another active ingredient.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or the pharmaceutical composition according to claim 18 or 19, in the manufacture of a medicament for treating or preventing rheumatoid arthritis.

21. The use according to claim 20, wherein the medicament is formulated for administration by a parenteral route of administration or by another route of administration.

22. Use according to claim 21, wherein the parenteral administration route is intravenous, subcutaneous, intraocular, intramuscular, intradermal, intraperitoneal, spinal route or by injection or infusion.

23. The use according to claim 21, wherein the other route of administration is rectal, oral, ocular, epidermal, mucosal, topical, by intracavitary infusion, intravesicular administration, or by inhalation.

24. The use according to claim 23, wherein the local route is the margin of tumor resection, intralesional, perilesional, peritumoral, paratumoral or intratumoral.

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