Antibodies binding to citrullinated histone 2A and / or 4

Abstract

The present invention provides antibodies or binding fragments thereof directed against citrulline-containing epitopes. The antibodies or binding fragments thereof described herein are useful in therapy, for example in the treatment or prevention of neutrophil extracellular trap (NET)-related pathologies.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201980070130.3 filed on August 20, 2019, and invention name “Antibodies binding to citrullinated histone 2A and / or 4”. Technical Field

[0002] 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 present invention can be used to treat or prevent NET-associated 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, Sjögren's disease, antiphospholipid syndrome, Behçet'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, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, bronchitis or other NET-associated 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, Behçet's disease, spondylitis, spondyloarthropathies, multiple system atrophy, Parkinson's disease, asthma with Lewy bodies, asthma exacerbated by allergic rhinovirus, allergic asthma, cystic fibrosis, fibrosis and idiopathic pulmonary fibrosis, dry eye, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, 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. Background Art

[0003] Inflammatory conditions, whether chronic or acute, are a significant problem in the healthcare industry. Briefly, chronic inflammation is considered to be inflammation of prolonged duration (weeks or months) in which active inflammation, tissue destruction, and attempts at healing occur simultaneously. Although chronic inflammation can occur secondary to an acute inflammatory episode, it can also begin as an insidious process that develops over time, such as due to a 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 directed against the body's own tissues (e.g., rheumatoid arthritis, systemic lupus erythematosus, vasculitis, multiple sclerosis, psoriasis).

[0004] 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 that 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 the neutrophil undergoes a process of chromatin decondensation that leads to the formation of NETs through NETosis (neutrophil net death, a form of neutrophil death).

[0005] 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, Behçet's disease, spondylitis, spondyloarthropathies, multiple system atrophy, Parkinson's disease, Lewy body dementia asthma, allergic rhinovirus-induced asthma exacerbations, cystic fibrosis, and idiopathic pulmonary fibrosis.

[0006] For example, NETs can lead to the exposure of autoantigens to the extracellular space and the subsequent production of pathological autoantibodies by the subject. Furthermore, NETs and NET remnants contain toxic histones, which can cause vascular damage and subsequent organ damage and failure. Therefore, in such diseases, interfering with NET formation and inducing the clearance of NETs and NET remnants from the circulation and tissues would have therapeutic benefits.

[0007] Neutrophils are also increasingly 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 crucial for tumor development. Research has also implicated NETs as promoters of tumor progression and metastasis. It has also been shown that neutrophils, through the production of NETs, ​​provide a scaffold and stimulus for platelet adhesion, thrombosis, and coagulation in tumors.

[0008] Furthermore, NETs have been implicated in reducing organ health after transplantation. NETs contribute to primary graft dysfunction and early mortality after lung transplantation. NETs have been shown to play a pathological role in solid organ transplantation.

[0009] Therefore, the identification of therapeutic agents that can prevent NET formation, clear NETs and / or prevent NETosis will have significant 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, Behçet'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, 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.

[0010] There remains a need for compounds useful in treating or preventing pathologies associated with NETs.

[0011] 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

[0012] 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.

[0013] The present 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 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's binding affinity to the tested histone-derived peptide to decrease over time. The inventors then conducted a thorough analysis of CDR1 light chain mutants to address the isomerization problem while attempting to retain the binding properties of the antibody. Multiple attempts resulted in antibodies with reduced binding affinity to the target peptide.

[0014] Finally, the inventors successfully identified 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.

[0015] Therefore, the present invention provides:

[0016] - 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:

[0017] 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

[0018] b) at least one CDR selected from SEQ ID NOs: 1 to 5.

[0019] The present invention also provides:

[0020] - 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:

[0021] a) CDR1 of SEQ ID NO: 13, 14, 15, 16 or 17; and

[0022] b) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12.

[0023] The present invention also provides:

[0024] - A polynucleotide encoding an antibody or binding fragment thereof as defined herein, a cloning or expression vector comprising said polynucleotide, or a host cell comprising said cloning or expression vector.

[0025] The present invention also provides:

[0026] - 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 a host cell as defined herein and isolating said antibody or binding fragment thereof from said cell.

[0027] The present invention also provides:

[0028] A pharmaceutical composition comprising an antibody or binding fragment thereof as defined herein and at least one pharmaceutically acceptable diluent or carrier.

[0029] The present invention also provides:

[0030] - An antibody or binding fragment thereof as defined herein or a pharmaceutical composition as defined herein, for use in therapy.

[0031] The present invention also provides:

[0032] - 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.

[0033] The present invention also provides:

[0034] - 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.

[0035] Brief Description of Sequence Listing

[0036] Antibody name

[0037] CDR = complementarity determining region.

[0038] VH = heavy chain variable domain.

[0039] VL = light chain variable domain.

[0040] CH = heavy chain constant domain.

[0041] CL = light chain constant domain.

[0042] msVH22.101 = mouse VH of therapeutic antibody.

[0043] msVL22.101 = mouse VL of therapeutic antibody.

[0044] hVH22.101x = humanized VH of therapeutic antibody, "x" refers to the heavy chain.

[0045] hVL22.101y = humanized VL of therapeutic antibody, "y" refers to the light chain.

[0046] hVH22.101(HC)x = optimized humanized VH of therapeutic antibody, "(HC)x" refers to the heavy chain.

[0047] hVL22.101(LC)y = optimized humanized VL of therapeutic antibody, "(LC)y" refers to the light chain.

[0048] hMQ22.101x / y = humanized therapeutic antibody, "x" refers to the heavy chain and "y" refers to the light chain.

[0049] hMQ22.101(HC)x / (LC)y=an optimized humanized therapeutic antibody of the present invention, where "(HC)x" refers to the heavy chain and "(LC)y" refers to the light chain.

[0050]

[0051] BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 - Accelerated stability test of hMQ22.101j / e and hMQ22.101f / g

[0053] 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 (Tris(hydroxymethyl)aminomethane hydrochloride) and pH 8.0 were stored at 37° C. for 8 weeks. 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).

[0054] 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.

[0055] 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.

[0056] Figures 2A-2C -Mass spectrometry analysis of hMQ22.101x / y antibodies

[0057] a) Mass spectrometry (MS) analysis of accelerated stability samples from antibody hMQ22.101j / e.

[0058] 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 aseptically removed from each glass tube weekly and stored at −80° C. until MS analysis.

[0059] 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.

[0060] b) Antigen binding assay was performed using a humanized antibody comprising an aspartic acid-mutated CDR1 of hVL22.101y.

[0061] As described in Example 1, the 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. The figure shows the optical density results for the three hVL22.101y CDR1 mutants (hVL22.101h with CDR1=DS mutation to AS; hVL22.101i with CDR1=DS mutation to ES; and hVL22.101j with CDR1=DS mutation to SS).

[0062] 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.

[0063] Figure 3A and 3B - Generation and affinity analysis of hMQ22.101 isomerization mutants

[0064] a) Table showing the 17 CDR1 mutation domains that were created for hVL22.101(LCy), and the unmutated CDR1s of hVL22.101e and hVL22.101g.

[0065] b) The graph shows the off-rates (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 off-rate indicates a higher affinity of the antibody for the antigen.

[0066] Figure 4 - Accelerated stability testing of hMQ22.101 isomerization mutants

[0067] 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 weekly 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.

[0068] 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 a percentage of Week 0 (100%) and plotted as a bar graph.

[0069] 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.

[0070] Figure 5 -Mass spectrometric analysis of hMQ22.101 isomerization mutants

[0071] 0.4 ml aliquots (glass tubes) containing the designated 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 weekly and stored at -80°C until further analysis. Mass spectrometry (MS) analysis of the VL CDR1 mutated hMQ22.101 antibodies (isomerization mutants) was performed as described in Example 2, except that accelerated stability samples from weeks 0 and 6 were used and compared to 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 the 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.

[0072] Figure 6 - Aggregation and degradation assays of preferred hMQ22.101 isomerization mutants

[0073] 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. 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. Measurements were performed on an Agilent 1200 system combined with an Agilent Zorbax GF-250 chromatographic column. Protein was detected using 240 nm 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 measured as 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.

[0074] Figure 7 - NETosis inhibition experiments using the preferred isomerization mutants hMQ22.101f / LC41 and hMQ22.101f / LC42

[0075] 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 that were 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 non-relevant antibody treatment.

[0076] Figure 8 - Dose response of hMQ22.101f / LC41, hMQ22.101f / LC42, and hMQ22.101f / g in the mouse CAIA model

[0077] The 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. On day 0, groups of 5 mice were treated with 2.8 mg of anti-collagen II antibody by intraperitoneal injection. 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; each was injected with an irrelevant isotype-matched control antibody (25 mg / kg of MQR2.201) at 6.25, 12.5 and 25 mg / kg or no antibody (placebo). The degree of swelling of the paw was scored for 2 weeks and is depicted in the figure as "mean arthritis score / mouse".

[0078] Figure 9A and 9B - In vitro NET inhibition and NET binding by hMQ22.101f / LC41

[0079] Bone marrow-derived mouse neutrophils were stimulated with A23187 to induce NET release in vitro. NET release was inhibited by hMQ22.101f / LC41, but not by MQR2.201 (Panel 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 NET clearance by macrophages (Panel B). Sytox Green was used to detect DNA, including NETs and pre-NETs, ​​while anti-hIgG was used to detect hMQ22.101f / LC41 bound to NETs and pre-NETs. Scale bar: 25 μm.

[0080] Figures 10A-10C - In vivo NET inhibition and NET binding using hMQ22.101f / LC41

[0081] 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, followed by another injection of 50 mg / kg of MQR2.201 or hMQ22.101f / LC41 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.

[0082] (Panel A) Representative images. (Panel B) Quantification of NET colocalization 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.

[0083] Scale bar: 50 μm (Panel A) or 25 μm (Panel C).

[0084] Figure 11 - NETs enriched with hMQ22.101f / LC41 are phagocytosed by mouse macrophages in vivo

[0085] A pristane-induced peritoneal cell influx mouse model was used to induce NET formation in vivo. MQR2.201 or hMQ22.101f / LC41 at 50 mg / kg was administered immediately after the injection of 500 μl of pristane oil, followed by another injection of MQR2.201 or hMQ22.101f / LC41 at 50 mg / kg 12 hours later. 24 hours later, cells were harvested and stained with nicotinate (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.

[0086] Figures 12A-12M -hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice

[0087] (Panel A) Schematic diagram of the CIA mouse model of RA. To induce chronic arthritis, mice were injected twice (days 0 and 21) with CII. Treatment began after disease onset (between days 21 and 28) when the MAS was ≥0.75. Treatment consisted of four injections (4 days apart) using a tapered dosing regimen of 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 was terminated 14 days after the initial injection. (Panel B) Mean arthritis scores (MAS) were assessed in CIA mice over 14 days (n = 10 mice per group; MQR2.201 was used for statistical significance). (Panel C) Bone lesions in the left and right hind knee and ankle joints were analyzed by X-ray 14 days after the first antibody injection (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) 14 days 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), the 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 NETs (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 scores (paw swelling) and NETs per joint. (Figure M) Significant correlation between macroscopic scores (paw swelling) and neutrophils (Ly6G) per joint. Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA using 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).

[0088] Figure 13 -hMQ22.101j / e does not bind to healthy leukocytes

[0089] 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, dendritic cells, monocytes, B cells, natural killer (NK) cells, and neutrophils, respectively. No binding of hMQ22.101j / e conjugated to HiLyte™ Fluor488 was detected to healthy, inactive T cells, B cells, monocytes, NK cells, dendritic cells, or neutrophils. Activated neutrophils (5 μM A23187 for 45 minutes), used as a positive control, showed increased binding to hMQ22.101j / e conjugated to HiLyte™ Fluor488. The results were analyzed by ordinary one-way ANOVA with Dunnett's multiple comparison test, ****P < 0.001. DETAILED DESCRIPTION

[0090] It will be appreciated that different applications of the disclosed invention may be tailored to suit 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.

[0091] In addition, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to the singular form "an antibody" includes the plural form "antibodies," etc.

[0092] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0093] 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 uses of such antibodies or binding fragments thereof, such as therapeutic uses.

[0094] 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, and their use in treating or preventing pathologies associated with NETs. The antibodies or binding fragments thereof of the present 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, Sjögren's disease, antiphospholipid syndrome, Behçet'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, 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.

[0095] Targets of the antibodies or binding fragments thereof of the present invention

[0096] Citrulline is an amino acid that is not incorporated into proteins during normal translation. However, it can be produced by post-translational modification of arginine residues by enzymes such as PAD (EC 3.5.3.15). To date, five PAD isoforms (PAD1-PAD6; "PAD4" and "PAD5" are used for the same isoform) have been identified in mammals (humans, mice, and rats), each encoded by a different gene.

[0097] 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 their own antigens to the extracellular space and subsequently produce pathological autoantibodies. NET-derived histones may be toxic to blood vessel 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 patients with systemic lupus erythematosus (SLE). NETs are also involved in metastasis and cancer progression.

[0098] The antibodies or binding fragments thereof according to the present invention specifically bind to citrullinated epitopes on deiminated human histone 2A and / or histone 4. The antibodies of the present invention may also specifically bind to citrullinated epitopes on deiminated human histone H3. In a specific embodiment, the antibodies or binding fragments thereof according to the present invention specifically bind to citrullinated epitopes on deiminated human histone 2A and / or histone 4, wherein the epitope comprises a peptide selected from SEQ ID NOs: 18, 19, 20, 21, and 22. The antibodies or binding fragments thereof may also bind to an epitope comprising a peptide of SEQ ID NOs: 53 or 54.

[0099] Antibodies or binding fragments thereof

[0100] 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".

[0101] As used herein, the antibody molecule refers to an antibody or its binding fragment. The term "antibody" as used herein generally refers to a complete (whole) antibody, i.e., an element comprising two heavy chains and two light chains. Antibodies may include other additional binding domains, such as the molecule DVD-Ig disclosed in WO 2007 / 024715, or the so-called (FabFv)2Fc described in WO2011 / 030107. Therefore, as used herein, "antibody" includes monovalent, bivalent, trivalent or tetravalent full-length antibodies.

[0102] 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 generating and manufacturing these antibody fragments are well known in the art (see, for example, 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 useful in the present invention include Fab and Fab' fragments. Multivalent antibodies may comprise multiple specificities, for example, bispecific or monospecific.

[0103] The antibody or its binding fragment can be selected from the group consisting of: single-chain antibodies, single-chain variable fragments (scFv), variable fragments (Fv), fragment antigen-binding regions (Fab), recombinant antibodies, monoclonal antibodies, fusion proteins comprising the antigenic domains of natural antibodies or aptamers, single-domain antibodies (sdAb), also known as VHH antibodies, nanobodies (camelid-derived single-domain antibodies), single-domain antibody fragments derived from shark IgNAR (called VNARs), diabodies, triabodies, anticalins, aptamers (DNA or RNA), and active ingredients or fragments thereof.

[0104] IgG1 (e.g., IgG1 / κ) antibodies having 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 kappa or lambda light chains. Similarly, all animal-derived antibodies of various isotypes can be used in the present invention. The antibodies can be full-size antibodies, or antigen-binding fragments of antibodies, including Fab, F(ab')2, single-chain Fv fragments, or single-domain VHH, VH, or VL single domains.

[0105] As used herein, the term "specifically binds to citrulline" or "specifically binds to a citrullinated epitope" means that an antibody or binding fragment thereof binds to a structure, such as a peptide containing citrulline residues, while the antibody or binding fragment thereof 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 correct circumstances so as to immunoreact with the antibody or binding fragment thereof described herein, preferably under the same circumstances as occur in the human or animal body, preferably in the context of a naturally occurring polypeptide.

[0106] The antibodies or binding fragments thereof of the present invention specifically bind to citrullinated epitopes on deiminated human histone 2A and / or histone 4. Binding of the antibodies or binding fragments thereof to citrullinated epitopes on deiminated human histone 2A and / or histone 4 prevents the formation of NETs. Citrullination of histones is associated with the formation of NETs.

[0107] The blocking of NET formation can be complete or partial. For example, the antibodies or binding fragments thereof 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, for example, by measuring NETosis in vitro (Kraaij T et.al, 2016, Autoimmun. Rev. 15, 577-584).

[0108] 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) for the antibody and its target. Similarly, the binding specificity of an antibody relative to its target can be defined by the comparative dissociation constant (Kd) of the antibody for its target compared to the dissociation constant for the antibody and another non-target molecule.

[0109] Typically, the Kd of an antibody for a target is less than 2-fold, preferably 5-fold, more preferably 10-fold, compared to the Kd of other non-target molecules (e.g., unrelated substances or accompanying substances in the environment). More preferably, the Kd will be less than 50-fold, even more preferably less than 100-fold, and still more preferably less than 200-fold.

[0110] The value of the dissociation constant can be determined directly by well-known methods and can be calculated, for example, by the method described in Catechims and Catechis WP (1984, Byte, 9, 340-362), even for complex mixtures. 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.

[0111] 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 and include, 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, for example, by Biacore. TM System analysis to evaluate.

[0112] 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.

[0113] 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).

[0114] 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 Applications" (Hurrell JGR, 1982 CRC Press).

[0115] The antibodies of the present invention or their binding fragments comprise a binding domain. The binding domain will typically 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.

[0116] Residues in antibody variable domains are generally 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.

[0117] IMGT residue designations 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 (framework or CDR structural components). The correct IMGT numbering of residues for a given antibody can be determined by aligning homologous residues in the antibody sequence with the "standard" IMGT numbered sequence.

[0118] 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).

[0119] 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).

[0120] 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.

[0121] 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).

[0122] 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 exhibits reduced isomerization, but retains the binding properties of the unmodified CDR1.

[0123] The amino acid sequences of the CDRs of VH of the specific antibodies or binding fragments 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.

[0124] 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.

[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 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.

[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 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.

[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 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.

[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: 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.

[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 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.

[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 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.

[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 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.

[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 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.

[0133] 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.

[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] 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.

[0136] The antibodies or binding fragments thereof of the present invention may include one or more of the CDR sequences of any one of the specific antibodies 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.

[0137] In addition to the VL CDR1, the antibodies or binding fragments thereof of the present invention may also include one or more VL CDR sequences of the specific antibody, and alternatively or additionally, include one or more VH CDR sequences. The antibodies or binding fragments thereof of the present invention may include one, two, or all three of the VH CDR sequences of the specific antibody or binding fragment described above, and alternatively or additionally, one, two, or all three of the VH chain CDR sequences of the specific antibody or binding fragment described above, including the VL CDR1. The antibodies or binding fragments thereof of the present invention may include all six CDR sequences of the specific antibody or binding fragment described above. For example, the antibodies of the present invention may include one of SEQ ID NOs: 6, 7, 8, 9, or 10 and one or more of SEQ ID NOs: 1, 2, 3, 4, and 5.

[0138] 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 exhibits 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 comprise 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 comprise 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 comprises 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.

[0139] 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.

[0140] Variant antibodies can 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. A "deletion" variant can 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. A "small amino acid group" can be defined as being continuous with each other, or close to but not continuous. A "substitution" variant preferably involves replacing one or more amino acids with the same number of amino acids and performing conservative amino acid substitutions. For example, an amino acid can be substituted with an alternative amino acid 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:

[0141] 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

[0142] 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, for example labeled, provided that the function of the antibody is not significantly adversely affected.

[0143] 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.

[0144] 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, for example, 75% or 80%, preferably greater than 85%, for example, greater than 90%, 95%, 96%, 97%, 98% or 99% amino acid identity with the VL and / or VH of the disclosed antibodies described herein or fragments thereof. Such amino acid identity levels may be found over the entire length of the relevant SEQ ID NO sequence or a portion of the sequence, for example, over 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

[0145] Preferably, the variant antibody comprises one or more of the CDR sequences described herein.

[0146] With respect to amino acid sequences, "sequence identity" means that the sequences have the recited values ​​when assessed using ClustalW (Thompson JD et al., 1994, Nucleic Acid Res., 22, 4673-4680) using the following parameters:

[0147] Pairwise alignment parameters-Method: slow / accurate, matrix: PAM, gap open penalty: 10.00, gap extension penalty: 0.10;

[0148] 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 that have been simply derivatized.

[0149] Thus, the present invention provides antibodies having specific VH and VL amino acid sequences and variants and fragments thereof that retain the function or activity of these VH and VL.

[0150] 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 a citrullinated epitope 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 a citrullinated epitope on deiminated human histone 2A and / or histone 4.

[0151] Polynucleotides, vectors and host cells

[0152] The present invention also encompasses polynucleotides, vectors, and expression vectors encoding the antibodies or binding fragments thereof described herein.

[0153] The present invention also relates to polynucleotides encoding antibodies of the present invention. Thus, 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 analogs thereof. 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.

[0154] A nucleic acid sequence that "encodes" 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. A transcription termination sequence may be located 3' to the coding sequence. In one embodiment, the polynucleotides of the present invention comprise a sequence encoding a VH or VL amino acid sequence as described above. As disclosed herein, the polynucleotides may encode a VH or VL sequence of a specific antibody or binding fragment thereof.

[0155] Therefore, the antibodies of the present invention or their binding fragments can be produced or delivered in the form of polynucleotides that encode them and can express them. When the antibody comprises two or more chains, the polynucleotides of the present invention can encode one or more antibody chains. For example, the polynucleotides 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.

[0156] 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).

[0157] Nucleic acid molecules of the present invention can provide in the form of expression cassette (expression cassette), and expression cassette comprises the control sequence that is operably connected to insertion sequence, thereby allows expressing antibody of the present invention in vivo.These expression cassettes are provided in carrier (for example plasmid or recombinant viral vector) conventionally again.This expression cassette can be directly applied to host experimenter.Or, can be applied to host experimenter by the carrier that comprises polynucleotide of the present invention.Preferably, use genetic vector to prepare and / or apply polynucleotide.Suitable carrier can be any carrier that can carry enough genetic information and allow expression polypeptide of the present invention.

[0158] 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, can 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 respect, 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).

[0159] 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 (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.

[0160] It is also well known in the art that one can 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.

[0161] 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 examples of cells that can be modified by insertion of 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 for mature glycosylation.

[0162] Such cell lines of the present invention can be cultured using conventional methods to produce the antibodies or binding fragments thereof of the present invention, or can be used therapeutically or prophylactically to deliver the antibodies or binding fragments thereof of the present invention to a subject. Alternatively, the polynucleotides, expression cassettes or vectors of the present invention can be administered to cells ex vivo from a subject and then the cells returned to the subject.

[0163] 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 binding fragment thereof from the cell.

[0164] Pharmaceutical composition

[0165] The present invention encompasses pharmaceutical compositions comprising the antibodies of the present invention or binding fragments thereof. The present invention encompasses pharmaceutical compositions comprising the antibodies of the present invention or binding fragments thereof and a pharmaceutically acceptable carrier.

[0166] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for parenteral administration (e.g., by injection or infusion), such as intravenous, intraocular, intramuscular, subcutaneous, intradermal, or intraperitoneal administration. In certain embodiments, the pharmaceutically acceptable carrier comprises 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 polymeric 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 further embodiments, 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, pentosans, keratan, alginates, cyclodextrins, and their salts and derivatives, including their esters and sulfates.

[0167] Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. The example of the suitable aqueous carrier that can be used in the pharmaceutical composition of the present invention includes water, buffered water and normal saline. The example of other carriers includes 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 keeping required particle size in the case of dispersion, and by using surfactants, suitable fluidity can be maintained. In many cases, it is preferred to include isotonic agents in the composition, such as sugar, polyols, such as mannitol, sorbitol or sodium chloride.

[0168] The pharmaceutical compositions of the present invention may also contain a pharmaceutically acceptable antioxidant. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Protection against the presence of microorganisms can be ensured by the above-mentioned sterilization procedures and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be necessary to include isotonic agents, such as sugars, sodium chloride, and the like, in the composition. In addition, absorption of the injectable drug form can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0169] 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.

[0170] Sterile injectable 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 ingredients as needed) 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 ingredients listed above. For sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying (lyophilization) to produce a powder of the active agent plus any other desired ingredients from a previously sterile-filtered solution thereof.

[0171] 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.

[0172] Depending on the route of administration, the antibody or binding fragment thereof can be coated in a material to protect the antibody from acids and other natural conditions that may inactivate or denature the antibody.

[0173] 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.

[0174] In a further embodiment, pharmaceutical compositions as herein described can be administered by, for example, 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.

[0175] Kits comprising the antibodies or other compositions of the invention and instructions for use are also within the scope of the invention. The kits may further comprise one or more other agents, such as other therapeutic or prophylactic agents described herein.

[0176] Therapeutic uses of the antibodies and binding fragments thereof of the present invention

[0177] The 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 disease or its symptoms. This therapeutic treatment may result in a decrease in the severity of the 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.

[0178] In certain embodiments, the antibodies or binding fragments thereof of the present 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 present invention may be bispecific antibodies.

[0179] 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. 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 carbodiimides, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) are particularly useful for coupling functional moieties to binding moieties.

[0180] Other methods can also be used to couple a moiety to the antibody. For example, sodium periodate oxidation can be used, followed by reductive alkylation with a suitable reactant, or glutaraldehyde cross-linking can 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 relevant function.

[0181] 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.

[0182] The antibody can be linked to a detectable label. A "detectable label" refers to an antibody linked to a moiety that, when located at the target site after administration of the antibody to a patient, can be detected non-invasively, typically from outside the body and at the target site. Thus, the antibody can be used for imaging and diagnosis.

[0183] 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. Obviously, a sufficient number of appropriate atomic isotopes must be attached to the antibody to make the molecule easy to detect.

[0184] 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 the polypeptide. 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.

[0185] In one embodiment, the antibodies of the invention are capable of selectively binding directly or indirectly to a cytotoxic moiety or a detectable label. Thus, in this embodiment, the antibody is linked to a moiety that selectively binds to a cytotoxic or other compound or component that is readily detectable.

[0186] The antibody or binding fragment of the present invention, or the composition comprising the antibody or fragment, can be administered by one or more of the various methods known in the art through one or more routes of administration. As recognized by those skilled in the art, the route of administration and / or mode will vary according to the desired result. Preferred routes of administration for the antibody or composition of the present invention include intravenous, subcutaneous, intraocular, intramuscular, intradermal, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, typically 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 suction 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.

[0187] The appropriate dosage of the antibody of the present invention or its binding fragment can be determined by a skilled medical practitioner. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that achieves the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the specific antibody used, route of administration, time of administration, antibody excretion rate, duration of treatment, other drugs, compounds, and / or materials used in conjunction with the specific composition used, the age, sex, weight, condition, overall health, and previous medical history of the treated patient, and similar factors well-known in the medical field.

[0188] Suitable dosages of the antibodies or binding fragments thereof 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.

[0189] 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.

[0190] The dosage regimen can be adjusted to provide the optimal 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 a physically discrete unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect together with the desired pharmaceutical carrier.

[0191] 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's body 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, a relatively low dose can be administered at relatively infrequent intervals over a long period of time. In therapeutic applications, a relatively high dose can be administered, for example, until the patient shows partial or complete improvement in the symptoms of the disease.

[0192] 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 separate compositions as part of a combination therapy. For example, the antibody or its binding fragment can be administered before, after, or simultaneously with the other agent.

[0193] Disease to be treated

[0194] 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.

[0195] 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.

[0196] The present 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.

[0197] The present invention also encompasses pharmaceutical compositions comprising the antibodies of the present invention or binding fragments thereof for use in treating or preventing pathologies associated with citrullination, such as NET-associated pathologies and inflammatory disorders.

[0198] 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 readily determined by a skilled artisan 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 using immunological tests such as Western blotting or ELISA, where the affected tissue is used as an antigen and citrullination of the antigen can be detected using anti-citrulline antibodies as described herein. Alternatively, one skilled in the art can use proteomic applications, such as mass spectrometry, to compare the level and type of citrullination in diseased tissue versus healthy tissue from an affected patient.

[0199] NET-related pathologies can be considered citrullination-related pathologies. A NET-related pathology 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 NET formation and NETosis play 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.

[0200] Therefore, the present invention relates to antibodies or binding fragments thereof for use in treating or preventing pathologies associated with NETs.

[0201] Thus, the present invention relates to methods of treating a patient in need thereof with a therapeutically effective amount of an antibody or binding fragment thereof of the present invention, wherein the patient suffers from a pathology associated with NETs.

[0202] Examples of NET-associated pathologies include inflammatory conditions or diseases, ocular inflammatory diseases, autoimmune diseases, cancer, and post-transplant organ health.

[0203] "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 response); infiltration of tissue by monocytes; destruction of tissue by inflammatory cells, connective tissue cells, and their cell products; and attempted repair by connective tissue replacement (e.g., chronic inflammatory response). 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, dementia with Lewy bodies, idiopathic pulmonary fibrosis, dry eye, 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.

[0204] NETs play a role in the pathology of autoimmune diseases including RA, SLE, and vasculitis. Therapeutic antibodies or their binding fragments may ameliorate disease by inhibiting NETosis, by clearing NET remnants (including toxic histones) and other autoantigens from tissues and circulation, and by clearing 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, suggesting a close correlation between the number of NETs and disease severity.

[0205] Thus, inflammatory conditions or diseases, as well as autoimmune diseases, can be treated by the antibodies and binding fragments thereof of the present invention.

[0206] 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, Behçet'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, 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.

[0207] 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, Behçet'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, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, bronchitis.

[0208] Further embodiments

[0209] The present invention is further described by the following examples:

[0210] 1. 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:

[0211] 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

[0212] b) at least one CDR selected from SEQ ID NOs: 1 to 5.

[0213] 2. The antibody or binding fragment thereof according to 1, wherein the antibody or binding fragment thereof comprises:

[0214] 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

[0215] b) CDRs of SEQ ID NO: 3 and SEQ ID NO: 5.

[0216] 3. The antibody or binding fragment thereof according to 2, wherein the antibody or binding fragment thereof comprises:

[0217] a) one of the CDRs of SEQ ID NOs: 6, 7, 8, 9, and 10; and

[0218] b) CDRs of SEQ ID NO: 3 and SEQ ID NO: 5.

[0219] 4. The antibody or binding fragment thereof according to 2, wherein the antibody or binding fragment thereof comprises:

[0220] 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

[0221] b) CDRs of SEQ ID NOs: 1 to 5.

[0222] 5. The antibody or binding fragment thereof according to any one of the preceding examples, wherein the antibody or binding fragment thereof comprises:

[0223] a) one of the CDRs of SEQ ID NOs: 6, 7, 8, 9, and 10;

[0224] b) CDRs of SEQ ID NOs: 1 to 5.

[0225] 6. The antibody or binding fragment thereof according to 1 or 2, wherein the antibody or binding fragment thereof comprises:

[0226] 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);

[0227] b) at least one of the CDRs of SEQ ID NOs: 4 and 5; and

[0228] c)

[0229] i) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12; or

[0230] 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

[0231] 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.

[0232] 7. The antibody or binding fragment thereof according to 6, wherein the antibody or binding fragment thereof comprises:

[0233] 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);

[0234] b) at least one of the CDRs of SEQ ID NOs: 4 and 5; and

[0235] c) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12.

[0236] 8. The antibody or binding fragment thereof according to 7, wherein the antibody or binding fragment thereof comprises:

[0237] 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);

[0238] b) the CDRs of SEQ ID NOs: 4 and 5; and

[0239] c) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12.

[0240] 9. The antibody or binding fragment thereof according to 8, wherein the antibody or binding fragment thereof comprises:

[0241] a) one of the CDRs of SEQ ID NOs: 6, 7, 8, 9, and 10;

[0242] b) the CDRs of SEQ ID NOs: 4 and 5; and

[0243] c) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12.

[0244] 10. The antibody or binding fragment thereof according to any one of the preceding examples, wherein the antibody or binding fragment thereof comprises:

[0245] 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;

[0246] 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;

[0247] 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;

[0248] 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;

[0249] 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;

[0250] 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;

[0251] 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;

[0252] 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;

[0253] 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

[0254] 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.

[0255] 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:

[0256] a) CDR1 of SEQ ID NO: 13, 14, 15, 16 or 17; and

[0257] b) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12.

[0258] 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.

[0259] 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.

[0260] 14. The antibody or binding fragment thereof according to any one of the preceding examples is selected from a recombinant antibody, a single-chain antibody, a single-chain variable fragment (scFv), a variable fragment (Fv), a fragment antigen-binding region (Fab), a single-domain antibody (sdAb), a VHH antibody, a nanobody, a single-domain antibody derived from camelids, a single-domain antibody fragment (VNAR) derived from shark IgNAR, a diabody, a triabody, an anti-lipid and an aptamer.

[0261] 15. The antibody or binding fragment thereof according to any one of 1 to 13, wherein the antibody is preferably a full-length antibody.

[0262] 16. The antibody or binding fragment thereof according to 15, comprising an Fc region, such as an IgG1, IgG2, IgG3 or IgG4 region.

[0263] 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.

[0264] 18. The antibody or binding fragment thereof according to any of the preceding examples, coupled to an additional moiety.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] 22. The pharmaceutical composition according to 21, further comprising other active ingredients.

[0269] 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.

[0270] 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.

[0271] 25. The use of the antibody, binding fragment thereof or 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, Sjögren's disease, antiphospholipid syndrome, Behçet'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, 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.

[0272] 26. The use of an antibody, a binding fragment thereof or a pharmaceutical composition for use according to any one of items 23 to 25, wherein the antibody, the binding fragment thereof or the 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.

[0273] 27. A method of 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.

[0274] 28. The method of 27, wherein the treatment is a NET-related pathology.

[0275] 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.

[0276] Example

[0277] Example 1: Accelerated stability test of hMQ22.101j / e and hMQ22.101f / g.

[0278] 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 were stored for 8 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 (ELISA and mass spectrometry).

[0279] 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 five 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 with PBS-T five more times, 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 five 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 1350 ng / well and then diluting 1:1 to a concentration of 0.66 ng / 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 high (HQC, 250 ng / ml), mid (MQC, 50 ng / ml), low (LQC 3, 75 ng / ml), and a lower limit quality control (LLQC, 1.25 ng / ml). These samples were diluted in PBS-T + 0.2% BSA and also added to the plate. Use these QC samples to validate the ELISA results.

[0280] 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 terminating the reaction with 2M H2SO4, the wells were incubated with TMB substrate for 10 minutes, and the optical density was measured at a wavelength of 450 nm. A sigmoidal 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 sigmoidal 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).

[0281] Accelerated stability testing showed that the binding affinity of hMQ22.101j / e and hMQ22.101f / g for histone-derived citrulline-containing peptides decreased over time.

[0282] Example 2: Mass spectrometric analysis of hMQ22.101j / e accelerated stability samples.

[0283] The reasons for the decrease in binding affinity of the hMQ22.101j / e antibody over time were investigated. hMQ22.101j / e has multiple potential aspartate isomerization sites in or near the VLCDR regions (CDR1 and CDR2). The purpose of this example was to determine the sensitivity of aspartate residues to isomerization by liquid chromatography (LC)-mass spectrometry (MS)-based peptide mapping.

[0284] Prior to 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 the tryptic digests was performed using reversed-phase liquid chromatography (RPLC) combined with UV and mass spectrometry detection (RPLC-UV-MS). Data were acquired using an Agilent Technologies 1290UHPLC system hyphenated to an Agilent Technologies 6540Q-TOF equipped with a jetstream electrospray ionization (ESI) source. Before UV 214nm and MS ( / MS) detection, use water, trifluoroacetic acid and acetonitrile as mobile phase components, on RPLC column (AdvanceBio peptide map C18, 250mm L, 2.1mm ID, 2.7 μ m dp, Agilent Technologies) separate sample.The amount of about 4.5 μ g is loaded on the chromatographic column. The MS system is operated under extended dynamic range mode (2GHz), and the resolution of the operation to mass 922.009798 is 20,000, and when not using reference mass, has higher mass accuracy (usually <10ppm). Acquire 2 spectra per second, and the acquisition range is 100-3000amu under MS and MS / MS mode. MS / MS data are obtained with data dependent mode. Collision energy has been optimized for peptide fragments. All MS measurements are carried out in positive ionization mode.

[0285] The signal measured was matched to the sequence using the Agilent MassHunter software comprising the BioConfirm algorithm. The mass tolerance for experimental data and sequence matching was set to 20ppm. The specified enzyme was trypsin (C-terminal cleavage of lysine or arginine), allowing 0-2 deletion cleavages. The peak area of ​​the extracted ion chromatogram obtained under 20ppm mass accuracy was used to quantify the modification. Taking into account the nearly complete sequence coverage, all candidate aspartate isomerization sites in the hCDR region were covered. These peptides were manually integrated. When present, peptides containing isoaspartate were eluted, followed by peptides containing aspartate. The relative isomerization level was then calculated in each case.

[0286] The relative aspartate (D) isomerization level of VL CDR1 of hMQ22.101j / e increased 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.

[0287] Example 3: Generation of hMQ22.101 antibody with three VL CDR1 aspartate mutations.

[0288] We then investigated whether deletion of the non-germline isomerization site in the VL CDR1 prevented isomerization. DNA containing three hVL22.101y domains containing a single aspartate mutation at each amino acid position 31 (L:Asp31) was synthesized by GeneArt. L:Asp31 was mutated to alanine, glutamine, or serine based on amino acid similarity, such as 1) size, 2) polarity, and 3) charge. 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 the 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 the culture supernatant 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 using 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, the antibodies were concentrated using a MicroSep Advance Centrifugal Device (10K MWCO).

[0289] Example 4: Antigen binding assay using hMQ22.101 antibody with VL CDR1 aspartic acid mutation.

[0290] As described in Example 1, the generated VL CDR1 aspartate-mutated antibodies hMQ22.101j / h, hMQ22.101j / i, and hMQ22.101j / j were compared to 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 ).

[0291] 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 achieved the highest optical density improvement.

[0292] Example 5: Mass spectrometry analysis of hMQ22.101j / i with VL CDR1 aspartate mutations after accelerated stability testing.

[0293] 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).

[0294] Mass spectrometry analysis was performed identically to that described in Example 2, except that only the accelerated stability samples from week 0 and week 4 were used. The isomerization percentages of hMQ22.101j / i were compared to those of the antibody hMQ22.101j / e ( Figure 2C ).

[0295] 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 largely resolve the isomerization problem.

[0296] 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.

[0297] Example 6: Generation of additional hMQ22.101 isomerization mutants.

[0298] A comprehensive mutational analysis of the isomerization sites in the VL CDR1 was then performed to investigate whether it was possible to remove the isomerization of 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.

[0299] 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, hVL22.101LC39, hVL22.101LC40, hVL22.101LC41, hVL22.101LC42 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.

[0300] Example 7: Dissociation rate analysis of hMQ22.101 isomerization mutants.

[0301] Off-rate screening of isomerization mutant antibodies was performed on an Octet RED96 instrument (Pall ForteBio). All measurements were performed at 30°C. Streptavidin (SA) biosensors were first washed with PBS for 50 seconds. 1 μg / 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 biosensors 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 washes in PBS, antibodies diluted in PBS at a concentration of 72 nM were bound to the biosensors for 200 seconds. The sensors were then placed in PBS for 4000 seconds to measure their off-rates.

[0302] Before plotting the dissociation curves for each antibody, the background signal generated by the uncoated biosensor exposed to each antibody and the signal generated by the coated biosensor 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, full fit).

[0303] The results are shown in Figure 3B Medium. Lower numbers indicate slower off-rates, meaning slower release of the antigen. 1×E-07 1 / s is the minimum value detected by Octet, meaning there is almost no measurable off-rate.

[0304] Several hMQ22.101 isomerization mutants exhibited 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.

[0305] Example 8: Accelerated stability testing of 9 best hMQ22.101 isomerization mutants.

[0306] 0.4 ml aliquots (glass tubes) of the mutant antibodies selected from the following (ranging from 2.06 to 4.29 mg / ml) in 25 mM Tris-HCl (pH 8.0) were each stored at 37°C for 6 weeks.

[0307] 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

[0308] 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).

[0309] 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 spiked QC samples.

[0310] 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, and hMQ22.101HC9 / LC42, boxed) were used to assess isomerization at weeks 0 and 6 by MS analysis.

[0311] Example 9: Mass spectrometric analysis of the five best hMQ22.101 isomerization mutants.

[0312] 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 the VL by MS analysis.

[0313]

[0314]

[0315] MS analysis was performed identically to that 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 to that of the 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 the HC chain f is more humanized than the HC9 and the difference in isomerization between week 0 and week 6 is smaller (1.9% vs. 2.6%).

[0316] Example 10: Aggregation and degradation analysis of the three best performing hMQ22.101 isomerization mutants.

[0317] Accelerated stability samples from three antibodies at 37°C showed less isomerization in the CDR1 of their VL (Example 6) and were further analyzed for aggregation and degradation levels.

[0318] hMQ22.101f / LC41 hMQ22.101f / LC42 hMQ22.101HC9 / LC42

[0319] 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 shoulder peaks before and after the main peak were quantified, and the brightness of the shoulder peaks was the aggregation and degradation levels, respectively. The results are shown in Figure 2. Figure 6 shown.

[0320] hMQ22.101f / LC41, hMQ22.101f / LC42, and hMQ22.101HC9 / LC42 showed acceptable aggregation and degradation profiles, indicating that they were acceptable for further development.

[0321] Example 11: Fragmentation analysis of the best performing hMQ22.101 isomerization mutants.

[0322] The analysis of complete mAb samples was carried out using reversed phase liquid chromatography (RP-HPLC) in conjunction 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 6540Q-TOF character equipped with a jet electrospray ionization (ESI) source. Samples were separated on a RPLC column (Zorbax300SB-C8, 100mm L, 2.1mm ID, 1.8 μm dp, Agilent Technologies), using 0.1% TFA in water as mobile phase A and 0.1% TFA in acetonitrile as mobile phase B. A concentration gradient of 15% to 80% B was applied in 65 minutes. Approximately 5 μg of sample was loaded onto the column. The MS system operated in high resolution mode (4 GHz), with a fragmentor voltage of 350 V and a Quad AMU setting of 300. One spectrum was acquired per second in positive MS mode, with an acquisition range of 300–3200 amu. Raw spectra were deconvoluted using the maximum entropy algorithm, which is integrated into Agilent Technologies MassHunter software with the BioConfirm accessory. The measured molecular weight was compared with the theoretical molecular weight determined from the full sequence, taking into account potential C-terminal lysine truncation and N-glycosylation.

[0323] Using RP-HPLC-UV-MS analysis, increased fragmentation was observed in 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.

[0324] Example 12: Human neutrophil extracellular trap assay.

[0325] Whole blood from 2 different healthy donors was collected in sodium heparin tubes (Beckton Dickinson). 30ml of each donor's blood was mixed with 15ml of 6% dextran in 0.9% NaCl and incubated at room temperature for 60 minutes. After incubation, 2 clear layers were observed, 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 room temperature at 300g for 10 minutes. The pellet was resuspended in 25ml of 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. The cells were counted using a Guava EasyCyte flow cytometer. 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 (Life Technologies) containing glutamine) 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 carefully twice using NET assay buffer. Extracellular DNA was then digested with S7 nuclease (7.5U / 0.5ml) at 37°C for 15 minutes, and then 10μl of 500mM EDTA was added to stop further digestion. NETs were collected from the wells and spun down at 20g for 5 minutes to remove intact cells. The number of NETs was quantified by measuring MPO activity in the samples by adding 50μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate to 50μl of harvested NETs. After incubation at room temperature for 10 minutes, 50μl of 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 ).

[0326] Surprisingly, the development candidate hMQ22.101f / LC41 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.

[0327] Example 13: Experimental mouse model for inflammation.

[0328] The goal of this study was to test a dose-response range using the designated development candidates hMQ22.101f / LC41 or hMQ22.101f / LC42 (in which isomerization issues are eliminated) compared to the earlier candidate hMQ22.101f / g and the isotype-matched control antibody MQR2.201 in a mouse model of collagen antibody-induced arthritis (CAIA). Paw and ankle swelling were quantified.

[0329] 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 cocktail 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 the mice. Simultaneously with LPS, the mice received tACPAs hMQ22.101f / g, hMQ22.101f / LC41, or hMQ22.101f / LC42 (6.25, 12.5, and 25 mg / kg), an unrelated isotype-matched control antibody MQR2.201 (25 mg / kg), or a placebo (0.9% NaCl in saline). Typically, inflammation of the forepaws and hind paws was visible starting 2 days after LPS injection (i.e., day 5). Macroscopic scoring of the paw swelling was performed starting on day 0 and continued for 13 days. The maximum swelling score (divided among the 4 paws) was 8. See the table below for the scoring system.

[0330]

[0331]

[0332] The results are shown in Figure 8 The paws of mice treated with the therapeutic antibody showed significantly reduced inflammation in a dose-dependent manner compared to mice treated with a control antibody or saline solution. The optimized lead antibodies hMQ22.101f / LC41 and hMQ22.101f / LC42 (in which the isomerization issue is eliminated) both prevented inflammation better than the previous lead candidate hMQ22.101f / g, as clearly demonstrated 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 outperformed 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.

[0333] Example 14: Further characterization of development candidate hMQ22.101f / LC41 in a mouse in vitro NET assay.

[0334] To further strengthen the concept 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 were studied as follows. Pre-NETs are defined as neutrophils with an amorphous, decondensed nuclear structure containing citrullinated chromatin that remains within the cell, while the nuclear membrane collapses.

[0335] 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 a mouse neutrophil enrichment kit (Stemcell Technologies). The purity of the isolated neutrophils was assessed by flow cytometry using a Ly6G antibody (Biolegend) and was found to be 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 the 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 12 hours. 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 for 12 hours at 4°C. 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 (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 ( Figure 9A Representative images showing hMQ22.101f / LC41 binding (hIgG; red) to NETs (yellow arrows) and pre-NETs (white arrows) are shown in Figure 5. Figure 9B Displayed in.

[0336] In vitro treatment of mouse bone marrow (BM)-derived neutrophils with hMQ22.101f / LC41 resulted in a reduction in A23187-induced NET extrusion compared to MQR2.201-treated mouse BM-derived neutrophils ( Figure 9A In addition, hMQ22.101f / LC41 was expressed in the mouse NETs ( Figure 9B ; yellow arrows) and anterior NET ( Figure 9B ; white arrows), which may be the first step in the clearance of NETs by macrophages.

[0337] Example 15: Further characterization of the development candidate hMQ22.101f / LC41 in an in vivo NET / macrophage assay using a pristane-induced peritonitis mouse model

[0338] Used previously by The ability of the developed candidate hMQ22.101f / LC41 to inhibit NET formation in vivo was tested in a pristane-induced peritoneal cell influx mouse model described by et al. (JCI Insight 2017;2(1):e92920).

[0339] Briefly, 50 mg / kg MQR2.201 or hMQ22.101f / LC41 was administered immediately after the injection of 500 μl of 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 the inflammatory cell count was adjusted to 1 × 10 6 Cells / ml were plated 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 three times with PBS and Cy5-conjugated goat anti-rabbit IgG secondary antibody (Jackson ImmunoResearch, 111-175-144; 1:400) was added for 1.5 hours at room temperature in the dark. The slides were washed again with PBS three times. A PBS staining solution containing 2.5 μM nicotinate was added for 15 minutes at room temperature. After washing with PBS, the samples were embedded in a mounting medium (BIOZOL). The slides were analyzed on a BZ-X710 microscope (Keyence). Figure 10A and C), and NET and pre-NET were quantitatively analyzed using Fiji imaging software ( Figure 10B ). Figure 10C Binding of hMQ22.101f / LC41 to NETs and pre-NETs is shown. Figure 11 The uptake of hMQ22.101f / LC41-enriched NETs by macrophages is shown.

[0340] 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 ( Figure 10AQuantification of NETs (colocalization of citH3 and DNA (nicotinate)) confirmed this observation ( Figure 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 ( Figure 10C ), which may be the first step in the clearance of NETs by macrophages. Indeed, F4 / 80-positive macrophages were observed in cell infiltrates containing phagocytosed hMQ22.101f / LC41 in combination with citH3 or neutrophil elastase ( Figure 11 ).

[0341] Example 16: CIA Mouse Model of RA

[0342] 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.

[0343] 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 of bovine CII. On day 21, mice received an intraperitoneal booster injection of 50 μg of bovine CII dissolved in PBS, and arthritis developed a few days later ( Figure 12A Mice were considered to have arthritis when significant changes in redness and / or swelling were noted in the paws or other areas. Joint inflammation in each paw was scored as described above (CAIA mouse model of RA). Treatment was initiated after disease onset (between 21 and 28 days) when the mean arthritis score (MAS) was ≥ 0.75 (0-2 per paw) on an arbitrary scale of 0-8. Therapeutic administration was performed with 4 repeated intravenous injections spaced 4 days apart, with the designated 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. Figure 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.

[0344] Notably, all hMQ22.101j / e treatments prevented disease progression within the first eight days, after which the 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 a MAS no greater than 0.75.

[0345] To further investigate the effect of tACPA on bone damage, X-rays of the knees and ankles of all hind paws were performed following hMQ22.101j / e and MQR2.201 treatment regimens. Consistent with the observed MAS, all hMQ22.101j / e treatments inhibited bone damage in the ankles and knees ( Figure 12C To further understand the protective effect of tACPA, histological analysis of ankle joints was performed using H&E and Safranin O (SO) staining. Compared with mice treated with MQR2.201, hMQ22.101j / e inhibited the influx of inflammatory cells ( Figure 12D Furthermore, hMQ22.101j / e significantly reduced bone and cartilage erosion, as well as cartilage proteoglycan depletion and chondrocyte death, compared with MQR2.201-treated mice ( Figure 12E These data suggest that tACPA can strongly alleviate arthritis symptoms, including joint damage.

[0346] We then investigated the presence of neutrophils and NETs in the paws of CIA mice that received 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 virtually absent in hMQ22.101j / e-treated mice ( Figure 12I DAPI was used as a nuclear and extracellular DNA stain ( Figure 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 tarsometatarsal joints. Neutrophils ( Figure 12J ) and NET( Figure 12K We found that the amount 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.

[0347] Example 17: hMQ22.101j / e does not bind to healthy leukocytes

[0348] 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) dextran in 0.9% NaCl and incubated at room temperature for 25 minutes. Subsequently, neutrophils were collected, exposed to ammonium chloride (ACK) buffer for 10 minutes at room temperature to dissolve the remaining red blood cells, and washed 2 times with RPMI 10%.

[0349] PBMCs and neutrophils were cultured at 2 × 10 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) at room temperature for 20 minutes to block Fc receptors. Subsequently, PBMCs were incubated with an antibody cocktail 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 for 45 minutes at room temperature, while neutrophils were incubated with an antibody cocktail 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 Fc receptor blocking. 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.

[0350] HiLyte™ 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 ( Figure 13 ). The hMQ22.101f / LC41 antibody is expected to have comparable results.

[0351] Sequence Listing

[0352] SEQ ID NO: 1 - CDR1 of msVH22.10 and hVH22.101(HC)x

[0353] GYTFTNYG

[0354] SEQ ID NO: 2 - CDR2 of msVH22.101 and hVH22.101(HC)x

[0355] INTYSGEA

[0356] SEQ ID NO: 3 - CDR3 of msVH22.101 and hVH22.101(HC)x

[0357] LRGYTYQSFDEGGDY

[0358] SEQ ID NO:4 - CDR2 of msVL22.101 and hVL22.101(LC)y

[0359] LVS

[0360] SEQ ID NO: 5 - CDR3 of msVL22.101 and hVL22.101(LC)y

[0361] WQGTHFPYT

[0362] SEQ ID NO: 6 - CDR1 of hVL22.101LC17

[0363] QSLLDTDGKTY

[0364] SEQ ID NO: 7 - CDR1 of hVL22.101LC21

[0365] QSLLDSDAKTY

[0366] SEQ ID NO:8—CDR1 of hVL22.101LC27

[0367] QSLLDTDAKTY

[0368] SEQ ID NO:9—CDR1 of hVL22.101LC41

[0369] QSLLDADGKTY

[0370] SEQ ID NO: 10—CDR1 of hVL22.101LC42

[0371] QSLLDNDGKTY

[0372] SEQ ID NO: 11-hVH22.101f

[0373] RIQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS

[0374] SEQ ID NO: 12-hVH22.101HC9

[0375] RIQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYVDDFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS

[0376] SEQ ID NO:13-hVL22.101LC17

[0377] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDTDGKTYLNWFQQRPGQSPRRLIYLVSKL DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0378] SEQ ID NO:14-hVL22.101LC21

[0379] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDAKTYLNWFQQRPGQSPRRLIYLVSKL DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0380] SEQ ID NO:15-hVL22.101LC27

[0381] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDTDAKTYLNWFQQRPGQSPRRLIYLVSKL DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0382] SEQ ID NO:16-hVL22.101LC41

[0383] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDADGKTYLNWFQQRPGQSPRRLIYLVSKL DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0384] SEQ ID NO:17-hVL22.101LC42

[0385] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDNDGKTYLNWFQQRPGQSPRRLIYLVSKL DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0386] SEQ ID NO: 18—SEQ ID NO 1 of histone 2A from WO2016092082 (used in Example 1 / 7)

[0387] SGXGKQGGKARA

[0388] Where X is citrulline

[0389] SEQ ID NO: 19—SEQ ID NO 2 of histone 4 from WO2016092082 (used in Example 7)

[0390] SGXGKGGKGLGKGGAKRHRKVLR

[0391] Where X is citrulline

[0392] SEQ ID NO: 20 - shortened SEQ ID NO 2 of histone 4 from WO2016092082 (used in Example 7)

[0393] SGXGKGGKGLGK

[0394] Where X is citrulline

[0395] SEQ ID NO: Peptide 21-4 (human histone 2A) (SEQ ID NO 24 from WO2011070172)

[0396] QFPVGXVHRLLR

[0397] Where X is citrulline

[0398] SEQ ID NO: 22-6 peptide: (human histone 2A) (SEQ ID NO 26 from WO2011070172)

[0399] VHRLLXKGNYSE

[0400] Where X is citrulline

[0401] SEQ ID NO: 23—Human heavy chain constant domain of IgG1

[0402] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0403] SEQ ID NO: 24 - Human κ-chain constant region

[0404] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0405] SEQ ID NO: 25 - msVH22.101

[0406] RIQLVQSGPELKKPGEAVKISCKASGYTFTNYGMHWMKQTPGKDFRWMGWINTYSGEATYVDDFKGRFAFSLGTSASTAYLQINNLKNDDTATYFCLRGYTYQSFDEGGDYWGQGTALTVSS

[0407] SEQ ID NO: 26 - hVH22.101j

[0408] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS

[0409] SEQ ID NO: 27 - hVH22.101HC7

[0410] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS

[0411] SEQ ID NO:28-hVH22.101HC8

[0412] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYVDDFQGRVTITADESTSTAYMELSSLRSEDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS

[0413] SEQ ID NO:29-hVH22.101HC10

[0414] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYVDDFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS

[0415] SEQ ID NO:30-msVL22.101

[0416] DVVMTQTPLTLSVTTGQPASISCKSSQSLLDSDGKTYLNWLFQRPGQSPKRLIYLVSKL DSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPYTFGGGTNLEIK

[0417] SEQ ID NO:31-hVL22.101e

[0418] DVVMTQSPLSLPVTLGQPASISCRSSQSLVDSDGKTYLNWFQQRPGQSPRRLIYLVSKL DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0419] SEQ ID NO:32-hVL22.101g

[0420] DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKL DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0421] SEQ ID NO: 33 - hVL22.101h

[0422] DVVMTQSPLSLPVTLGQPASISCRSSQSLVASDGKTYLNWFQQRPGQSPRRLIYLVSKL DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0423] SEQ ID NO: 34 - hVL22.101i[[ID=ten]]

[0424] DVVMTQSPLSLPVTLGQPASISCRSSQSLVESDGKTYLNWFQQRPGQSPRRLIYLVSKL DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0425] SEQ ID NO: 35 - hVL22.101j

[0426] DVVMTQSPLSLPVTLGQPASISCRSSQSLVSSDGKTYLNWFQQRPGQSPRRLIYLVSKL DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0427] SEQ ID NO: 36 - CDR1 of msVL22.101 and hVL22.101g

[0428] QSLLDSDGKTY

[0429] SEQ ID NO: 37 - CDR1 of hVL22.`101e

[0430] QSLVDSDGKTY

[0431] SEQ ID NO: 38 - CDR1 of hVL22.101h

[0432] QSLVASDGKTY

[0433] SEQ ID NO: 39—CDR1 of hVL22.101i

[0434] QSLVESDGKTY

[0435] SEQ ID NO: 40 - CDR1 of hVL22.101j

[0436] QSLVSSDGKTY

[0437] SEQ ID NO: 41 - CDR1 of hVL22.101LC16

[0438] QSLLESDGKTY

[0439] SEQ ID NO: 42 - CDR1 of hVL22.101LC19

[0440] QSLLDSEGKTY

[0441] SEQ ID NO: 43 - CDR1 of hVL22.101LC20

[0442] QSLLDSSGKTY

[0443] SEQ ID NO: 44 - CDR1 of hVL22.101LC22

[0444] QSLLESEGKTY

[0445] SEQ ID NO: 45 - CDR1 of hVL22.101LC23

[0446] QSLLESSGKTY

[0447] SEQ ID NO: 46 - CDR1 of hVL22.101LC24

[0448] QSLLESDAKTY

[0449] SEQ ID NO: 47 - CDR1 of hVL22.101LC25

[0450] QSLLDTEGKTY

[0451] SEQ ID NO: 48 - CDR1 of hVL22.101LC26

[0452] QSLLDTSGKTY

[0453] SEQ ID NO: 49 - CDR1 of hVL22.101LC37

[0454] QSLLDSAGKTY

[0455] SEQ ID NO: 50 - CDR1 of hVL22.101LC38

[0456] QSLLESAGKTY

[0457] SEQ ID NO: 51 - CDR1 of hVL22.101LC39

[0458] QSLLDAEGKTY

[0459] SEQ ID NO: 52 - CDR1 of hVL22.101LC40

[0460] QSLLDNEGKTY

[0461] SEQ ID NO: 53-msFibβXG (SEQ ID NO 37 from WO2011070172)

[0462] EPTDSLDAXGHRPVDRR

[0463] Where X is citrulline

[0464] SEQ ID NO: 54 - msVim XS / XL (SEQ ID NO 38 from WO2011070172)

[0465] YVTXSSAVXLXSSVP

[0466] Where X is citrulline

[0467] SEQ ID NO: 55 - CDR2 vicinity of msVL22.101 and hVL22.101(LC)y

[0468] LVSKLDS

[0469] SEQ ID NO: 56 - Heavy chain constant domain of hCH22.101f

[0470] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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 CDR 1 of SEQ ID NO:10 (QSLLDNDGKTY), SEQ ID NO:6 (QSLLDTDGKTY), SEQ ID NO:7 (QSLLDSDAKTY) or SEQ ID NO:8 (QSLLDTDAKTY); and b) VH CDR 1 of SEQ ID NO:1 (GYTFTNYG), VH CDR 2 of SEQ ID NO:2 (INTYSGEA), VH CDR 3 of SEQ ID NO:3 (LRGYTYQSFDEGGDY), VL CDR 2 of SEQ ID NO:4 (LVS) and VL CDR 3 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:6, 7, 8 or 10; b) VL CDR2 of SEQ ID NO:4 and VL CDR3 of SEQ ID NO:5; and c) the amino acid sequence of the heavy chain variable domain 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 amino acid sequence of the heavy chain variable domain of SEQ ID NO:11 and the amino acid sequence of the light chain variable domain of SEQ ID NO:17; b) the amino acid sequence of the heavy chain variable domain of SEQ ID NO:12 and the amino acid sequence of the light chain variable domain of SEQ ID NO:17; c) the amino acid sequence of the heavy chain variable domain of SEQ ID NO:11 and the amino acid sequence of the light chain variable domain of SEQ ID NO:13; d) the amino acid sequence of the heavy chain variable domain of SEQ ID NO:11 and the amino acid sequence of the light chain variable domain of SEQ ID NO:14; e) the amino acid sequence of the heavy chain variable domain of SEQ ID NO:11 and the amino acid sequence of the light chain variable domain of SEQ ID NO:15; f) the amino acid sequence of the heavy chain variable domain of SEQ ID NO:12 and the amino acid sequence of the light chain variable domain of SEQ ID NO:13; g) the amino acid sequence of the heavy chain variable domain of SEQ ID NO:12 and the amino acid sequence of the light chain variable domain of SEQ ID NO:14; or h) the amino acid sequence of the heavy chain variable domain of SEQ ID NO:12 and the amino acid sequence of the light chain variable domain of SEQ ID NO:

15.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises the following CDRs: a) CDR1 present in SEQ ID NO:13, 14, 15 or 17; and b) The amino acid sequence of the heavy chain variable domain 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 NO: 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 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, camelid-derived single-domain antibodies, shark-derived IgNAR-derived single-domain antibody fragments (VNAR), diabodies, triabodies, anticalins, and aptamers.

8. 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 includes an Fc region.

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

11. 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, which includes a heavy chain constant region that includes SEQ ID NO: 23 or 56, and / or a light chain constant region that includes SEQ ID NO:

24.

15. The antibody or antigen-binding fragment thereof according to claim 14, wherein the antibody includes the amino acid sequence of the heavy chain variable domain of SEQ ID NO: 11 or SEQ ID NO: 12, the amino acid sequence of the light chain variable domain of SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 17, the amino acid sequence of the heavy chain constant region of SEQ ID NO: 23 or 56, and the amino acid sequence of the light chain constant region 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 of 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 an antigen-binding fragment thereof according to any one of claims 1 to 15, and isolating the antibody or an antigen-binding fragment thereof from the cell.

18. A pharmaceutical composition comprising an antibody or an 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 an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 18 or 19 in the preparation of a medicament for the treatment or prevention of a NET-related pathology, wherein the NET-related pathology 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, spondyloarthropathy, 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 the health of transplanted organs in vivo or in vitro.

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

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

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

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

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