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

Abstract

To provide antibodies or binding fragments thereof directed against citrulline-containing epitopes.SOLUTION: An antibody or binding fragment thereof that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4 comprises CDR having a particular amino acid sequence. The antibodies or binding fragments thereof of the present invention can be used in therapy, for example, in the treatment or prevention of Neutrophil Extracellular Trap (NET)-associated pathologies.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention provides an antibody or a binding fragment thereof against a citrulline-containing epitope. The antibody or the binding fragment thereof of the present invention can be used in therapeutic methods, for example, in the treatment or prevention of neutrophil extracellular trap (NET)-related disorders. The antibody or the binding fragment thereof of the present invention can be used in the treatment or prevention of other NET-related disorders such as NET-related disorders, for example, 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, 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 wound healing in diabetes, cancer, cancer metastasis, and the health of transplanted organs in vivo or ex vivo. The present invention also provides a pharmaceutical composition and a method for treating or preventing NET-related disorders, for example, 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, 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 disorders such as wound healing in diabetes, cancer, cancer metastasis, and the health of transplanted organs in vivo or ex vivo.

Background Art

[0002] Inflammatory states, whether chronic or acute, are a significant issue in the healthcare industry. In short, chronic inflammation is considered a long-term (weeks or months) inflammation characterized by simultaneous active inflammation, tissue destruction, and attempts at healing. While chronic inflammation may follow an acute inflammatory episode, it can also begin as a latent, insidious process progressing over time, resulting from, for example, persistent infections causing delayed hypersensitivity reactions (e.g., tuberculosis, syphilis, fungal infections), prolonged exposure to endogenous (e.g., elevated plasma lipids) or exogenous (e.g., silica, asbestos, tobacco tar, surgical sutures) toxins, or autoimmune reactions against the body's own tissues (e.g., rheumatoid arthritis, systemic lupus erythematosus, vasculitis, multiple sclerosis, psoriasis).

[0003] One consequence of inflammation is the formation of neutrophil extracellular traps (NETs). NETs are also known to cause inflammation. NETs are DNA and histone-containing structures produced by neutrophils as part of the host's defense mechanism against pathogens. NETs can capture and kill various bacterial, fungal, viral, and protozoan pathogens, and the release of NETs is one of the first lines of defense against pathogens. Following activation by microorganisms or cytokines, histones are hypercitrullinated, and the neutrophil nucleus undergoes a process of chromatin decondensation, which leads to NET formation in NETosis, a form of neutrophil cell death.

[0004] NETs play a pathological role in various diseases, for example, by causing abnormal inflammation. Therefore, NETs are involved in 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, and Behçet's disease. It is involved in the pathogenesis of spondylitis, spondyloarthropathy, multiple system atrophy, Parkinson's disease, Lewy body dementia, asthma, allergic rhinovirus exacerbated asthma, cystic fibrosis, and idiopathic pulmonary fibrosis.

[0005] For example, NETs can cause exposure of autoantigens to the extracellular space and subsequent production of pathological autoantibodies by the subject. Furthermore, NETs and NET remnants possess toxic histones that induce 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 circulation and tissues would have therapeutic benefits.

[0006] Neutrophils are also increasingly recognized as an important component in tumor progression. Neutrophils have been shown to exert a significant effect at almost every stage of tumor progression, and numerous studies have demonstrated that their presence is crucial for tumor development. Studies have also indicated that NETs are associated with factors that promote tumor progression and metastasis. Furthermore, neutrophils have been shown to provide a scaffold and stimulus for platelet adhesion, thrombus formation, and coagulation in tumors through the generation of NETs.

[0007] Furthermore, NETs are also involved in the decline of organ health after transplantation. NETs contribute to primary graft failure and are a cause of early death after lung transplantation. NETs have been shown to play a pathogenic role in solid organ transplantation.

[0008] Therefore, identifying therapeutic agents that can block NET formation, clear NETs, ​​and / or prevent NETosis is crucial for inflammatory diseases, such as inflammatory arthritis, rheumatoid arthritis, and osteoarthritis, as well as other NET-related conditions, 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, and spinal cord disease. It may have clinical utility in spondyloarthritis, 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, wound healing in diabetes, cancer, cancer metastasis, and the health of transplanted organs in vivo or ex vivo.

[0009] Compounds for treating or preventing NET-related conditions are still needed.

[0010] Antibodies that bind to citrullinated epitopes in deiminated human histone 2A and histone 4 are described in Patent Documents 1, 2, and 3. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] International Publication No. 2009147201 [Patent Document 2] International Publication No. 2011070172 [Patent Document 3] International Publication No. 2016092082 [Overview of the project]

[0012] The inventors have created improved antibodies that bind to citrullinated epitopes at the amino terminus of histone 2A and / or histone 4. These antibodies can be used to treat diseases or conditions associated with citrullination, such as NET-related conditions and inflammatory states.

[0013] The inventors have produced antibodies exhibiting improved properties compared to the therapeutic antibodies disclosed in Patent Documents 1, 2, and 3. Through accelerated stability testing and mass spectrometry, the inventors found that the binding affinity of the antibodies to the tested histone-derived peptides decreased over time as a result of isomerization of specific amino acid residues in the complementarity-determining region 1 (CDR1) of the light chain of the antibodies disclosed in Patent Documents 1, 2, and 3. Subsequently, the inventors conducted a thorough analysis of CDR1 light chain mutants in an attempt to resolve the isomerization problem while maintaining the binding properties of the antibodies. As a result of several attempts, antibodies with reduced binding affinity to target peptides were obtained.

[0014] Finally, the inventors succeeded in identifying a group of mutations in the light chain CDR1 that eliminated the isomerization problem while maintaining the binding properties of the original antibody. Surprisingly, this mutant antibody showed improved properties compared to the original antibody both in vitro and in vivo.

[0015] Therefore, the present invention is - An antibody or its binding fragment that specifically binds to a citrullinated epitope in deiminated human histone 2A and / or histone 4, a) 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 neither QSLLLDSDGKTY (SEQ ID NO: 36) nor QSLVDSDGKTY (SEQ ID NO: 37), but has a light chain variable domain (VL) CDR1; b) At least one CDR selected from sequence numbers 1-5 and The present invention provides an antibody or a conjugated fragment thereof containing such an antibody.

[0016] The present invention also relates to - an antibody or a binding fragment thereof that specifically binds to a citrullinated epitope in deiminated human histone 2A and / or histone 4, a) CDR1 of SEQ ID NO: 13, 14, 15, 16, or 17; and b) the heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12 and provides an antibody or a binding fragment thereof containing the CDR.

[0017] The present invention also relates to - a polynucleotide encoding an antibody or a binding fragment thereof as defined herein, a cloning or expression vector containing the polynucleotide, or a host cell containing the cloning or expression vector.

[0018] The present invention also relates to - a process for producing an antibody or a binding fragment thereof that specifically binds to a citrullinated epitope in deiminated human histone 2A and / or histone 4, the process comprising culturing a host cell as defined herein and isolating the antibody or a binding fragment thereof from the cell.

[0019] The present invention also relates to - a pharmaceutical composition comprising an antibody or a binding fragment thereof as defined herein and at least one pharmaceutically acceptable diluent or carrier.

[0020] The present invention also relates to - an antibody or a binding fragment thereof as defined herein, or a pharmaceutical composition as defined herein for use in therapy.

[0021] The present invention also relates to - an antibody or a binding fragment thereof as defined herein, or a pharmaceutical composition as defined herein for use in a method of treating or preventing a NET-related disorder.

[0022] The present invention also provides - A method of treating a patient, comprising administering to the patient a therapeutically effective amount of an antibody or a binding fragment thereof as defined herein or a pharmaceutical composition as defined herein.

[0023] Brief Description of the Sequence Listing Nomenclature of Antibodies CDR = Complementary Determining Region. VH = Variable Domain of Heavy Chain VL = Variable Domain of Light Chain CH = Constant Domain of Heavy Chain CL = Constant Domain of Light Chain msVH22.101 = Mouse VH of Therapeutic Antibody msVL22.101 = Mouse VL of Therapeutic Antibody hVH22.101x = Humanized VH of Therapeutic Antibody, "x" refers to the heavy chain. hVL22.101y = Humanized VL of Therapeutic Antibody, "y" refers to the light chain. hVH22.101(HC)x = Optimized Humanized VH of Therapeutic Antibody, "(HC)x" refers to the heavy chain. hVL22.101(LC)y = Optimized Humanized VL of Therapeutic Antibody, "(LC)y" refers to the light chain. hMQ22.101x / y = Humanized Therapeutic Antibody, "x" refers to the heavy chain, "y" refers to the light chain. [[ID=三十六]] hMQ22.101(HC)x / (LC)y = Optimized Humanized Therapeutic Antibody of the Present Invention, "(HC)x" refers to the heavy chain, "(LC)y" refers to the light chain.

[0024] [Table 1-1] [Table 1-2] Brief Description of the Drawings

[0025] [Figure 1]Accelerated Stability Test of hMQ22.101j / e and hMQ22.101f / g 0.75 mL aliquots (glass tubes) containing either hMQ22.101j / e (12.5 mg / mL) or hMQ22.101f / g (3.31 mg / mL) in 25 mM Tris-HCl, pH 8.0 were stored at 37°C for 8 weeks. Each week, several 10 μL and 20 μL samples were taken from each glass tube under sterile conditions and stored at -80°C until further analysis (ELISA and mass spectrometry) was performed. 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 in-house validated CMC ELISA to evaluate binding to a histone-derived peptide (SEQ ID NO: 18). The antibody binding affinity of the accelerated stability sample at week 0 was set to 100%, and all other binding affinity values ​​of the accelerated stability samples (weeks 2, 3, 4, 6, and 8) were recalculated as a percentage of week 0 (100%) and plotted as a bar graph. [Figure 2]Mass Spectrometry of hMQ22.101x / y Antibody Figure 2A Mass spectrometry (MS) analysis of accelerated stability samples from the antibody hMQ22.101j / e. 0.75 mL aliquots (glass tubes) containing hMQ22.101j / e (12.5 mg / mL) were stored at 37°C for 8 weeks. Samples were removed from each glass tube under sterile conditions weekly and stored at -80°C until MS analysis was performed. MS analysis was performed as described in Example 2. The table shows the relative aspartic acid (D) isomerization levels within and near CDR2 of hVL22.101e. Figure 2B Antigen-binding assay using a humanized antibody containing a mutated aspartic acid CDR1 of hVL22.101y. The generated CDR1 aspartate mutant antibodies hMQ22.101j / h, hMQ22.101j / i, and hMQ22.101j / j were compared with the aspartate-containing antibody hMQ22.101j / e using the CMC ELISA validated in-house as described in Example 1. The graph shows the optical density results for the three hVL22.101y CDR1 mutants (mutation of the CDR1=DS site to AS in hVL22.101h; mutation of the CDR1=DS site to ES in hVL22.101i; mutation of the CDR1=DS site to SS in hVL22.101j). Figure 2C 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 aspartic acid (D) isomerization levels within CDR1 and near CDR2 of hVL22.101i. [Figure 3A] Generation and affinity analysis of hMQ22.101 isomerized mutants. The table shows 17 mutated domains in the CDR1 of hVL22.101(LC)y, which were generated in addition to the unmutated CDR1 of hVL22.101e and hVL22.101g. [Figure 3B] Generation and affinity analysis of hMQ22.101 isomerized mutants. Graph showing the dissociation rate (kdis × E-07 (1 / s)) of isomerized mutants to citrullinated H2A-derived peptide (SEQ ID NO: 18) and H4-derived peptide (SEQ ID NO: 20), measured using the Octet RED96 instrument. A lower dissociation rate indicates higher antibody affinity to the antigen. [Figure 4]Accelerated Stability Testing of hMQ22.101 Isomerized Mutants 0.4 mL aliquots (glass tubes) containing specified mutant antibodies (ranging from 2.06 to 4.29 mg / mL) were stored at 37°C for 6 weeks. Each week, samples were removed from each glass tube under sterile conditions and stored at -80°C until further analysis. Samples from weeks 0, 3, and 6 were subjected to an in-house validated CMC ELISA to evaluate binding to a citrullinated H2A-derived peptide (SEQ ID NO: 18). The recalculated antibody binding affinity from the week 0 accelerated stability sample was set to 100%, and all other binding affinity values ​​of the accelerated stability samples were recalculated as a percentage of week 0 (100%) and plotted as a bar graph. The preferred heavy chains used in the accelerated stability testing were hVH22.101f and hVH22.101HC9. Nine combinations of the heavy chain and the CDR1-mutated light chain were tested. hMQ22.101f / LC41, hMQ22.101f / LC42, hMQ22.101HC9 / LC21, hMQ22.101HC9 / LC27, and hMQ22.101HC9 / LC42 showed the highest stability after 6 weeks. [Figure 5] Mass spectrometry of hMQ22.101 isomerized mutants 0.4 mL aliquots (glass tubes) containing the specified mutant antibody (range 2.06–4.29 mg / mL) were stored at 37°C for 6 weeks. Each week, samples were removed from each glass tube under sterile conditions and stored at -80°C until further analysis. Using accelerated stability samples from week 0 and week 6, mass (MS) analysis of hMQ22.101 antibodies (isomerized mutants) with mutated VL CDR1 was performed as described in Example 2, except for comparison with the isomerization level of hMQ22.101j / e. The table shows the relative aspartic acid (D) isomerization levels within CDR1 of hVL22.101(LC)y. MS analysis of the hMQ22.101 isomerized mutants indicates that hMQ22.101f / LC41 showed the least isomerization over time (0.5%) and was therefore the most preferred candidate. Other preferred candidates were hMQ22.101f / LC42 and hMQ22.101HC9 / LC42. [Figure 6]Agglutination and Degradation Assay of Preferred hMQ22.101 Isomerized Mutants 0.4 mL aliquots (glass tubes) containing the specified mutant antibody (range 2.06–4.29 mg / mL) were stored at 37°C for 6 weeks. Each week, samples were removed from each glass tube under sterile conditions and stored at -80°C until further analysis. Stable samples from week 0 and week 6 of the hMQ22.101f / LC41, hMQ22.101f / LC42, and hMQ22.101HC9 / LC42 isomerized mutants were used for agglutination and degradation analysis as described in Example 10. Measurements were performed using an Agilent 1200 system combined with an Agilent Zorbax GF-250 column. Proteins were detected using 240 nm ultraviolet light. The main antibody peak was detected in approximately 4.25 minutes. The shoulders before and after the main peak were quantified to indicate the levels of agglutination and degradation, respectively. hMQ22.101f / LC41, hMQ22.101f / LC42, and hMQ22.101HC9 / LC42 exhibited acceptable aggregation and degradation profiles, indicating that they are acceptable for further development. [Figure 7]NETosis inhibition experiments using preferred isomerized mutants hMQ22.101f / LC41 and hMQ22.101f / LC42. Neutrophils from healthy volunteers (donors 154 and 155) were stimulated with the calcium ionophore A23187 for 4 hours. The effect of neutrophil extracellular trap (NET) reduction antibodies was tested by adding 25 μg / mL of antibody or assay buffer 15 minutes before adding A23187 to the cells. After incubation at 37°C and 5% CO2 for 4 hours, the cells were washed, and then extracellular DNA was digested with S7 nuclease. NET fragments were collected from the wells and quantified by measuring the MPO activity in the sample by adding 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate to 50 μL of the collected NET. After incubation for 10 minutes in RT, 450 μL of H2SO was added, and the optical density was measured at 450 nm. Background signals from neutrophils not treated with A23187 were subtracted, and the signal from neutrophils treated with A23187 + an unrelated antibody was set to 100%. Signals from all other treatment groups were set to the percentage of the unrelated antibody treatment. [Figure 8] Dose-response efficacy of hMQ22.101f / LC41, hMQ22.101f / LC42, and hMQ22.101f / g in a mouse CAIA model: Lead-optimized candidate antibodies prevent the onset of inflammation. The dose-response efficacy of hMQ22.101f / LC41, hMQ22.101f / LC42, or hMQ22.101f / g was tested using a collagen antibody-induced arthritis (CAIA) model. A group of five mice were treated by intravenous injection of 2.8 mg of anti-collagen-II antibody on day 0. On day 3, LPS (25 μg / mouse) was administered via intravenous injection at doses of 6.25, 12.5, and 25 mg / kg of hMQ22.101f / LC41, hMQ22.101f / LC42, or hMQ22.101f / g, respectively, simultaneously with an unrelated isotype-matched control antibody (MQR2.201 at 25 mg / kg), or without antibody (placebo). The degree of limb swelling was scored over two weeks and graphed as the "mean arthritis score / mouse". [Figure 9A]In vitro NET inhibition and NET binding by hMQ22.101f / LC41. Mouse neutrophils derived from bone marrow were stimulated with A23187 to induce NET release in vitro. NET release was inhibited by hMQ22.101f / LC41 but not by MQR2.201 (Figure A; left bar graph, quantification of colocalization of Hoechst (DNA) and citrullinated histone 3 (citH3); right bar graph, quantification of Hoechst only). Sytox Green was used to detect DNA including NET and pre-NET, and anti-hIgG was used to detect hMQ22.101f / LC4 bound to NET and pre-NET. Scale bar: 25 μm. [Figure 9B] In vitro NET inhibition and NET binding by hMQ22.101f / LC41. Mouse neutrophils derived from bone marrow were stimulated with A23187 to induce NET release in vitro. Furthermore, hMQ22.101f / LC41 bound to the released NETs (yellow arrows) and pre-NETs (white arrows), which may be the first step toward NET clearance by macrophages (Figure B). Sytox Green was used to detect DNA containing NETs and pre-NETs, ​​and anti-hIgG was used to detect hMQ22.101f / LC4 bound to NETs and pre-NETs. Scale bar: 25 μm. [Figure 10A]In vivo NET inhibition and NET binding by hMQ22.101f / LC41. A pristane-induced peritoneal cell influx mouse model was used to induce NET formation in vivo. 50 mg / kg MQR2.201 or hMQ22.101f / LC41 was administered immediately after injection of 500 μL of pristane oil, followed by a second injection of 50 mg / kg MQR2.201 or hMQ22.101f / LC41 12 hours later. Cells were collected after 24 hours. Inhibition of NET release was observed in vivo when mice were treated with hMQ22.101f / LC41, but not with MQR2.201. Representative photograph. Sytox Green was used to detect DNA including NETs and pre-NETs, ​​and anti-hIgG was used to detect hMQ22.101f / LC4 bound to NETs and pre-NETs. Scale bar: 50 μm. [Figure 10B] In vivo NET inhibition and binding by hMQ22.101f / LC41: A pristane-induced peritoneal cell influx mouse model was used to induce NET formation in vivo. 500 μL of pristane oil was injected, followed immediately by 50 mg / kg MQR2.201 or hMQ22.101f / LC41. A second injection of 50 mg / kg MQR2.201 or hMQ22.101f / LC41 was administered 12 hours later. Cells were collected after 24 hours. Inhibition of NET release was observed in vivo when mice were treated with hMQ22.101f / LC41, but not with MQR2.201. NET quantification was performed using co-localized Hoechst (DNA) and citrullinated histone 3 (citH3). (Sytox Green is used to detect DNA including NETs and pre-NETs, ​​and anti-hIgG is used to detect hMQ22.101f / LC4 bound to NETs and pre-NETs.) [Figure 10C]In vivo NET inhibition and NET binding by hMQ22.101f / LC41: A pristane-induced peritoneal cell influx mouse model was used to induce NET formation in vivo. 50 mg / kg MQR2.201 or hMQ22.101f / LC41 was administered immediately after injection of 500 μL of pristane oil, followed by a second injection of 50 mg / kg MQR2.201 or hMQ22.101f / LC41 12 hours later. Cells were collected after 24 hours. Inhibition of NET release was observed in vivo when mice were treated with hMQ22.101f / LC41, but not with MQR2.201. Binding of hMQ22.101f / LC41 to NETs and pre-NETs may be the first step toward macrophage-mediated NET clearance. Sytox Green is used to detect DNA containing NETs and pre-NETs, ​​and anti-hIgG is used to detect hMQ22.101f / LC4 bound to NETs and pre-NETs. Scale bar: 25 μm. [Figure 11] NETs containing high levels of hMQ22.101f / LC41 are phagocytosed by mouse macrophages in vivo. A pristane-induced peritoneal cell influx mouse model was used to induce NET formation in vivo. 500 μL of pristane oil was injected, followed immediately by 50 mg / kg MQR2.201 or hMQ22.101f / LC41, and then a second injection of 50 mg / kg MQR2.201 or hMQ22.101f / LC41 was administered 12 hours later. After 24 hours, cells were collected and stained with Hoechst (DNA: blue), macrophage markers 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) are present in macrophages (F4 / 80). Scale bar: 10 μm. [Figure 12A]hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice. Schematic diagram of a CIA mouse model of RA. To induce chronic arthritis, mice were injected with CIII twice (on days 0 and 21). If the MAS was ≥0.75, therapeutic treatment was initiated after disease onset (days 21-28). Treatment consisted of four injections (4-day intervals) using a tapering 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). Mice were discontinued 14 days after the start of treatment. [Figure 12B] hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice. The mean arthritis score (MAS) of CIA mice was evaluated over 14 days (n=10 mice / group; statistical differences were calculated using MQR2.201). Results are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-way ANOVA with Dunnett's multiple comparison test. [Figure 12C] hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice. Bone damage at the knee and ankle of both hind limbs was analyzed by X-ray 14 days after the first antibody injection (n=10). Results are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, independent two-tailed Student's t-test was used. [Figure 12D] hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice. Histological analysis of the ankle joints using H&E and SO staining revealed influx of inflammatory cells 14 days after the first antibody injection (n=16-20 mouse ankles). Results are shown as mean ± SEM. Two-sided Mann-Whitney U test was used. [Figure 12E]hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice. Histological analysis of the ankle joints using H&E and SO staining revealed bone erosion 14 days after the first antibody injection (n=16-20 mouse ankles). Results are shown as mean ± SEM. *P<0.05, two-sided Mann-Whitney test used. [Figure 12F] hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice. Histological analysis of the ankle joints using H&E and SO staining revealed cartilage erosion 14 days after the first antibody injection (n=16-20 mouse ankles). Results are shown as mean ± SEM. **P<0.01, ***P<0.001, two-sided Mann-Whitney test used. [Figure 12G] hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice. Histological analysis of the ankle joints using H&E and SO staining revealed depletion of cartilage prostaglandins (PGs) 14 days after the first antibody injection (n=16-20 mouse ankles). Results are shown as mean ± SEM. *P<0.05, **P<0.01, two-sided Mann-Whitney test used. [Figure 12H] hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice. Histological analysis of the ankle joints using H&E and SO staining revealed chondrocyte death 14 days after the first antibody injection (n=16-20 mouse ankles). Results are shown as mean ± SEM. **P<0.01, two-sided Mann-Whitney test used. [Figure 12I] hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice. Representative immunofluorescence and H&E images of NET release in the joint of the right hindlimb, showing citrullinated histone 3 (citH3; red), DAPI (blue), neutrophil marker Ly6G (green), and myeloperoxidase (MPO; yellow). DAPI was used for nuclear and extracellular DNA staining. Scale bar: 100 μm. [Figure 12J] hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice. Ly6G was quantified in the tibiotarsal joint, proximal intertarsal joint, distal intertarsal joint, and tarsometatarsal joint of the right hindlimb of mice (n=10). Results are shown as mean ± SEM. **P<0.01, two-sided Mann-Whitney test used. [Figure 12K] hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice. Quantitative analysis of NETs (colocalization of citH3 and MPO) was performed in the tibiotarsal joint, proximal intertarsal joint, distal intertarsal joint, and tarsometatarsal joint of the right hindlimb of mice (n=10). Results are shown as mean ± SEM. *P<0.05, two-sided Mann-Whitney test. [Figure 12L] hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice. A significant correlation was observed between macroscopic scores (limb swelling) and NETs per joint. Spearman's r-test was used. [Figure 12M] hMQ22.101j / e prevents NET-mediated tissue damage and disease progression in chronic CIA mice. A significant correlation was observed between macroscopic scores (limb swelling) and neutrophils per joint (Ly6G). Spearman's r-test was used. [Figure 13]hMQ22.101j / e does not bind to healthy leukocytes. PBMCs and neutrophils were isolated from the blood of healthy volunteers. CD45 was used to distinguish leukocytes from red blood cells and platelets, and CD3, CD11c, CD14, CD20, CD56, and CD66b were used to mark T cells, DCs, monocytes, B cells, NK cells, and neutrophils, respectively. No binding of HiLyte® Fluor488 conjugate hMQ22.101j / e to healthy, quiescent T cells, B cells, monocytes, NK cells, DCs, and neutrophils was observed. Activated neutrophils (5 μM A23187, 45 minutes) were used as a positive control, showing increased binding of HiLyte® MFluor488 conjugate hMQ22.101j / e. ****P<0.001, standard one-way ANOVA with Dunnett's multiple comparison test was used. [Modes for carrying out the invention]

[0026] It should be understood that the various applications of the disclosed invention can be adapted to specific needs in the art. Furthermore, it should be understood that the terminology used herein is intended solely to describe specific embodiments of the invention and is not intended to limit them.

[0027] Furthermore, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless otherwise explicitly indicated. Thus, for example, a reference to "an antibody" includes "multiple antibodies," etc.

[0028] Whether above or below, all publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

[0029] The present invention relates to an antibody or its binding fragment that specifically binds to citrullinated epitopes in deiminated human histone 2A and / or histone 4. Deiminolysis of human histones 2A and 4 can be carried out by enzymes, such as peptidylarginine deiminases (PADs) such as PAD2 and PAD4. The antibody of the present invention can also specifically bind to citrullinated epitopes in human histone 3. The antibody of the present invention can specifically bind to citrullinated epitopes in human histone 2A and / or histone 4 and / or histone 3. The present invention also relates to the use of such an antibody or its binding fragment, for example, for therapeutic use.

[0030] The present invention relates to an antibody or a binding fragment thereof that specifically binds to citrullinated epitopes in deiminoated human histone 2A and / or histone 4 for use in the treatment or prevention of NET-related conditions. The antibodies or their conjugated fragments of the present invention can be used in the treatment or prevention of NET-related conditions, 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, 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 conditions, such as wound healing in diabetes, cancer, cancer metastasis, and the health of transplanted organs in vivo or ex vivo.

[0031] Target of the antibody or its conjugated fragment of the present invention Citrulline is an amino acid that is not incorporated into proteins during normal translation, but can be produced by post-translational modification of arginine residues by enzymes such as PAD (EC3.5.3.15). In mammals (humans, mice, and rats), five PAD isotypes (PAD1-PAD6; "PAD4" and "PAD5" are used for the same isotype) have been identified so far, each encoded by a different gene.

[0032] Citrullination of histone 2A and / or histone 4 is associated with NET formation. Numerous downstream pathological effects of NET formation may occur. For example, these effects could include exposure of autoantigens to the extracellular space and subsequent production of pathological autoantibodies by the subject. Histones derived from NETs can be toxic to vascular walls and organs, leading to vascular damage and organ failure. NETs can also trigger the formation of autoantigen / autoantibody immune complexes that further exacerbate inflammation, for example, in the kidneys of SLE patients. NETs are also involved in metastasis during cancer progression.

[0033] The antibody or its conjugated fragment according to the present invention specifically binds to citrullinated epitopes in deiminated human histone 2A and / or histone 4. Furthermore, the antibody may also specifically bind to citrullinated epitopes in deiminated human histone H3. In certain embodiments, the antibody or its conjugated fragment specifically binds to citrullinated epitopes in deiminated human histone 2A and / or histone 4, and the epitope contains a peptide selected from the group consisting of SEQ ID NOs. 18, 19, 20, 21, and 22. The antibody or its conjugated fragment may also bind to epitopes containing the peptide of SEQ ID NO. 53 or 54.

[0034] Antibodies or their conjugated fragments The terms “multiple antibodies,” “antibodies,” or “their binding fragments” as used herein refer to structures, preferably protein or polypeptide structures, that can specifically bind to a target molecule, often referred to as an “antigen.”

[0035] As used herein, the term "antibody molecule" refers to an antibody or its binding fragment. As used herein, the term "antibody" generally refers to an intact (whole) antibody, i.e., an antibody comprising two heavy chains and two light chains. The antibody may further include additional binding domains, such as the molecule DVD-Ig disclosed in International Publication No. 2007 / 024715 or the so-called (FabFv)2Fc described in International Publication No. 2011 / 030107. Therefore, as used herein, "antibody" includes monovalent, divalent, trivalent, or tetravalent full-length antibodies.

[0036] Examples of antibody binding fragments 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, VL, or VHH), scFv, monovalent, divalent, trivalent, or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and any of the above epitope binding fragments (e.g., Holliger P and Hudson PJ, 2005, Nat. Biotechnol., 23, :1126-1136; Adair JR and Lawson). See ADG, 2005, Drug Design Reviews-Online, 2, 209-217). Methods for preparing 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 International Publication No. 2009 / 040562, and its disulfide-stabilized version Fab-dsFv was first disclosed in International Publication No. 2010 / 035012. Other antibody fragments for use in the present invention include Fab and Fab' fragments. The polyvalent antibody may have multiple specificity, for example, it may be bispecific or monospecific.

[0037] Antibodies or their binding fragments may 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 containing the antigen-binding domain or aptamer of a native antibody, single-domain antibodies (sdAb), also known as VHH antibodies, nanobodies (single-domain antibodies derived from camels), single-domain antibody fragments derived from shark IgNAR called VNAR, diabodies, triabodies, antikalin, aptamers (DNA or RNA), and their active components or fragments.

[0038] IgG1 antibodies having IgG1 heavy and light chains (e.g., IgG1 / κ) can be advantageously used in the present invention. However, other human antibody isotypes, including IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE combined with κ or λ light chains, are also included in the present invention. Furthermore, all animal-derived antibodies of various isotypes can be used in the present invention. The antibody may be a full-size antibody, or it may be a Fab, F(ab')2, single-chain Fv fragment, or an antigen-binding fragment of an antibody containing a single-domain VHH, VH, or VL single domain.

[0039] The terms "specifically binds to citrulline" or "specifically binds to a citrullinated epitope" mean that, in this context, the antibody or its binding fragment binds to structures such as peptides containing citrulline residues, while the antibody or its binding fragment binds less weakly, or preferably not at all, to the same structures containing arginine residues instead of citrulline residues. The term peptide should be interpreted as a structure capable of presenting citrulline residues in appropriate immunoreactivity with the antibody or its binding fragment described herein, preferably in the same context as that present in the human or animal body, preferably in the context of a native polypeptide.

[0040] The antibody or its binding fragment of the present invention specifically binds to citrullinated epitopes in deiminated human histone 2A and / or histone 4. Binding of the antibody or its binding fragment to citrullinated epitopes in deiminated human histone 2A and / or histone 4 blocks NET formation. Histone citrullination is associated with NET formation.

[0041] Blocking of NET formation may be total or partial. For example, the antibody or its conjugated fragment of the present invention can reduce NET formation by 10-50%, 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).

[0042] The terms "binding activity" and "binding affinity" are intended to refer to the tendency of an antibody molecule to bind to or not bind to its target. Binding affinity can be quantified by determining the dissociation constant (Kd) for the antibody and its target. Similarly, the specificity of an antibody's binding to its target can be defined in terms of the comparative dissociation constant (Kd) of the antibody to its target, compared to the dissociation constant for the antibody and another non-target molecule.

[0043] Typically, the Kd of an antibody against a target is determined by unrelated or coexisting substances in the environment. The Kd is 2 times, preferably 5 times, and more preferably 10 times lower than that for other non-target molecules. More preferably, the Kd is 50 times or less, even more preferably 100 times or less, and even more preferably 200 times or less.

[0044] The value of this dissociation constant can be directly determined by well-known methods, and even for complex mixtures, it can be calculated by computer using methods such as those described in Caceci MS and Cacheris WP (1984, Byte, 9, 340-362). For example, Kd can be established using a double-filter nitrocellulose filter-bound assay, such as that disclosed by Wong I and Lohman TM (1993, Proc. Natl. Acad. Sci. USA, 90, 5428-5432), or by using, for example, Octet surface plasmon resonance.

[0045] One method for evaluating the binding affinity to deiminoized human histone 2A and / or histone 4 is by ELISA. Other standard assays for evaluating the binding ability of ligands, such as antibodies, to targets are known in the art, including, for example, Western blotting, RIA, and flow cytometry analysis. The antibody binding reaction rate (e.g., binding affinity) can also be evaluated by standard assays known in the art, such as surface plasmon resonance, for example, by Biacore® system analysis.

[0046] Preferably, the antibody of the present invention has a binding affinity of 1 nM or less to deiminated human histone 2A and / or histone 4. Preferably, the antibody of the present invention has 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 to deiminated human histone 2A and / or histone 4, and / or deiminated human histone H3.

[0047] Furthermore, the antibody or its binding fragment may be a fusion protein containing the antigen-binding domain of the native antibody, or it may be an aptamer such as an aptamer in the form of DNA or RNA.

[0048] Preferably, the antibody or its binding fragment of the present invention is a monoclonal antibody. Monoclonal antibodies are immunoglobulin molecules that are identical to one another and have a single binding specificity and affinity for a specific epitope. The monoclonal antibodies (mAbs) of the present invention are used in various techniques, including conventional monoclonal antibody methods, e.g., “Monoclonal Antibodies: a manual of techniques” (Zola H, 1987, CRC Press) and “Monoclonal Hybridoma They can be produced by those disclosed in “Antibacters: Techniques and Applications” (Hurrell JGR, 1982 CRC Press).

[0049] The antibody or its binding fragment of the present invention includes a binding domain. The binding domain generally contains six CDRs (three in the case of VHH), three from the heavy chain and three from the light chain. In one embodiment, the CDRs are within a framework and together form a variable region or domain. Accordingly, in one embodiment, the antibody or binding fragment includes an antigen-specific binding domain comprising a light chain variable region or domain and a heavy chain variable region or domain.

[0050] Residues in the antibody variable domain are conventionally numbered according to IMGT (http: / / www.imgt.org). This system is described in Lefranc MP (1997, J, Immunol. Today, 18, 509). In this specification, in particular, Unless otherwise specified, this numbering system will be used.

[0051] IMGT residue designations do not necessarily directly correspond to linear numbering of amino acid residues. Actual linear amino acid sequences, whether frameworks or CDRs, may contain fewer or additional amino acids than the strict IMGT numbering corresponding to the shortening or insertion of structural components of the basic variable domain structure. The correct IMGT numbering of residues for a given antibody can be determined by aligning homologous residues in the antibody sequence with a "standard" IMGT numbering sequence.

[0052] The CDRs of the heavy chain variable domain are located at residues 27-38 (CDR1 of VH), 56-65 (CDR2 of VH), and 105-117 (CDR3 of VH), according to the IMGT numbering system.

[0053] The CDRs of the light chain variable domain are located at residues 27-38 (VL CDR1), 56-65 (VL CDR2), and 105-117 (VL CDR3), according to the IMGT numbering system.

[0054] The antibody or its conjugate fragment of the present invention is disclosed herein by the primary amino acid sequence of its CDR region. The antibody or its conjugate fragment of the present invention is disclosed herein by the primary amino acid sequences of its heavy chain and light chain.

[0055] The present invention is based on the finding that a modified VL CDR1 of an antibody or its conjugate that specifically binds to citrullinated epitopes in deiminoated human histone 2A and / or histone 4 provides the antibody or its conjugate with improved properties compared to an antibody or its conjugate containing an unmodified version of the VL CDR1. The unmodified VL CDR1 of the antibody used to derive the present invention comprises or consists of the amino acid sequence QSLLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37).

[0056] The modified VL chain CDR1 of the antibody or its binding fragment 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 neither QSLLLDSDGKTY (SEQ ID NO: 36) nor QSLVDSDGKTY (SEQ ID NO: 37). The modified VL chain CDR1 of the antibody or its binding fragment of the present invention exhibits reduced isomerization compared to the unmodified CDR1 of SEQ ID NO: 36 or 37, but maintains the binding properties of the unmodified CDR1.

[0057] The amino acid sequences of the VH CDR of the specific antibody or its binding fragment of the present invention are shown in SEQ ID NOs: 1, 2, and 3. The VL CDRs 2 and 3 are shown in SEQ ID NOs: 4 and 5.

[0058] The amino acid sequences of VH and VL of the specific antibody or its conjugated fragment of the present invention are given in SEQ ID NOs: 11 and 13. The CDR of VH is shown in SEQ ID NOs: 1, 2, and 3. The CDR of VL is shown in SEQ ID NOs: 6, 4, and 5.

[0059] The amino acid sequences of VH and VL of another antibody or its conjugated fragment of the present invention are given in SEQ ID NOs: 11 and 14. The CDR of VH is shown in SEQ ID NOs: 1, 2, and 3. The CDR of VL is shown in SEQ ID NOs: 7, 4, and 5.

[0060] The amino acid sequences of VH and VL of another antibody or its conjugated fragment of the present invention are given in SEQ ID NOs: 11 and 15. The CDR of VH is shown in SEQ ID NOs: 1, 2, and 3. The CDR of VL is shown in SEQ ID NOs: This is shown in sections 8, 4, and 5.

[0061] The amino acid sequences of VH and VL of another antibody or its conjugated fragment of the present invention are given in SEQ ID NOs: 11 and 16. The CDR of VH is shown in SEQ ID NOs: 1, 2, and 3. The CDR of VL is shown in SEQ ID NOs: 9, 4, and 5.

[0062] The amino acid sequences of VH and VL of another antibody or its conjugated fragment of the present invention are given in SEQ ID NOs: 11 and 17. The CDR of VH is shown in SEQ ID NOs: 1, 2, and 3. The CDR of VL is shown in SEQ ID NOs: 10, 4, and 5.

[0063] The amino acid sequences of VH and VL of another antibody or its conjugated fragment of the present invention are given in SEQ ID NOs: 12 and 13. The CDR of VH is shown in SEQ ID NOs: 1, 2, and 3. The CDR of VL is shown in SEQ ID NOs: 6, 4, and 5.

[0064] The amino acid sequences of VH and VL of another antibody or its conjugated fragment of the present invention are given in SEQ ID NOs: 12 and 14. The CDR of VH is shown in SEQ ID NOs: 1, 2, and 3. The CDR of VL is shown in SEQ ID NOs: 7, 4, and 5.

[0065] The amino acid sequences of VH and VL of another antibody or its conjugated fragment of the present invention are given in SEQ ID NOs: 12 and 15. The CDR of VH is shown in SEQ ID NOs: 1, 2, and 3. The CDR of the VL chain is shown in SEQ ID NOs: 8, 4, and 5.

[0066] The amino acid sequences of VH and VL of another antibody or its conjugated fragment of the present invention are given in SEQ ID NOs: 12 and 16. The CDR of VH is shown in SEQ ID NOs: 1, 2, and 3. The CDR of VL is shown in SEQ ID NOs: 9, 4, and 5.

[0067] The amino acid sequences of VH and VL of another antibody or its conjugated fragment of the present invention are given in SEQ ID NOs: 12 and 17. The CDR of VH is shown in SEQ ID NOs: 1, 2, and 3. The CDR of VL is shown in SEQ ID NOs: 10, 4, and 5.

[0068] In embodiments of the present invention, the antibody of the present invention comprises the heavy chain variable domain amino acid sequence of SEQ ID NO: 11, the light chain variable domain amino acid sequence of SEQ ID NO: 16, the heavy chain constant region amino acid sequence including SEQ ID NO: 23 or 56, and the light chain constant region amino acid sequence of SEQ ID NO: 24.

[0069] In embodiments of the present invention, the antibody of the present invention comprises the heavy chain variable domain amino acid sequence of SEQ ID NO: 11, the light chain variable domain amino acid sequence of SEQ ID NO: 16, the heavy chain constant region amino acid sequence of SEQ ID NO: 23 or 56, and the light chain constant region amino acid sequence of SEQ ID NO: 24.

[0070] The antibody or its conjugated fragment of the present invention exists such that the VL CDR1 always contains 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), except that the amino acid sequence is neither QSLLLDSDGKTY (SEQ ID NO: 36) nor QSLVDSDGKTY (SEQ ID NO: 37), or contains or consists of SEQ ID NOs. 6, 7, 8, 9, or 10, and may contain one or more of the CDR sequences of any one of the specific antibodies described above.

[0071] The antibody or its conjugated fragment of the present invention may, in addition to VL CDR1, comprise one or more VH CDR sequences of the particular antibody, and alternatively or additionally, one or more VL CDR sequences. The antibody or its conjugated fragment of the present invention may comprise one, two, or all of the VH CDR sequences of the particular antibody or its conjugated fragment described above, and alternatively or additionally, VL CDR The antibody or its conjugated fragment may contain one, two, or all three of the VL chain CDR sequences of the particular antibody or its conjugated fragment containing R1. The antibody or its conjugated fragment of the present invention may contain all six CDR sequences of the particular antibody or conjugated fragment described above. For example, the antibody of the present invention may contain 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.

[0072] In embodiments of the present invention, the modified VL chain CDR1 of the antibody or its binding fragment 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 neither QSLLLDSDGKTY (SEQ ID NO: 36) nor QSLVDSDGKTY (SEQ ID NO: 37). The modified VL chain CDR1 of the antibody or its binding fragment exhibits reduced isomerization compared to the unmodified CDR1 of SEQ ID NO: 36 or 37, while maintaining the binding properties of the unmodified CDR1. The modified CDR1 of the VL chain of the antibody or its binding fragment of the present invention may include, or consist of, SEQ ID NOs: 6, 7, 8, 9, 10, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52. In embodiments of the present invention, the antibody of the present invention may include one of SEQ ID NOs: 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 NOs: 1, 2, 3, 4, and 5. In embodiments of the present invention, the antibody of the present invention includes one of SEQ ID NOs: 6, 7, 8, 9, 10, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 and all of SEQ ID NOs: 1, 2, 3, 4, and 5.

[0073] The antibody or its binding fragment of the present invention may alternatively contain one of these heavy chain variable domains or a variant of the CDR sequence of CDR2 or 3 of the VL. For example, the variant may be a substitution, deletion, or addition variant of any of the above amino acid sequences.

[0074] Variant antibodies may include substitutions and / or deletions of one, two, three, four, five, ten or fewer, twenty or fewer, thirty or more amino acids from the specific sequences and fragments described herein, while maintaining the activity of the antibodies described herein. A “deletion” variant may include, for example, the deletion of one, two, three, four, or five individual amino acids, or a small group of one or more amino acids such as two, three, four, or five amino acids. A “small group of amino acids” can be defined as those that are consecutive with each other or adjacent but not consecutive. A “substitution” variant preferably includes substituting one or more amino acids with the same number of amino acids and performing conservative amino acid substitutions. For example, an amino acid may be substituted with another amino acid having similar properties, such as 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 very small amino acid, another small amino acid, or another large amino acid. Some properties of the 20 main amino acids that can be used to select suitable substituents are as follows:

[0075] [Table 2]

[0076] Preferred "derivatives" or "mutants" include those in which an amino acid present in the sequence is a structural analog of a naturally occurring amino acid. Furthermore, the amino acids used in the sequence may be derivatized or modified, for example, labeled, as long as the antibody function is not significantly adversely affected.

[0077] The above derivatives and variants can be prepared during antibody synthesis, by modification after generation, or, if the antibody is in recombinant form, by known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or ligation of nucleic acids.

[0078] Preferably, the variant antibody according to the present invention has an amino acid sequence having more than 60%, or more than 70%, for example 75%, or 80%, more preferably more than 85%, for example more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% amino acid identity with respect to the VL and / or VH or fragments thereof of the antibody disclosed herein. This level of amino acid identity may be observed over the entire length of the sequence of the relevant SEQ ID NO: or over 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.

[0079] Preferably, the variant antibody comprises one or more of the CDR sequences described herein.

[0080] In relation to amino acid sequences, "sequence identity" refers to a sequence that has the specified values ​​when evaluated using ClustalW (Thompson JD et al., 1994, Nucleic Acid Res., 22, 4673-4680) with the following parameters: Pairwise alignment parameter method: Slow / Precise, Matrix: PAM, Gap Open Penalty: 10.00, Gap Extension Penalty: 0.10; Multiple alignment parameters - Matrix: PAM, Gap open penalty: 10.00, Delay identity %: 30, End gap penalty: On, Gap separation distance: 0, Negative matrix: None, Gap extension penalty: 0.20, Residue-specific gap penalty: On, Hydrophilic gap penalty: On, Hydrophilic residue: G, P, S, N, D, Q, E, K, R. Sequence identity at specific residues is intended to include only identical residues that have been derivatized.

[0081] Accordingly, the present invention provides antibodies having specific VH and VL amino acid sequences and their variants and fragments, which maintain the function or activity of these VH and VL.

[0082] Accordingly, the present invention encompasses antibodies or their binding fragments comprising variants of VH that retain the ability to specifically bind to citrullinated epitopes in deiminated human histone 2A and / or histone 4. Heavy chain variants 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 sequence identity with unmodified VH. The variants of VH may include at least seven amino acid fragments of hVH22.101f or hVH22.101HC9 (SEQ ID NOs. 11 and 12, respectively) that retain the ability of the antibody or its binding fragment to specifically react with citrullinated epitopes in deiminated human histone 2A and / or histone 4; or The antibody or its binding fragment may include variants of hVH22.101f or hVH22.101HC9 (sequences 11 and 12, respectively) that have at least 70% amino acid sequence identity with hVH22.101f or hVH22.101HC9 (sequences 11 and 12, respectively), and which retain the ability for the antibody or its binding fragment to specifically react with citrullinated epitopes in deiminoized human histone 2A and / or histone 4.

[0083] Polynucleotides, vectors, and host cells The present invention also includes polynucleotides, vectors, and expression vectors encoding antibodies or their conjugated fragments as described herein.

[0084] The present invention also relates to polynucleotides encoding antibodies of the present invention. Accordingly, the polynucleotides of the present invention may encode any antibody or fragment described herein. The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to polymeric forms of nucleotides of any length, which are either deoxyribonucleotides, 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 may be provided in isolated or purified forms.

[0085] The nucleic acid sequence "coding" the selected polynucleotide is a nucleic acid molecule that, when controlled by an appropriate regulatory sequence, is transcribed (in the case of DNA) and translated (in the case of mRNA) in vivo into a polypeptide. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation termination codon at the 3' (carboxy) terminus. For the purposes of the present invention, such nucleic acid sequences may, but are not limited to, viral cDNA, prokaryotic or eukaryotic mRNA, genomic sequences derived from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. The transcription termination sequence may be located on the 3' side of the coding sequence. In one embodiment, the polynucleotide of the present invention comprises a sequence encoding the above-described VH or VL amino acid sequence. The polynucleotide may encode the VH or VL sequence of a particular antibody or its conjugated fragment disclosed herein.

[0086] Accordingly, the antibody or its conjugated fragment of the present invention may be generated from a polynucleotide that encodes and can express it, or may be delivered in the form of the polynucleotide. If the antibody contains two or more chains, the polynucleotide of the present invention may encode one or more antibody chains. For example, the polynucleotide of the present invention may encode an antibody light chain, an antibody heavy chain, or both. Two polynucleotides may be provided, one encoding an antibody light chain and the other encoding the corresponding antibody heavy chain. Linucleotides or polynucleotide pairs can be expressed together to produce the antibody of the present invention.

[0087] The polynucleotides of the present invention are, for example, those described in Sambrook J et al. (1989, Molecular cloning: a laboratory manual; Cold Spring Harbor: New York: Cold Spring As described in Harbor Laboratory Press, it can be synthesized according to methods well known in the art.

[0088] The nucleic acid molecules of the present invention may be provided in the form of an expression cassette containing a control sequence operably ligated to an inserted sequence, thereby enabling the expression of the antibody of the present invention in vivo. These expression cassettes are then typically provided within a vector (e.g., a plasmid or recombinant viral vector). Such an expression cassette may be administered directly to a host subject. Alternatively, a vector containing the polynucleotide of the present invention may be administered to a host subject. Preferably, the polynucleotide is prepared and / or administered using a gene vector. A suitable vector may be any vector that contains a sufficient amount of genetic information and is capable of expressing the polypeptide of the present invention.

[0089] Accordingly, the present invention includes an expression vector comprising such a polynucleotide sequence. Such an expression vector is routinely constructed in the art of molecular biology and may, for example, involve the use of plasmid DNA, as well as appropriate initiators, promoters, enhancers, and other elements, such as polyadenylation signals, which may be necessary and positioned in a precise orientation, in order to express the peptide of the present invention. Other suitable vectors will be apparent to those skilled in the art. As a further example thereof, see Sambrook J. See et al. (1989, Molecular cloning: a laboratory manual; Cold Spring Harbor: New York: Cold Spring Harbor Laboratory Press).

[0090] Those skilled in the art can use the sequences described herein to clone or generate cDNA or genomic sequences, for example, as described in the following examples. These sequences are cloned into a suitable eukaryotic expression vector or derivative thereof, such as pcDNA3 (Invitrogen), and then mammalian cells (such as CHO cells) are transfected with a suitable combination of light and heavy chain-containing vectors, resulting in the expression and secretion of antibodies described herein.

[0091] Those skilled in the art can also produce analogues of the antibodies or their binding fragments described herein by using the specific binding domain of the antibody sequence, and express them in different contexts, such as polypeptides including fusion proteins. This is well known in the art.

[0092] Furthermore, the present invention includes cells modified to express the antibody of the present invention. Examples of 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 the antibody of the present invention include mammalian HEK293, CHO, HeLa, NS0, and COS cells. Preferably, the selected cell line is not only stable but also capable of mature glycosylation.

[0093] Such cell lines of the present invention may be cultured using routine methods to produce the antibody or its conjugated fragment of the present invention, or the antibody or its conjugated fragment of the present invention may be used as a test subject. It may be used therapeutically or prophylactically to deliver to the target. Alternatively, the polynucleotide, expression cassette, or vector of the present invention may be administered ex vivo to cells from a subject, and then the cells may be returned to the subject's body.

[0094] Furthermore, the present invention comprises a process for producing an antibody or a bound fragment thereof that specifically binds to a citrullinated epitope in deiminoated human histone 2A and / or histone 4, the process comprising culturing the host cells described herein and isolating the antibody or a bound fragment thereof from the cells.

[0095] Pharmaceutical composition The present invention encompasses pharmaceutical compositions comprising the antibody of the present invention or a conjugated fragment thereof. The present invention also encompasses pharmaceutical compositions comprising the antibody of the present invention or a conjugated fragment thereof and a pharmaceutically acceptable carrier.

[0096] When used herein, “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc. Preferably, the carrier is suitable for parenteral administration, for example, intravenous, intraocular, intramuscular, subcutaneous, intradermal, or intraperitoneal administration (e.g., by injection or infusion). In certain embodiments, the pharmaceutically acceptable carrier includes at least one carrier selected from the group consisting of cosolvent solutions, liposomes, micelles, liquid crystals, nanocrystals, nanoparticles, emulsions, fine particles, microspheres, nanospheres, nanocapsules, polymers or polymer carriers, surfactants, suspensions, complexing agents such as cyclodextrins, or adsorbent molecules such as albumin, surface-active particles, and chelating agents. In further embodiments, the polysaccharides include hyaluronic acid and its derivatives, dextran and its derivatives, cellulose and its derivatives (e.g., methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose phthalate acetate, cellulose succinate acetate, cellulose butyrate acetate, hydroxypropylmethylcellulose phthalate), chitosan and its derivatives, [β]-glucan, arabinoxylan, carrageenan, pectin, glycogen, fucoidan, chondrothin, dermatan, heparan, heparin, pentosan, keratan, alginate, cyclodextrin, and salts and derivatives containing esters and sulfates thereof.

[0097] Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, buffer water, and physiological saline. Examples of other carriers include ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using a coating material such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using a surfactant. In many cases, it is preferable to include an isotonic agent in the composition, such as sugar, polyalcohols such as mannitol or sorbitol, or sodium chloride.

[0098] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants. These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. The presence of microorganisms can be reliably prevented by the sterilization procedure described above, or by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Furthermore, sustained absorption of injectable pharmaceutical forms can be achieved by including absorption-delaying agents such as aluminum monostearate and gelatin.

[0099] Therapeutic compositions are typically sterile and stable under manufacturing and storage conditions. Pharmaceutical compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0100] Sterile injectable solutions can be prepared by compounding the required amount of an activator (e.g., an antibody) in a suitable solvent, along with one or a combination of the components listed above as needed, followed by sterile microfiltration. Generally, dispersions are prepared by compounding the activator in a sterile vehicle containing a basic dispersion medium and other necessary components listed above. In the case of sterile powders for preparing sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying, which yield any further desired components from the already sterile-filtered solution in addition to the activator powder.

[0101] The pharmaceutical composition of the present invention may contain additional active ingredients in addition to the antibody of the present invention. As described above, the composition of the present invention may contain one or more antibodies of the present invention. The composition may also contain additional therapeutic or prophylactic activators.

[0102] Depending on the route of administration, the antibody or its conjugated fragment may be coated with a material to protect the antibody from the action of acids and other natural conditions that may inactivate or denature the antibody.

[0103] In a preferred embodiment, the pharmaceutical composition according to the present invention is in a form selected from the group consisting of aqueous solutions, gels, hydrogels, films, pastes, creams, sprays, ointments, or wraps.

[0104] In further embodiments, the pharmaceutical compositions described herein may be administered by routes such as intravenous, subcutaneous, intraocular, intramuscular, intraarticular, intradermal, intraperitoneal, or spinal cord, or by other parenteral routes of administration, such as injection or infusion. Administration may be carried out via rectal, oral, ocular, topical, epidermal, or mucosal routes. Administration may be local, including peritumoral, peritumoral, intratumoral, intra-tumoral, intra-surgical margin of tumor, intralesional, peri-lesional, intracavitary injection, intravesicular administration, or inhalation. In preferred embodiments, the pharmaceutical composition is administered intravenously or subcutaneously.

[0105] Furthermore, a kit comprising the antibody or other composition of the present invention and instructions for use is also within the scope of the present invention. The kit may further contain one or more additional reagents, such as additional therapeutic or prophylactic agents, as discussed herein.

[0106] Therapeutic use of the antibody and its conjugated fragments of the present invention The antibodies or their conjugated fragments according to the present invention can be used in therapeutic applications. In therapeutic applications, a subject already suffering from a disorder or condition is administered an amount of the antibody or composition sufficient to cure, alleviate, or partially cessate one or more of the condition or its symptoms. As a result of such therapeutic treatment, the severity of the symptoms of the disease may decrease or the frequency or duration of asymptomatic periods may increase. An amount appropriate to achieve this is defined as a "therapeutably effective amount." An effective amount for a given purpose depends on the severity of the disease or injury, as well as the subject's weight and overall condition. As used herein, the term "subject" includes any human being.

[0107] In certain embodiments, the antibody or its binding fragment of the present invention may be linked (directly or indirectly) to another portion. The other portion may be a therapeutic agent such as a drug. The other portion may be a detectable label. The other portion may be a binding portion such as a binding domain of an antibody or polypeptide specific to a therapeutic target. The antibody or its binding fragment of the present invention may be a bispecific antibody.

[0108] Therapeutic agents or detectable labels can be directly conjugated to the antibody or its binding fragment of the present invention, for example, by chemical conjugation. Methods for conjugating agents or labels to antibodies are known in the art. For example, carbodiimide conjugation (Bauminger S and Wilchek M, 1980, Methods Enzymol., 70, 151-159) can be used to conjugate various agents, including doxorubicin, to antibodies or peptides. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a water-soluble carbodiimide, is particularly useful for conjugating functional moieties to binding sites.

[0109] Other methods can also be used to conjugate the portion to the antibody. For example, reductive alkylation following sodium periodate oxidation of the appropriate reactant may be used, as well as crosslinking of glutaraldehyde. However, regardless of which method is chosen to generate the conjugate of the present invention, it is recognized that it must be confirmed that the antibody retains its targeting ability and the functional portion retains its relevant function.

[0110] Therapeutic agents linked to antibodies may contain therapeutically beneficial polypeptides or polynucleotides encoding polypeptides. Examples of such polypeptides include antiproliferative or anti-inflammatory cytokines.

[0111] Antibodies may be ligated to detectable labels. “Detectable labels” means that the antibody is ligated to a portion that can typically be detected non-invasively from outside the body and from the site where the target is located, after the antibody has been administered to a patient and is positioned at the target site. Therefore, antibodies may be useful in imaging and diagnostics.

[0112] Typically, the label is or contains a radioactive atom useful in imaging. Suitable radioactive atoms include 99mTc and 123I for scintigraphy 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. Clearly, a sufficient amount of the appropriate atomic isotope must be linked to the antibody for easy detection of the molecule.

[0113] Radioactive labels or other labels can be incorporated by known methods. For example, antibodies or fragments thereof may be biosynthesized or synthesized by chemical amino acid synthesis using, for example, a suitable amino acid precursor containing fluorine-19 instead of hydrogen. Labels such as 99mTc, 123I, 186Rh, 188Rh, and 111In can be attached, for example, via cysteine ​​residues in polypeptides. Yttrium-90 can be attached via lysine residues. Preferably, detectable labels include radioactive atoms such as technetium-99m or iodine-123. Alternatively, detectable labels may be selected from the group including iodine-123; iodine-131; indium-111; fluorine-19; carbon-13; nitrogen-15; oxygen-17; gadolinium; manganese; and iron.

[0114] In one embodiment, the antibody of the present invention can selectively bind directly or indirectly to a cytotoxic moiety or a detectable label. Therefore, in this embodiment, the antibody is linked to a moiety that selectively binds to a further cytotoxic or readily detectable compound or component.

[0115] The antibody or conjugated fragment of the present invention, or a composition containing the antibody or fragment, may be administered via one or more routes of administration using one or more methods known in the art. As will be understood by those skilled in the art, the route of administration and / or mode of administration will vary depending on the desired result. A preferred route of administration for the antibody or composition of the present invention is intravenous. Subcutaneous, intraocular, intramuscular, intradermal, intraperitoneal, spinal, or other parenteral administration routes include injection or infusion. The term "parenteral administration," as used herein, means modes of administration other than enteral and topical administration, usually by injection. Administration may be via rectal, oral, ocular, topical, epidermal, or mucosal routes. Administration may be local, including peritumoral, peritumoral, intratumoral, intrasurgical, intra-surgical, intra-surgical margin, intralesional, peri-lesional, intracavitary injection, intravesicular, or inhalation. In preferred embodiments, the pharmaceutical composition is administered intravenously or subcutaneously.

[0116] The preferred dosage of the antibody or its conjugated fragment of the present invention can be determined by a skilled physician. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level depends on a variety of pharmacokinetic factors, including the activity of the particular antibody used, the route of administration, the time of administration, the rate of antibody excretion, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, overall health, and medical history of the patient being treated, and similar factors well known in the medical field.

[0117] The preferred dose of the antibody or its conjugated fragment of the present invention may range, for example, from about 0.1 μg / kg (body weight of the patient being treated) to about 100 mg / kg. For example, preferred doses may be about 1 μg / kg (body weight) to about 50 mg / kg per week, about 100 μg / kg (body weight) to about 25 mg / kg per week, or about 10 μg / kg (body weight) to about 12.5 mg / kg per week.

[0118] Suitable dosages may be approximately 1 μg / kg (body weight) to 50 mg / kg per day, approximately 100 μg / kg (body weight) to 25 mg / kg per day, or approximately 10 μg / kg (body weight) to 12.5 mg / kg per day.

[0119] The administration regimen may be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, it may be administered as a single rapid dose, or in several divided doses over time, or the dose may be proportionally reduced or increased if appropriate depending on the urgency of the treatment situation. It is particularly advantageous to formulate the parenteral composition into unit dosage forms to facilitate administration and ensure uniformity of the dose. A unit dosage form, as used herein, refers to a physically separate unit suitable as a single dose for the subject being treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in conjunction with the necessary pharmaceutical carrier.

[0120] Antibodies may be administered as a single dose or in multiple doses. In the case of multiple doses, they may be administered via the same or different routes and at the same or different locations. Alternatively, antibodies may be administered as a sustained-release formulation, in which case the required frequency of administration will be reduced. The dosage and frequency may vary depending on the half-life of the antibody in the patient and the desired duration of treatment. The dosage and frequency may also vary depending on whether the treatment is prophylactic or therapeutic. For prophylactic use, relatively low doses may be administered over a long period at relatively wide intervals. For therapeutic use, relatively high doses may be administered, for example, until the patient shows partial or complete remission of the disease symptoms.

[0121] The combined administration of two or more agents can be achieved in numerous different ways. In one embodiment, the antibody or its conjugate fragment and the other agent may be administered together in a single composition. In another embodiment, the antibody and the other agent may be administered in separate compositions as part of a combination therapy. For example, the antibody or its conjugate fragment may be administered before, after, or simultaneously with the other agent.

[0122] Diseases to be treated The antibodies or their conjugated fragments of the present invention, or the pharmaceutical compositions defined herein, are particularly suitable for use in the treatment or prevention of citrullination-related conditions such as NET-related conditions and inflammatory states.

[0123] The present invention also optionally includes a method of treating a patient, comprising administering to the patient a therapeutically effective amount of an antibody or its conjugated fragment or a pharmaceutical composition as defined herein, for the treatment or prevention of citrullination-related conditions such as NET-related conditions and inflammatory conditions.

[0124] The present invention also includes antibodies or their conjugated fragments or pharmaceutical compositions as defined herein, for use in the manufacture of pharmaceuticals for preventing or treating citrullination-related conditions such as NET-related conditions and inflammatory conditions.

[0125] The present invention also encompasses pharmaceutical compositions comprising the antibody or its conjugated fragment for treating or preventing citrullination-related conditions such as NET-related conditions and inflammatory states.

[0126] A citrullination-related pathology 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 a disease can be readily determined by those skilled in the art using routine tests available in the art. For example, these diseases may be characterized by the presence of abnormal levels of citrullinated proteins in the affected tissue or tissue associated with the disease. This can be achieved by immunological tests such as Western blotting or ELISA, in which the affected tissue is used as an antigen and citrullination of the antigen can be detected using the anti-citrulline antibodies described herein. Alternatively, those skilled in the art can use proteomics applications such as mass spectrometry to compare the level and type of citrullination in affected tissue to that of healthy tissue from an affected patient.

[0127] NET-related conditions can be considered conditions associated with citrullination. NET-related conditions can be defined as diseases or conditions in which NET formation and NETosis are associated with the pathological state of the disease or condition. Whether NET formation and NETosis play a role in the pathogenesis of a disease can be readily determined by those skilled in the art using routine tests available in the art. For example, these diseases may be characterized by the presence of NETs in the relevant tissues.

[0128] Accordingly, the present invention relates to an antibody or a conjugated fragment thereof for use in the treatment or prevention of NET-related pathological conditions.

[0129] Accordingly, the present invention relates to a method for treating a patient in need of such treatment with a therapeutically effective amount of the antibody or a conjugated fragment thereof, wherein the patient is suffering from a NET-related condition.

[0130] Examples of NET-related conditions include inflammatory states or diseases, inflammatory eye diseases, autoimmune diseases, cancer, and organ health after transplantation.

[0131] "Inflammatory state" or "inflammatory disease" refers to any of the many conditions or diseases characterized by changes in blood vessels: edema and neutrophil infiltration (e.g., acute inflammatory response); tissue infiltration by mononuclear cells; tissue destruction by inflammatory cells, connective tissue cells, and their products; and attempts at repair by replacement of connective tissue (e.g., chronic inflammatory response). Such diseases include, for example, inflammatory arthritis, including rheumatoid arthritis and osteoarthritis, SLE, lupus, sepsis, vasculitis, These include multiple sclerosis, psoriatic arthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, spondyloarthritis, multiple system atrophy, Parkinson's disease, Lewy body dementia, idiopathic pulmonary fibrosis, dry eye diseases, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, and lung diseases such as COPD and bronchitis. Non-granulomatous uveitis may be associated with neutrophil-dominant inflammation, while granulomatous uveitis may be associated with macrophage-dominant inflammation.

[0132] NETs play a role in the pathogenesis of autoimmune diseases, including RA, SLE, and vasculitis. The pathways by which therapeutic antibodies or their conjugate fragments improve disease are likely via inhibition of NETosis, clearance of NET remnants, including toxic histones, and other autoantigens from tissues and circulation, and clearance of toxic histones from tissues and circulation. For many autoimmune diseases, improvement in disease state has been shown in wild-type animals treated with PAD knockout models or PAD inhibitors, which suggests a strong correlation with the amount of NETs and disease severity.

[0133] Thus, the antibodies and their conjugated fragments of the present invention can be used to treat inflammatory conditions or inflammatory diseases and autoimmune diseases.

[0134] In preferred embodiments, the diseases to be treated are NET-related conditions, 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, 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 diseases, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, bronchitis, or other NET-related conditions, such as wound healing in diabetes, cancer, cancer metastasis, and the health of transplanted organs in vivo or ex vivo.

[0135] In preferred embodiments, the diseases to be treated are inflammatory conditions 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, spondyloarthropathy, multiple system atrophy, Parkinson's disease, Lewy body dementia, asthma, allergic rhinovirus exacerbated asthma, allergic asthma, cystic fibrosis, fibrosis, idiopathic pulmonary fibrosis, dry eye diseases, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, and bronchitis.

[0136] Further Embodiments The present invention is further described by the following embodiments: 1. An antibody or its binding fragment that specifically binds to a citrullinated epitope in deiminoized human histone 2A and / or histone 4, a) 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), but is VL CDR1; b) At least one CDR selected from sequence numbers 1-5 and An antibody or its conjugated fragment containing such an antibody.

[0137] 2. a) Containing 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) or It consists of, however, the amino acid sequence is neither QSLLDSDGKTY (sequence number 36) nor QSLVDSDGKTY (sequence number 37), but VL CDR1; b) CDRs of Sequence ID 3 and Sequence ID 5 The antibody or its conjugated fragment as described in 1, comprising:

[0138] 3. a) One of the CDRs of sequence numbers 6, 7, 8, 9, and 10; b) CDRs of Sequence ID 3 and Sequence ID 5 The antibody or its conjugated fragment as described in 2, including the antibody described in 2.

[0139] 4. a) 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), but is VL CDR1; b) CDRs with sequence numbers 1-5 and The antibody or its conjugated fragment as described in 2, including the antibody described in 2.

[0140] 5. a) One of the CDRs of sequence numbers 6, 7, 8, 9, and 10; b) CDRs of sequence numbers 1-5 An antibody or its conjugated fragment as described in any of 1 to 4, including the above.

[0141] 6. a) 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), but is VL CDR1; b) At least one of the CDRs of Sequence IDs 4 and 5; c) i) The heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12; or ii) A fragment of at least seven amino acids of (i) wherein the antibody or its binding fragment retains the ability to specifically react with citrullinated epitopes in deiminated human histone 2A and / or histone 4; or iii) A variant of (i) having at least 70% amino acid sequence identity with the sequence of (i), wherein the antibody or its binding fragment retains the ability to specifically react with citrullinated epitopes in deiminoized human histone 2A and / or histone 4. An antibody or its conjugated fragment according to 1 or 2, comprising:

[0142] 7. a) 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), but is VL CDR1; b) At least one of the CDRs of Sequence IDs 4 and 5; c) The heavy chain variable domain amino acid sequence of SEQ ID NO. 11 or 12 and The antibody or its conjugated fragment as described in 6, including the antibody described in 6.

[0143] 8. a) 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), but is VL CDR1; b) CDRs of sequence numbers 4 and 5; c) The heavy chain variable domain amino acid sequence of SEQ ID NO. 11 or 12 and The antibody or its conjugated fragment as described in 7, including the antibody described in 7.

[0144] 9. a) One of the CDRs of sequence numbers 6, 7, 8, 9, and 10; b) CDRs of sequence numbers 4 and 5; c) The heavy chain variable domain amino acid sequence of SEQ ID NO. 11 or 12 and The antibody or its conjugated fragment as described in 8, including the antibody described in 8.

[0145] 10. a) Amino acid sequences of the heavy chain variable domain of Sequence ID No. 11 and the light chain variable domain of Sequence ID No. 13; 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; c) Amino acid sequence of the heavy chain variable domain of SEQ ID NO: 11 and the light chain variable domain of SEQ ID NO: 15; d) Amino acid sequences of the heavy chain variable domain of SEQ ID NO: 11 and the light chain variable domain of SEQ ID NO: 16; e) Amino acid sequences of the heavy chain variable domain of Sequence ID No. 11 and the light chain variable domain of Sequence ID No. 17; f) Amino acid sequences of the heavy chain variable domain of SEQ ID NO: 12 and the light chain variable domain of SEQ ID NO: 13; g) Amino acid sequence of the heavy chain variable domain of SEQ ID NO: 12 and the light chain variable domain of SEQ ID NO: 14; h) Amino acid sequences of the heavy chain variable domain of SEQ ID NO: 12 and the light chain variable domain of SEQ ID NO: 15; 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 j) Amino acid sequence of the heavy chain variable domain of SEQ ID NO: 12 and the light chain variable domain of SEQ ID NO: 17 An antibody or its conjugated fragment as described in any one of 1 to 9, including the above.

[0146] 11. An antibody or its binding fragment that specifically binds to a citrullinated epitope in deiminoized human histone 2A and / or histone 4, a) CDR1 of sequence number 13, 14, 15, 16, or 17; and b) Amino acid sequence of the heavy chain variable domain of SEQ ID NO: 11 or 12 An antibody or its conjugated fragment containing a CDR.

[0147] 12. An antibody or a conjugated fragment thereof according to any one of 1 to 11, which specifically binds to a peptide selected from the group consisting of SEQ ID NOs: 18, 19, 20, 21, and 22, and binds to deiminoized human histone 2A and / or histone 4.

[0148] 13. An antibody or a conjugated fragment thereof according to any one of 1 to 12, which specifically binds to a citrullinated epitope in deiminoated human histone 2A and / or histone 4 with an affinity of at least 1 nM or less.

[0149] 14. An antibody or its conjugated fragment as described in any one of 1 to 13, 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, single-domain antibodies derived from camels, single-domain antibody fragments derived from shark IgNAR (VNAR), diabodies, triabodies, antikalin, and aptamers.

[0150] 15. The antibody or its conjugated fragment according to any one of 1 to 13, wherein the antibody is preferably a full-length antibody.

[0151] 16. The antibody or its conjugated fragment according to 15, comprising an Fc region such as the IgG1, IgG2, IgG3, or IgG4 region.

[0152] 17. The antibody or its conjugated fragment 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.

[0153] 18. An antibody or its conjugated fragment described in any one of 1-17, conjugated to the additional portion.

[0154] 19. A polynucleotide encoding an antibody or a conjugated fragment thereof as described in any one of 1 to 17, a cloning or expression vector containing the polynucleotide, or a host cell containing the cloning or expression vector.

[0155] 20. A process for producing an antibody or a binding fragment thereof that specifically binds to a citrullinated epitope in deiminoized human histone 2A and / or histone 4, comprising culturing the host cells described in 19 and isolating the antibody or a binding fragment thereof from the cells.

[0156] 21. A pharmaceutical composition comprising an antibody or a conjugated fragment thereof as described in any one of 1 to 18, and at least one pharmaceutically acceptable diluent or carrier.

[0157] 22. The pharmaceutical composition according to 21, further comprising other active ingredients.

[0158] 23. An antibody or a conjugated fragment thereof according to any one of 1 to 18, or a pharmaceutical composition according to 21 or 22, for use in therapeutic purposes.

[0159] 24. An antibody or a conjugated fragment thereof according to any one of 1 to 18, or a pharmaceutical composition according to 21 or 22, for use in a method for treating or preventing NET-related conditions.

[0160] 25. Antibodies, conjugated fragments thereof, or pharmaceutical compositions for use in accordance with 24, wherein the NET-related condition 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, 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 conditions, such as wound healing in diabetes, cancer, cancer metastasis, and the health of transplanted organs in vivo or ex vivo.

[0161] 26. Antibodies, conjugated fragments thereof, or pharmaceutical compositions for use according to any one of 23-25, administered by parenteral routes, such as intravenous, subcutaneous, intraocular, intramuscular, intradermal, intraperitoneal, or spinal routes, or by injection or infusion; or by other routes, such as rectal, oral, ocular, topical, epidermal, mucous membrane, local, peritumoral, near-tumor, intratumoral, to the resection margin of a tumor, intralesional, perilesional, intracavitary injection, intravesicular administration, or inhalation.

[0162] 27. A method for treating a patient, comprising administering to the patient a therapeutically effective amount of an antibody or a conjugated fragment thereof described in any one of 1 to 18, or a pharmaceutical composition described in 21 or 22.

[0163] 28. The method according to 27, wherein the treatment is for the treatment of a NET-related condition.

[0164] The present invention is further illustrated by the following embodiments, which should not be construed as further limitations. All figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. [Examples]

[0165] Example 1: Accelerated stability test of hMQ22.101j / e and hMQ22.101f / g. 0.75 mL aliquots (glass tubes) containing either hMQ2 2.101 j / e (12.5 mg / mL) or hMQ2 2.101 f / g (3.31 mg / mL) in 25 mM Tris-HCl, pH 8.0 were stored at 37°C for 8 weeks. Each week, several 10 μL and 20 μL samples were taken from each glass tube under sterile conditions and stored at -80°C until further analysis (ELISA and mass spectrometry) was performed.

[0166] 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 in-house validated CMC ELISA. 96-well ELISA plates were coated with neutraavidin (0.1 μg / well) by incubation overnight at 4°C. Wells were washed five times with PBS-Tween20 (PBS-T) and blocked by incubation at room temperature (RT) for 2 hours with PBS-T + 1% bovine serum albumin (BSA). After further washing with PBS-T five times, wells were incubated at RT for 1 hour with a histone-derived peptide (SEQ ID NO: 18: SGXGKQGGKARA) containing 3-position citrulline (X) and C-terminal biotin (40 ng / well) in PBS-T + 0.2% BSA. After five further washes with PBS-T, a starting point of 1350 ng / well was used, and the sample was further diluted at a 1:1 ratio in PBS-T + 0.2% BSA until a concentration of 0.66 ng / mL was reached. Calibration curves were then prepared from the reference lot of hMQ22.101j / e or hMQ22.101f / g. Spike quality control (QC) samples prepared from the same reference lot of hMQ22.101j / e or hMQ22.101f / g at high (HQC, 250 ng / mL), medium (MQC, 50 ng / mL), low (LQC, 3.75 ng / mL), and lower limit quality control (LLQC, 1.25 ng / mL) concentrations were diluted with PBS-T + 0.2% BSA and similarly added to plates. These QC samples were used to validate the ELISA results.

[0167] Finally, 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 at RT for 2 hours. The wells were washed 5 times with PBS-T and incubated at RT for 1 hour with rabbit anti-human HRP antibody (1:12.000 in PBS-T + 0.2% BSA), followed by 3 washes with PBS-T and PBS The samples were washed three times. After incubating the wells with TMB substrate for 10 minutes, the reaction was stopped with 2M H2SO4, and then the optical density was measured at a wavelength of 450 nm. Sigmoid calibration curves were plotted and fitted using values ​​from serially diluted reference antibodies. The concentrations of the QC samples and accelerated stability samples were recalculated using the equation from the sigmoid approximation curve. The recalculated antibody concentration of the accelerated stability sample at week 0 was set to 100%, and all other accelerated stability recalculated concentrations (weeks 2, 3, 4, 6, and 8) were calculated as percentages of week 0 (100%) and plotted in a bar graph (upper panel of Figure 1 for hMQ22.101j / e, lower panel of Figure 1 for hMQ22.101f / g).

[0168] Accelerated stability testing showed that the binding affinity of hMQ22.101j / e and hMQ22.101f / g to histone-derived citrulline-containing peptides decreased over time.

[0169] Example 2: Mass spectrometry of an accelerated stability sample of hMQ2 2.101 j / e The cause of the decrease in binding affinity of the hMQ22.101j / e antibody over time was investigated. hMQ22.101j / e has several potential aspartic acid isomerization sites within or near the VL CDR region (CDR1 and CDR2). The objective of this example was to determine the sensitivity of the aspartic acid residue to isomerization by liquid chromatography (LC)-mass spectrometry (MS)-based peptide mapping.

[0170] Prior to digestion, 50 μg of each accelerated stability sample (weeks 0, 4, and 8) was subjected to desalting, reduction with dithiothreitol, and alkylation with iodoacetamide. Following 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 the C-terminus of arginine or lysine. The trypsin digests were analyzed using reversed-phase liquid chromatography (RPLC) (RPLC-UV-MS) combined with UV and mass spectrometry detection. Agilent Technologies 1290 hyphenated on Agilent Technologies 6540 Q-TOF with Jetstream electrospray ionization (ESI) source. Data was acquired using a UHPLC system. Before UV214nm and MS( / MS) detection, water, trifluoroacetic acid, and acetonitrile were used as mobile phase components, and the data was collected on an RPLC column (AdvanceBio Peptide Map C18, 250mm). Samples were separated using a column (L, 2.1 mm ID, 2.7 μm dp, Agilent Technologies). Approximately 4.5 μg was loaded onto the column. The MS system was operated in extended dynamic range mode (2 GHz) with a resolution of 20,000 for a mass of 922.009798, without using a reference mass, achieving high mass accuracy (typically <10 ppm). Two spectra were acquired per second, with an acquisition range of 100–3000 amu in MS and MS / MS modes. MS / MS data were acquired in data-dependent mode. Collision energy was optimized for peptide fragmentation. All MS measurements were performed in positive ionization mode.

[0171] The measured signals were matched to sequences using the BioConfirm algorithm integrated into Agilent MassHunter software. A mass tolerance of 20 ppm was used to match experimental data to sequences. The specified enzyme was trypsin (C-terminal cleavage with lysine or arginine), with 0-2 cleavage errors being tolerated. Peak areas from extracted ion chromatograms obtained with a mass precision of 20 ppm were used for modification quantification. Given the nearly complete sequence coverage, all aspartic acid isomerization candidate sites in the hCDR region were covered. These peptides were manually integrated. Where present, peptides containing isoaspartic acid were integrated with peptides containing aspartic acid. It elutes immediately beforehand. Next, the relative isomerization level was calculated in each case.

[0172] The relative aspartic acid (D) isomerization level of VL CDR1 in hMQ22.101j / e increased over time (Figure 2A). The isomerization sites tested in VL CDR1 and CDR2 are shown in Figure 2A. The isomerization of VL CDR1 was considered to be the cause of the loss of antibody binding affinity over time.

[0173] Example 3: Generation of hMQ22.101 antibodies in which the aspartic acid of three types of VL CDR1 is mutated. Next, we investigated whether deletion of the non-germline isomerization site in VL CDR1 prevented isomerization. Three types of hVL22.101y domain DNA, each containing a single aspartic acid mutation at amino acid position 31 (L:Asp31) of CDR1, were synthesized by GeneArt. L:Asp31 was mutated to alanine, glutamine, or serine based on amino acid similarities such as 1) size, 2) polarity, and 3) charge. These aspartic acid-mutated VL domains were cloned into mammalian expression vectors encoding full-length human light chains. Subsequently, these light chain constructs (hVL22.101h, hVL22.101i, and hVL22.101j) were all used 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. In all cases, full-size antibodies were purified from the culture supernatant using a MabSelect SuRe affinity column in an Akta-FPLC system, and then the buffer was changed to 25 mM Tris-HCl, pH 8.0 using a desalting column. Next, the antibodies were polished using an ion-exchange spin column to remove host cell proteins and residual protein A-derived resin, followed by an endotoxin removal process using a high-volume endotoxin removal resin. Finally, the antibodies were enriched using a MicroSep Advance Centrifugal Device (10K MWCO).

[0174] Example 4: Antigen-binding assay using an hMQ22.101 antibody in which the aspartic acid of VL CDR1 has been mutated. The generated VL CDR1 antibodies hMQ22.101j / h, hMQ22.101j / i, and hMQ22.101j / j, in which the aspartic acid was mutated, were compared with the aspartic acid-containing antibody hMQ22.101j / e using CMC ELISA validated in-house as described in Example 1. Here, the hMQ22.101j / e reference lot was used for calibration curves at 5ng / mL, 10ng / mL, 20ng / mL, 30ng / mL, 40ng / mL, 60ng / mL, 80ng / mL, and 100ng / mL, as well as for separate spike QC samples at 10ng / mL, 20ng / mL, 60ng / mL, and 80ng / mL. hMQ22.101j / h, hMQ22.101j / i, hMQ22.101j / j, and hMQ22.101j / e were tested at 10ng / mL, 20ng / mL, 30ng / mL, 40ng / mL, 80ng / mL, and 100ng / mL, and the dose-response curves were plotted on a graph (Figure 2B).

[0175] Figure 2B shows the optical density results for three VL CDR1 variants (hVL22.101h with a mutation to AS at the CDR1=DS site, hVL22.101i with a mutation to ES at the CDR1=DS site, and hVL22.101j with a mutation to SS at the CDR1=DS site). The most improved optical density results were achieved with the hMQ22.101j / i antibody.

[0176] Example 5: Mass spectrometry following accelerated stability testing of hMQ22.101j / i, in which the aspartic acid of VL CDR1 has mutated. hMQ22.101j / i (12.5mg) in 25mM Tris-HCl, pH 8.0 0.75 mL aliquots (glass tubes) containing ( / mL) were stored at 37°C for 4 weeks. Each week, several 10 μL and 20 μL samples were taken from each glass tube under sterile conditions and stored at -80°C until further analysis (mass spectrometry) was performed.

[0177] Mass spectrometry was performed in the same manner as in Example 2, except that only accelerated stability samples from week 0 and week 4 were used. The percentage of isomerization of hMQ22.101j / i was compared with the percentage of isomerization-containing antibody hMQ22.101j / e (Figure 2C).

[0178] Mass spectrometry data for the hMQ22.101j / i antibody (Figure 2C) still shows a slight increase in isomerization at the CDR1 of the VL over time, but the isomerization problem was largely resolved by deleting the non-germline DS isomerization site at the CDR1 of the VL.

[0179] However, hMQ22.101j / i was not suitable as a therapeutic antibody candidate because it had lower affinity for the target (SEQ ID NO: 18) compared to hMQ22.101j / e.

[0180] Example 6: Generation of other hMQ22.101 isomerized mutants. Next, we performed a comprehensive mutational analysis of the isomerization site in the CDR1 of VL to investigate whether it is possible to remove CDR1 isomerization while maintaining the affinity of the mutant antibody against the target. Seventeen mutant CDR1 domains were generated for hVL22.101. The sequences of these 17 VL CDR1 mutants, as well as the sequences of the non-mutant CDR1 of hVL22.101e and hVL22.101g, are shown in Figure 3A.

[0181] DNA for 17 mutant VL CDR1 domains of hVL22.101 and 4 mutant VH domain variants of hVH22.101 were synthesized using 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, hVL22.101 LC37, hVL22.101LC38, hVL22.101LC39, hVL22.101LC40, hVL22.101LC41, and hVL22.101LC42) were combined with the non-variant heavy chain hVH22.101j or with four variant heavy chains (hVH22.101HC7, hVH22.101HC8, hVH22.101HC9, and hVH22.101HC10). Therefore, the light chain (hVL22.101e, hVL22.101LC16, hVL22.101LC17, hVL22.101LC19, hVL22.101LC20, hVL22.101LC21, hVL22.101LC22, hVL22.101LC23, hVL22.101LC24, hVL22.101LC25, hVL22.101LC26, hVL22.101LC27, hVL22.101LC37, hVL22.101LC38, h HEK293 cells were transiently transfected to generate full-size antibodies (isomerized mutants) using all possible combinations of constructs of VL22.101LC39, hVL22.101LC40, hVL22.101LC41, and hVL22.101LC42) and heavy chains (hVH22.101j, hVH22.101HC7, hVH22.101HC8, hVH22.101HC9, hVH22.101HC10). The antibodies were purified, desalted, polished, and concentrated as described in Example 3.

[0182] Example 7: Analysis of the dissociation rate of the hMQ22.101 isomerized mutant. Off-rate screening for isomerized mutant antibodies was performed using the Octet RED96 instrument (Pall ForteBio). All measurements were performed at 30°C. The streptavidin (SA) biosensor was first washed with PBS for 50 seconds. Citrulline at position 3. Peptides containing both N-terminal histone 2A (SEQ ID NO: 18) and histone 4 (SEQ ID NO: 20), at a concentration of 1 μg / mL and containing both C-terminal biotin, were immobilized on an SA biosensor over 200 seconds. The biosensor was then washed with PBS for 50 seconds, and excess reactive streptavidin molecules were blocked with EZ-link biocitin for 200 seconds. After two more 50-second washes with PBS, a 72 nM antibody diluted in PBS was bound to the biosensor for 200 seconds. The sensor was then immersed in PBS for 4000 seconds to measure their dissociation rates.

[0183] Before plotting the dissociation curves for each antibody, background signals from uncoated biosensors exposed to various antibodies and signals from coated biosensors not exposed to various antibodies were subtracted. Using ForteBio data analysis software 8.1, a 1:1 interaction model (fitting local, full) was applied to calculate the histone 2A and histone 4 dissociation rate constants (kdis × E-07 (1 / s)) for each antibody.

[0184] The results are shown in Figure 3B. Smaller numbers indicate a slower off-rate, which means a slower release of the antigen. 1 × E-07 1 / s is the minimum value detectable by Octet, meaning there is almost no measurable off-rate.

[0185] Several hMQ22.101 isomerized mutants exhibited a dissociation rate 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.

[0186] Example 8: Accelerated stability testing of nine best hMQ22.101 isomerized mutants.

[0187] 0.4 mL aliquots (glass tubes) of the following selected mutant antibodies (ranging from 2.06 to 4.29 mg / mL) in 25 mM Tris-HCl, pH 8.0 were stored at 37°C for 6 weeks.

[0188] [Table 3]

[0189] Each week, several 10 μL and 20 μL samples were taken from each glass tube under sterile conditions and stored at -80°C until further analysis (ELISA and MS analysis) was performed.

[0190] Except for the calibration curve and the use of only the hMQ2 2.101 f / g reference lot for the spike QC samples of HQC, MQC, LQC, and LLQC, antibody samples at weeks 0, 3, and 6 were used in-house validated CMC, as described in Example 1. It was subjected to ELISA.

[0191] The results are shown in Figure 4. Using five of the best-performing isomerized mutants (hMQ22.101f / LC41, hMQ22.101f / LC42, hMQ22.101HC9 / LC21, hMQ22.101HC9 / LC27, hMQ22.101HC9 / LC42, boxed), isomerization at weeks 0 and 6 was evaluated via MS analysis.

[0192] Example 9: Mass spectrometry of five of the best hMQ22.101 isomerized mutants. The 37°C accelerated stability samples from the five antibodies that exhibited the best performance in the accelerated stability test (Example 5) were further analyzed by MS analysis for the isomerization level in VL CDR1.

[0193] [Table 4]

[0194] MS analysis was performed in the same manner as in Example 2, except that only accelerated stability samples from week 0 and week 6 were used. The percentage of isomerization was compared with that of the antibody hMQ22.101j / e (Figure 5). hMQ22.101f / LC41 showed almost no 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 was closer to human than HC9, and the difference in isomerization between week 0 and week 6 was smaller (1.9% vs. 2.6%).

[0195] Example 10: Aggregation and degradation analysis of three high-performance hMQ22.101 isomerized mutants. 37°C accelerated stability samples from three antibodies that showed less VL CDR1 isomerization (Example 6) were further analyzed for their levels of aggregation and degradation.

[0196] [Table 5]

[0197] Measurements were performed using an Agilent 1200 system equipped with an Agilent Zorbax GF-250, 4 μm, 9.4 × 250 mm column, G1311A quaternary pump, G1322A degasser, G1329A autosampler, G1330B thermostat, G1316A column oven, and G1314B VWD detector (Agilent Technologies). 10 μL of antibody was injected and flowed for 10 minutes at a rate of 2 mL / min using a mobile phase consisting of 200 mM NaH2PO4 in H2O, pH 7.0. Proteins were detected using 240 nm ultraviolet light. The main peak of the antibody was detected at approximately 4.25 minutes. The shoulders before and after the main peak were quantified, serving as indicators of aggregation and degradation levels, respectively. The results are shown in Figure 6.

[0198] hMQ22.101f / LC41, hMQ22.101f / LC42, and hMQ22.101HC9 / LC42 exhibited acceptable aggregation and degradation profiles, indicating that they are acceptable for further development.

[0199] Example 11: Fragmentation analysis of the highest-performing hMQ22.101 isomerized mutant. Reverse-phase liquid chromatography (RP-H) combined with UV and mass spectrometry (MS) detection Intact mAb samples were analyzed using PLC (RP-HPLC-UV-MS). Data were acquired using an Agilent Technologies 1290 UHPLC system hyphenated with an Agilent Technologies 6540 Q-TOF equipped with a Jetstream electrospray ionization (ESI) source. Samples were separated using an RPLC column (Zorbax 300 SB-C8, 100 mm L, 2.1 mm ID, 1.8 μm dp, Agilent Technologies) with 0.1% TFA in water as mobile phase A and 0.1% TFA in acetonitrile as mobile phase B. A gradient from 15% B to 80% B was applied over 65 minutes. Approximately 5 μg was loaded into the column. The MS system was operated in high-resolution mode (4 GHz), with a fragmenter voltage of 350 V and a Quad AMU setting of 300. One spectrum per second was acquired in the acquisition range of 300–3200 amu in positive MS mode. The raw spectra were deconvolved using the maximal entropy algorithm integrated into Agilent Technologies' MassHunter software with the BioConfirm add-on. The measured MW was compared to the theoretical MW determined from the complete sequence, taking into account potential C-terminal lysine cleavage and N-glycosylation.

[0200] 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 profiles observed for other therapeutic antibodies used in clinical trials and is acceptable.

[0201] Example 12: Human neutrophil extracellular trap assay. Whole blood was collected from two different healthy donors into heparin sodium tubes (Beckton Dickinson). 30 mL of blood from each donor was mixed with 15 mL of 6% dextran in 0.9% NaCl and incubated at RT for 60 minutes. After incubation, two clear layers were observed: a lower layer containing most red blood cells and an upper layer containing neutrophils. The upper layer was collected and spun down at 300 g for 10 minutes at RT. The pellet was resuspended in 25 mL of PBS, and neutrophils were separated by density gradient centrifugation using a Ficoll-Paque Plus (GE Healthcare), followed by a 10-minute erythrocyte lysis at RT. Cells were counted using a Guava EasyCyte flow cytometer. 900,000 neutrophils per well were seeded in 24-well tissue culture plates (Greiner bio-one) in neutrophil extracellular trap (NET) assay buffer (RPMI 1640 medium (Life Technologies) containing glutamax) supplemented with 1% heat-inactivated fetal bovine serum and 1 mM CaCl2. Neutrophils were stimulated with calcium ionophore A23187 (Molecular Probes) for 4 hours. The effect of NET-reducing 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 at 37°C and 5% CO2 for 4 hours, the cells were very gently washed twice with NET assay buffer. Extracellular DNA was then digested with S7 nuclease (7.5 U / 0.5 mL) at 37°C for 15 minutes, followed by the addition of 10 μL of 500 mM EDTA to halt further digestion. NET was collected from the wells and spun down at 20 g for 5 minutes to remove intact cells. The amount of NET was quantified by measuring the MPO activity in the sample by adding 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate to 50 μL of collected NET.After incubation in RT for 10 minutes, 450 μL of H2SO was added, and the optical density was measured at 450 nm. The background graph was generated from neutrophils that had not been treated with A23187. After subtracting the und signal, the signal from neutrophils treated with A23187+MQR2.201 was set to 100%. The signals from all other antibody treatment groups were compared with those from the A23187+MQR2.201 treatment group (Figure 7).

[0202] Surprisingly, the development candidate hMQ22.101f / LC41 performed better than 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-mutant antibody but also improved them.

[0203] Example 13: A mouse model for experimental inflammation. The purpose of this study was to test the dose-response range of the designated development candidate hMQ22.101f / LC41 or hMQ22.101f / LC42 (with the isomerization issue removed) in a collagen antibody-induced arthritis (CAIA) mouse model, compared with the previous candidate hMQ22.101f / g and the isotype-matched control antibody MQR2.201. Limb and ankle swelling was quantified.

[0204] Arthritis was induced in mice using the CAIA mouse model (catalog number MQ18.101) commercially available from ModiQuest Research BV, according to the manufacturer's specifications. For this purpose, DBA / J1 mice were intravenously injected with 2.8 mg of an anti-collagen-II antibody mix. Three days later, the mice were given another intravenous injection containing 25 μg of LPS to synchronize inflammation among the mice. Simultaneously with the LPS injection, the mice were administered tACPA hMQ22.101f / g, hMQ22.101f / LC41, or hMQ22.101f / LC42 (6.25 mg / kg, 12.5 mg / kg, and 25 mg / kg), an unrelated isotype-matched control antibody MQR2.201 (25 mg / kg), or a placebo (0.9% NaCl saline). Typically, inflammation of the forelimbs and hindlimbs was visible from two days after the LPS injection (i.e., day 5). The degree of limb swelling was macroscopically scored from day 0 to day 13. The highest score for the degree of swelling was 8 (divided by the number of limbs). See the table below for the scoring system.

[0205] [Table 6]

[0206] The results are shown in Figure 8. Mice treated with the therapeutic antibody showed a dose-dependent, significant reduction in limb inflammation compared to mice treated with the control antibody or saline. Both the optimized lead antibodies hMQ22.101f / LC41 and hMQ22.101f / LC42 (with the isomerization issue removed) were superior to the previous lead candidate hMQ22.101f / g. Furthermore, It prevented inflammation, which is clearly demonstrated at a dose of 25 mg / kg (Figure 8, upper panel). No adverse effects were observed. At the lowest dose of 6.25 mg / kg (Figure 8, lower panel), hMQ22.101f / LC41 was superior to all other antibodies, and the p-value of Student's t-test at day 13 was higher for hMQ22 compared to the placebo group. For 101f / LC41, hMQ22.101f / LC42, and hMQ22.101f / g, the results were p<0.001, p<0.05, and p=0.46, respectively.

[0207] Example 14: Further characterization of the development candidate hMQ22.101f / LC41 in a mouse in vitro NET assay. To further reinforce the concept that hMQ22.101f / LC41 is a potent inhibitor of NET formation, in addition to the binding of hMQ22.101f / LC41 to mouse NETs and pre-NETs, ​​the inhibition of mouse NET formation was studied as described below. Pre-NETs are defined as neutrophils with a disintegrated nuclear membrane and an amorphous decondensed nuclear structure containing citrullinated chromatin still present within the cell.

[0208] The purpose of this study was to test whether the designated development candidate hMQ22.101f / LC41 could inhibit NET formation in mice. Neutrophils were isolated from the bone marrow of C57BL / 6J mice by negative selection using the EasySep® Mouse Neutrophil Concentration Kit (Stemcell Technologies) according to the manufacturer's instructions. The purity of the isolated neutrophils was confirmed to be over 90% by flow cytometry using an antibody against Ly6G (Biolegend). The isolated bone marrow neutrophils were divided into 2 × 10⁶ units in HBSS containing calcium and magnesium. 6The concentration was adjusted to cells / mL. A total of 100 μL of cell suspension was added to each well of an 8-well chamber slide (Thermo Fisher Scientific). 150 μL of HBSS containing 25 μg / mL hMQ22.101f / LC41, MQR2.201, or no antibody was incubated with the cells for 15 minutes before adding HBSS containing 1 μg / mL A23187 or vehicle control. 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. Samples were blocked at room temperature for 1 hour with 10% fetal bovine serum (FCS; Biochrome) in PBS. Primary rabbit anti-citH3 antibody (Abcam, ab5103; 1:200) or TRITC-conjugated goat anti-human IgG (Jackson Immunoresearch, 109-025-003; 1:100) was added to 10% FCS in PBS over 12 hours at 4°C. The slides were washed three times with PBS, and secondary Cy5-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch, 111-175-144; 1:400) was added over 1.5 hours at room temperature in the dark. The slides were washed three times again with PBS. A staining solution containing 2.5 μM Hoechst in PBS was added over 15 minutes at room temperature. After washing with PBS, the samples were embedded in mounting medium (BIOZOL). The slides were analyzed using a BZ-X710 microscope (Keyence), and NET and pre-NET were quantified using Fiji imaging software (Figure 9A). Figure 9B shows representative images illustrating the binding of hMQ22.101f / LC41 (hIgG; red) to NET (yellow arrow) and pre-NET (white arrow).

[0209] In vitro treatment of mouse bone marrow (BM) neutrophils with hMQ22.101f / LC41 resulted in reduced NET efflux induced by A23187 compared to mouse BM neutrophils treated with MQR2.201 (Figure 9A). Furthermore, hMQ22.101f / LC41 bound to effluxed mouse NETs (Figure 9B; yellow arrows) and pre-NETs (Figure 9B; white arrows), suggesting that this may be the first step toward macrophage-mediated NET clearance.

[0210] Example 15: Further characterization of the development candidate hMQ22.101f / LC41 in a mouse in vivo NET / macrophage assay using a mouse model of pristane-induced peritonitis.

[0211] Kienhofer et al.(JCI Insight 2017;2(1): Using a pristane-induced mouse model of peritoneal cell influx already described by e92920), we tested the ability of the development candidate hMQ22.101f / LC41 to inhibit NET formation in vivo.

[0212] In short, 500 μL of pristane oil (Sigma-Aldrich) was injected, followed immediately by administration of 50 mg / kg MQR2.201 or hMQ22.101f / LC41. A second injection of 50 mg / kg MQR2.201 or hMQ22.101f / LC41 was administered 12 hours later. After a total of 24 hours, inflammatory cells were isolated from the peritoneum and measured at 1 × 10⁶. 6The cells were adjusted to the required concentration (cells / mL) and transferred to either flow chamber slides or cytospin slides for analysis via 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 conjugate rat anti-mouse F4 / 80 (Biologend, 123120; 1:200), or TRITC conjugate goat anti-human IgG (Jackson ImmunorenoResearch, 109-025-003; 1:100). The slides were washed three times with PBS, and secondary Cy5 conjugate goat anti-rabbit IgG antibody (Jackson ImmunoResearch, 111-175-144; 1:400) was added over 1.5 hours at room temperature in the dark. The slides were washed three times again with PBS. A staining solution containing 2.5 μM Hoechst in PBS was added at room temperature over 15 minutes. After washing with PBS, the samples were embedded in mounting medium (BIOZOL). The slides were analyzed using a BZ-X710 microscope (Keyence) (Figures 10A and C), and NETs and pre-NETs were quantified using Fiji imaging software (Figure 10B). Figure 10C shows the binding of hMQ22.101f / LC41 to NETs and pre-NETs. Figure 11 shows the uptake of NETs containing a large amount of hMQ22.101f / LC41 by macrophages.

[0213] Compared to peritoneal cells from MQR2.201-treated mice, peritoneal cells from hMQ22.101f / LC41-treated mice showed a decrease in NET filaments containing DNA and citrullinated histone 3 (citH3) (Figure 10A). This observation was confirmed by quantification of NETs (colocalization of citH3 and DNA (Hoechst)) (Figure 10B). Colocalization of DNA and citH3 is characteristic of NET formation. Furthermore, hMQ22.101f / LC41 bound to excreted mouse NETs and mouse pre-NETs (Figure 10C), which may be the first step toward NET clearance by macrophages. In fact, F4 / 80-positive macrophages were observed in cell infiltrations containing hMQ22.101f / LC41 phagocytosed in combination with citH3 or neutrophil elastase (Figure 11).

[0214] Example 16: RA CIA mouse model To investigate the efficacy of tACPA against NET-induced tissue damage, various tapering tACPA strategies were used in a mouse model of chronic collagen-induced arthritis (CIA) in rheumatoid arthritis (RA).

[0215] To induce chronic arthritis, bovine collagen II was diluted to a concentration of 2 mg / mL in 0.05 M acetic acid and emulsified with an equal volume of Freund's complete adjuvant. On day 0, the tail base of 10-12 week old male DBA / J1 mice was intradermally immunized with 100 μg of bovine CII. On day 21, mice were injected with an ip booster 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 observed in the fingers or other parts of the limbs. Arthritis in each limb was scored as described above (CAIA mouse model of RA). If the mean arthritis score (MAS) was ≥0.75 on an arbitrary scale of 0-8 (0-2 per limb), the disease was considered to be Therapeutic treatment was initiated early after the onset of symptoms (days 21-28). Therapeutic administration of the specified doses of hMQ22.101j / e (50 / 10 / 10, 30 / 30 / 30 / 10, and 50 / 50 / 50 / 15 mg / kg) administered via repeated IV injections four times with four days between doses resulted in a 38%, 52%, and 81% reduction in MAS at day 14 compared to 50 / 50 / 50 / 50 mg / kg MQR2.201, respectively (Figure 12B). Mice were discontinued 14 days after the start of treatment. Ankle and knee joints were excised and preserved in formalin for histological analysis.

[0216] Of particular note is that all hMQ22.101j / e treatments prevented disease onset for the first 8 days, after which MAS levels 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 the disease for a total of 14 days without MAS levels exceeding 0.75.

[0217] To further investigate the effects of tACPA on bone injury, radiographic analyses of the knee and ankle of all hindlimb mice treated with hMQ22.101j / e and MQR2.201 were performed. Consistent with the observed MAS, all hMQ22.101j / e treatments suppressed bone injury in both the ankle and knee (Figure 12C). To gain further insight into the protective effects of tACPA, histological analyses of the ankle joint were performed using H&E and safranin O(SO) staining. Compared to MQR2.201-treated mice, hMQ22.101j / e inhibited the influx of inflammatory cells (Figure 12D). Furthermore, compared to MQR2.201-treated mice, hMQ22.101j / e significantly reduced bone and cartilage erosion, as well as cartilage proteoglycan depletion and chondrocyte death (Figures 12E-H). These data demonstrate that tACPA strongly reduces symptoms of arthritis, including joint damage.

[0218] Next, the presence of neutrophils and NETs in the limbs of CIA mice administered 50 / 50 / 50 / 15 mg / kg hMQ22.101j / e or 50 / 50 / 50 / 50 mg / kg MQR2.201 was investigated. Mouse neutrophil markers Ly6G, citrullinated histone 3 (citH3), and myeloperoxidase (MPO) were shown in MQR2.201-treated animals, but these markers were almost absent in hMQ22.101j / e-treated mice (Figure 12I). DAPI was used for nuclear and extracellular DNA staining (Figure 12I). Neutrophils (Ly6G) and NETs (co-localization of citH3 and MPO) were quantified by analysis of multiple joints in the right hind limb of each animal, including the tibiotarsal joint, proximal intertarsal joint, distal intertarsal joint, and tarsometatarsal joint. Compared to MQR2.201-treated mice, hMQ22.101j / e-treated mice showed reduced levels of both neutrophils (Figure 12J) and NETs (Figure 12K) in their joints. The amount of NETs in the joints was found to be significantly correlated with macroscopic limb swelling (Figure 12L; r=0.6120, P=0.0041). Similarly, a significant correlation was observed between limb swelling and the presence of neutrophils in the joints (Figure 12M; r=0.8729, P<0.0001). In summary, these data indicate that tACPA treatment eradicates NETs in inflammatory tissue in vivo, thereby preventing severe bone and tissue destruction.

[0219] Example 17: hMQ22.101j / e does not bind to healthy leukocytes. Blood samples from healthy volunteers (HVs), collected using lithium heparin tubing, were obtained from the Sanquin Blood Bank in Nijmegen, Netherlands. Informed consent was obtained from all donors. Peripheral blood mononuclear cells (PBMCs) and neutrophils were separated by Ficol density gradient centrifugation. PBMCs were collected and washed three times with RPMI1640 (hereinafter referred to as RPMI 10%) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FCS) and 50 U / mL penicillin-streptomycin to remove platelets. The neutrophil / erythrocyte suspension was mixed with 6% (w / v) dextran in 0.9% NaCl and incubated at room temperature for 25 minutes. Incubated. Thereafter, neutrophils were collected and exposed to ammonium chloride-potassium (ACK) buffer for 10 minutes at room temperature to lyse the remaining red blood cells, and washed twice with 10% RPMI.

[0220] PBMCs and neutrophils were seeded in 96-well V-bottom plates at a density of 2×10 5 cells / well. To block Fc receptors, cells were incubated with Human Trustain FcX (diluted 1:50 in FACS buffer) for 20 minutes at room temperature. Thereafter, PBMCs were incubated with an antibody mix containing 6.25 μg / mL HiLyte™ Fluor488-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 mix containing 6.25 μg / mL HiLyte™ Fluor488-conjugated hMQ22.101j / e, 83 ng / mL anti-CD45, and 83 ng / mL anti-CD66b. As a positive control for HiLyte™ Fluor488-conjugated hMQ22.101j / e binding, neutrophils were stimulated with 5 μM A23187 for 45 minutes before blocking Fc receptors. After antibody incubation, PBMCs and neutrophils were fixed with 4% formaldehyde for 15 minutes at room temperature, washed with FACS buffer, and analyzed on a CytoFLEX flow cytometer.

[0221] HiLyte™ Fluor488-conjugated hMQ22.101j / e did not bind to healthy resting T cells, B cells, monocytes, natural killer (NK) cells dendritic cells (DCs), or neutrophils, but bound to activated neutrophils (Figure 13). Equivalent results are expected for the hMQ22.101f / LC41 antibody.

[0222] Sequence Listing CDR1 of sequence numbers 1-msVH22.101 and hVH22.101(HC)x GYTFTNYG CDR2 of sequence numbers 2-msVH22.101 and hVH22.101(HC)x INTYSGEA CDR3 of sequence numbers 3-msVH22.101 and hVH22.101(HC)x LRGYTYQSFDEGGDY CDR2 of sequence numbers 4-msVL22.101 and hVL22.101(LC)y LVS CDR3 of sequence numbers 5-msVL22.101 and hVL22.101(LC)y WQGTHFPYT CDR1 of sequence number 6-hVL22.101LC17 QSLLDTDGKTY CDR1 of sequence number 7-hVL22.101LC21 QSLLDSDAKTY CDR1 of sequence number 8-hVL22.101LC27 QSLLDTDAKTY CDR1 of sequence number 9-hVL22.101LC41 QSLLDADGKTY CDR1 of sequence number 10-hVL22.101LC42 QSLLDNDGKTY Sequence ID 11-hVH22.101f RIQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYAQKFQGRVTMTRDTSISTAY MELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS Sequence ID 12-hVH22.101HC9 RIQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYVDDFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS Sequence ID 13-hVL22.101LC17 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDTDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Sequence ID 14-hVL22.101LC21 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDAKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Sequence ID 15-hVL22.101LC27 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDTDAKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Sequence ID 16-hVL22.101LC41 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDADGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Sequence ID 17-hVL22.101LC42 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDNDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Sequence ID 18 - Sequence ID 1 from International Publication No. 2016092082, derived from histone 2A (used in Example 1 / 7) SGXGKQGGKARA In the formula, X is citrulline. Sequence ID 19 - Sequence ID 2 from International Publication No. 2016092082, derived from histone 4 (used in Example 7) SGXGKGGKGLGKGGAKRHRKVLR In the formula, X is citrulline. Sequence ID 20 - abbreviated version of Sequence ID 20, derived from histone 4, International Publication No. 2016092082 (used in Example 7) SGXGKGGKGLGK In the formula, X is citrulline. Sequence ID 21 - Peptide number 4 (Human Histone 2A) (Sequence ID 24 from International Publication No. 2011070172) QFPVGXVHRLLR In the formula, X is citrulline. Sequence ID 22 - Peptide number 6 (Human Histone 2A) (Sequence ID 26 from International Publication No. 2011070172) VHRLLXKGNYSE In the formula, X is citrulline. Human heavy chain constant domain of SEQ ID NO: 23-IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Sequence ID 24 - Human κ chain constant domain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 25-msVH22.101 RIQLVQSGPELKKPGEAVKISCKASGYTFTNYGMHWMKQTPGKDFRWMGWINTYSGEATYVDDFKGRFAFSLGTSASTAYLQINNLKNDDTATYFCLRGYTYQSFDEGGDYWGQGTALTVSS Sequence number 26 - hVH22.101j QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS Sequence number 27 - hVH22.101HC7 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS Sequence number 28 - hVH22.101HC8 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYVDDFQGRVTITADESTSTAYMELSSLRSEDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS Sequence number 29 - hVH22.101HC10 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYVDDFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS Sequence number 30 - msVL22.101 DVVMTQTPLTLSVTTGQPASISCKSSQSLLDSDGKTYLNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPYTFGGGTNLEIK Sequence number 31 - hVL22.101e DVVMTQSPLSLPVTLGQPASISCRSSQSLVDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK SEQ ID NO: 32-hVL22.101g DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Sequence ID 33-hVL22.101h DVVMTQSPLSLPVTLGQPASISCRSSQSLVASDGKTYLNW FQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Sequence ID 34-hVL22.101i DVVMTQSPLSLPVTLGQPASISCRSSQSLVESDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Sequence ID 35-hVL22.101j DVVMTQSPLSLPVTLGQPASISCRSSQSLVSSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK CDR1 of sequence number 36-msVL22.101 and hVL22.101g QSLLDSDGKTY CDR1 of sequence number 37-hVL22.101e QSLVDSDGKTY CDR1 of sequence number 38-hVL22.101h QSLVASDGKTY CDR1 of sequence number 39-hVL22.101i QSLVESDGKTY CDR1 of sequence number 40-hVL22.101j QSLVSSDGKTY CDR1 of sequence number 41-hVL22.101LC16 QSLLESDGKTY CDR1 of sequence number 42-hVL22.101LC19 QSLLDSEGKTY CDR1 of sequence number 43-hVL22.101LC20 QSLLDSSGKTY CDR1 of sequence number 44-hVL22.101LC22 QSLLESEGKTY CDR1 of sequence number 45-hVL22.101LC23 QSLLESSGKTY CDR1 of sequence number 46-hVL22.101LC24 QSLLESDAKTY CDR1 of sequence number 47-hVL22.101LC25 QSLLDTEGKTY CDR1 of sequence number 48-hVL22.101LC26 QSLLDTSGKTY CDR1 of sequence number 49-hVL22.101LC37 QSLLDSAGKTY CDR1 of sequence number 50-hVL22.101LC38 QSLLESAGKTY CDR1 of sequence number 51-hVL22.101LC39 QSLLDAEGKTY CDR1 of sequence number 52-hVL22.101LC40 QSLLDNEGKTY Sequence ID 53-msFibβ XG (Sequence ID 37 from International Publication No. 2011070172) EPTDSLDAXGHRPVDRR In the formula, X is citrulline. Sequence ID 54 - msVim XS / XL (Sequence ID 38 from International Publication No. 2011070172) YVTXSSAVXLXSSVP In the formula, X is citrulline. CDR2 peripheral region of sequence numbers 55-msVL22.101 and hVL22.101(LC)y LVSKLDS Heavy chain constant domain of SEQ ID NO: 56-hCH22.101f astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyi cnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgv evhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk

Claims

1. An antibody or its binding fragment that specifically binds to citrullinated epitopes in deiminated human histone 2A and / or histone 4, a) CDR1 of VL consisting of the sequence of sequence numbers 6 (QSLLLDTDGKTY), 7 (QSLLDSDAKTY), 8 (QSLLLDTDAKTY), or 10 (QSLLNDGKTY); b) VH CDR1 consisting of sequence number 1 (GYTFTNYG), VH CDR2 consisting of sequence number 2 (INTYSGEA), VH CDR3 consisting of sequence number 3 (LRGYTYQSFDEGGDY), VL CDR2 consisting of sequence number 4 (LVS), and VL CDR3 consisting of sequence number 5 (WQGTHFPYT) An antibody or its conjugated fragment containing such an antibody.

2. a) CDR1 of VL consisting of sequence 6, 7, 8, or 10; b) CDR2 of VL consisting of the sequence of sequence number 4 and CDR3 of VL consisting of the sequence of sequence number 5; c) The heavy chain variable domain amino acid sequence of SEQ ID NO: 11 or 12 and The antibody or its conjugated fragment according to claim 1, comprising:

3. 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; 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; 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; 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: 17; e) 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; 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: 14; 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: 15; or h) Amino acid sequence of the heavy chain variable domain of SEQ ID NO: 12 and amino acid sequence of the light chain variable domain of SEQ ID NO: 17 The antibody or its conjugated fragment according to claim 1 or 2, comprising:

4. An antibody or a conjugated fragment thereof according to any one of claims 1 to 3, which specifically binds to a peptide selected from the group consisting of SEQ ID NOs: 18, 19, 20, 21, and 22, and binds to deiminoized human histone 2A and / or histone 4.

5. An antibody or a conjugated fragment thereof according to any one of claims 1 to 4, which specifically binds to a citrullinated epitope in deiminoated human histone 2A and / or histone 4 with an affinity of at least 1 nM or less.

6. An antibody or its conjugated fragment according to any one of claims 1 to 5, selected from the group consisting of recombinant antibody, single-chain antibody, single-chain variable fragment (scFv), variable fragment (Fv), fragment antigen-binding region (Fab), diabody, and triabody.

7. An antibody or a conjugated fragment thereof according to any one of claims 1 to 6, which is a full-length antibody.

8. The antibody or its conjugated fragment according to claim 7, comprising an Fc region selected from the IgG1, IgG2, IgG3, or IgG4 region.

9. The antibody or its conjugated fragment according to claim 8, wherein the heavy chain constant region includes SEQ ID NO: 23 or 56, and / or the light chain constant region includes SEQ ID NO:

24.

10. An antibody or a conjugated fragment thereof according to any one of claims 1 to 9, conjugated to an additional portion.

11. A polynucleotide encoding an antibody or a conjugated fragment thereof according to any one of claims 1 to 9, a cloning or expression vector containing the polynucleotide, or a host cell containing the cloning or expression vector.

12. A process for producing an antibody or a binding fragment thereof that specifically binds to a citrullinated epitope in deiminoized human histone 2A and / or histone 4, comprising culturing the host cells described in claim 11 and isolating the antibody or a binding fragment thereof from the cells.

13. A pharmaceutical composition comprising an antibody or a conjugated fragment thereof according to any one of claims 1 to 10, and at least one pharmaceutically acceptable diluent or carrier.

14. The pharmaceutical composition according to claim 13, further comprising other active ingredients.

15. A drug for the treatment or prevention of NET-related pathological conditions, comprising, as an active ingredient, an antibody or a conjugated fragment thereof as described in any one of claims 1 to 10, or the pharmaceutical composition as described in claim 13 or 14.

16. The agent according to claim 15, wherein the NET-related pathological condition is selected from the group consisting of 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, 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, and bronchitis.

17. The agent according to claim 15, wherein the NET-related pathological condition is selected from the group consisting of wound healing in diabetes, cancer, cancer metastasis, and the health of transplanted organs in vivo or ex vivo.

18. The agent according to any one of claims 15 to 17, which is administered by a parenteral route or by another route of administration.

19. The agent according to claim 18, wherein the parenteral administration route is administered intravenously, subcutaneously, intraocularly, intramuscularly, intradermally, intraperitoneally, or by spinal cord, or by injection or infusion.

20. The drug according to claim 18, wherein the other route of administration is administered by rectal, oral, ocular, topical, epidermal, mucous membrane, local, peritumoral, near-tumor, intratumoral, to the resection margin of the tumor, intralesional, peri-lesional, intracavitary injection, intravesical administration, or inhalation.

Images