Dosage

Abstract

The present invention provides an antibody or a conjugated fragment of a citrulline-containing epitope used for the treatment or prevention of diseases associated with the release of extracellular traps from cells, such as neutrophil extracellular trap (NET)-associated pathology (NET-associated pathology) or eosinophil extracellular trap (EET)-associated pathology (EET-associated pathology), and provides a method comprising administering at least one dose of the antibody at a specific concentration. The present invention also provides the method itself. NET-related pathologies include systemic lupus erythematosus (SLE), lupus, sepsis, vasculitis, inflammatory arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, myositis (polymyositis and dermatomyositis), Sjögren's disease, antiphospholipid syndrome, Behçet's disease, spondylitis, spondyloarthritis, multiple system atrophy, Parkinson's disease, Lewy body dementia, asthma, allergic rhinovirus exacerbated asthma, allergic asthma, acute respiratory distress syndrome, cystic fibrosis, fibrosis and idiopathic pulmonary fibrosis, heart failure, atherosclerosis, dry eye disease, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, bronchitis, or wound healing in diabetes, cancer, cancer metastasis, or wound healing in diabetes, cancer, cancer metastasis, and in This includes other NET-related pathologies, such as the health of transplanted organs in vivo or ex vivo.The present invention also relates to wound healing in SLE, lupus, sepsis, vasculitis, inflammatory arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, myositis (polymyositis and dermatomyositis), Sjögren's disease, antiphospholipid antibody syndrome, Behçet's disease, spondylitis, spondyloarthritis, multiple system atrophy, Parkinson's disease, Lewy body dementia asthma, allergic rhinovirus exacerbating asthma, allergic asthma, acute respiratory distress syndrome, cystic fibrosis, fibrosis and idiopathic pulmonary fibrosis, heart failure, atherosclerosis, dry eye disease, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, bronchitis, thrombotic disease, cardiovascular disease, or diabetes, cancer, cancer metastasis, or wound healing in diabetes, cancer, cancer metastasis, and in vivo or ex vivo The present invention provides pharmaceutical compositions and methods for treating and preventing NET-related pathologies, including other NET-related pathologies such as the health of transplanted organs in vivo. NET-related pathologies include eosinophilic diseases or conditions of the skin, respiratory eosinophilic diseases or conditions, gastrointestinal eosinophilic diseases or conditions, allergic diseases or conditions, or eosinophilic diseases or conditions such as helminthic, fungal, viral or bacterial infections.

Description

Technical Field

[0001] Field of Invention The present invention relates to an antibody or a binding fragment thereof against a citrulline-containing epitope for use in a method for treating or preventing a disease associated with extracellular trap release from cells, such as neutrophil extracellular trap (NET)-related pathology or eosinophil extracellular trap (EET)-related pathology, and particularly relates to the dosage of the antibody or a binding fragment thereof used. Furthermore, the present invention relates to a method for treating or preventing neutrophil extracellular trap (NET)-related pathology or eosinophil extracellular trap (EET)-related pathology, which comprises administering an antibody or a binding fragment thereof against a citrulline-containing epitope to a subject in need thereof at a specific dosage.

Background Art

[0002] Background of the Invention The inflammatory process plays an important role in the normal response of the body to infection, while in inflammatory disorders, for example, when the immune system is activated against the body's own tissues, or when the nature of the immune response to a pathogen is too strong and causes damage to the body, it forms part of the underlying pathogenesis of the disorder. Considering the large number of disorders involving inflammation, inflammation is a major problem in the medical industry. Inflammation is often subdivided into acute inflammation and chronic inflammation. Chronic inflammation is considered to be an inflammation of an extended period (weeks to months) in which active inflammation, tissue destruction, and attempts at healing proceed simultaneously. Chronic inflammation may follow an acute inflammatory episode, but for example, it may start as a insidious process that progresses over time as a result of a persistent infection that causes a delayed hypersensitivity reaction (e.g., tuberculosis, syphilis, fungal infection), long-term exposure to endogenous (e.g., elevated plasma lipids) or exogenous (e.g., silica, asbestos, tobacco tar, surgical sutures) toxins, or an autoimmune reaction against the body's own tissues (e.g., rheumatoid arthritis, systemic lupus erythematosus, vasculitis, multiple sclerosis, psoriasis).

[0003] Inflammatory disorders include those in which research has likely focused on other aspects of the disorder. One such condition is Parkinson's disease (PD), in which much research has focused on the formation of tau protein neurofibrillary tangles. However, the underlying pathogenesis of PD also involves inflammation, which is thought to be the cause of the underlying damage to the central nervous system (CNS), along with neutrophils, which are thought to play a role in its inflammation.

[0004] 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. They can capture and kill various bacterial, fungal, viral, and protozoan pathogens, and their release is one of the first lines of defense against pathogens. After activation by microorganisms or cytokines, histones undergo hypercitrullination, and the neutrophil nucleus undergoes chromatin decondensation, leading to NET formation through netosis, a form of neutrophil cell death.

[0005] NETs play a pathological role in various diseases, for example, by causing abnormal inflammation. Therefore, NETs are involved in the pathology of various inflammatory conditions, including systemic lupus erythematosus (SLE), lupus, sepsis, vasculitis, inflammatory arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, hidradenitis suppurativa, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, myositis (polymyositis and dermatomyositis), Sjögren's disease, Behçet's disease, spondylitis, spondyloarthritis, multiple system atrophy, Parkinson's disease, Lewy body dementia asthma, allergic rhinovirus exacerbating asthma, COPD, acute respiratory distress syndrome, cystic fibrosis, idiopathic pulmonary fibrosis, heart failure, and atherosclerosis. For example, NETs can cause exposure of autoantigens to the extracellular space, followed by the production of pathological autoantibodies by the subject. Furthermore, NETs and NET residues contain toxic histones that induce vascular damage and subsequent organ damage and organ failure. Therefore, inhibiting NET formation and inducing the removal of NETs and NET residues from circulation and tissues are therapeutic benefits in these diseases. Neutrophils are also increasingly recognized as an important component in tumor progression. Numerous studies demonstrating the essential role of neutrophils in tumor development have shown that neutrophils play a significant role at almost every stage of tumor progression. Studies have also suggested that NETs are promoters of tumor progression and metastasis. It has also been shown that neutrophils, through NET development, promote (intratumoral platelet activation), providing a scaffold and stimulus for platelet adhesion, thrombus formation, and coagulation within the tumor.

[0006] NETs are also involved in the decline of organ health after transplantation. NETs contribute to primary graft failure, which is a cause of early death after lung transplantation. NETs have been shown to play a pathogenic role in solid organ transplantation.

[0007] A promising approach to treating inflammatory diseases targeting NET production is the use of antibodies that bind to citrullinated epitopes at the amino terminus of histone 2A and / or histone 4. These antibodies may be used to treat citrullination-related diseases or pathologies, such as NET-associated pathologies and inflammatory conditions. Antibodies that bind to citrullinated epitopes in deiminated human histone 2A and histone 4 are described in WO2009147201, WO2011070172, WO2016092082, and in particular WO2020 / 038963.

[0008] Eosinophils are a type of circulating white blood cell that normally makes up about 1-3% of white blood cells (WBCs) in healthy individuals. Eosinophils play a wide variety of roles in homeostasis and various diseases, including allergies and infections. It has been revealed that a specific mechanism of activated cytolytic eosinophil cell death releases eosinophil extracellular traps (EETs) and the entire cellular contents. This is called eosinophil extracellular trap cell death (EETosis). Charcot-Leyden crystals (a classic pathological marker of eosinophilic inflammation) have also been shown to be associated with EETosis, and the presence of EETosis and EETs has been reported in multiple diseases.

[0009] An antibody capable of inhibiting EETosis by binding to a citrullinated epitope at the amino terminus of histone 2A and / or histone 4 is disclosed in WO2022233931. WO2022233931 discloses a method for treating or preventing EET-related pathologies using the antibody. Such pathologies may include: eosinophilic diseases of the skin; eosinophilic diseases of the respiratory tract; eosinophilic diseases of the gastrointestinal tract; allergic diseases; or infections of helminths, fungi, viruses, or bacteria. In addition, arteriosclerosis may also be treated or prevented. In another embodiment, vasculitis may also be treated.

[0010] Further optimization of the treatment of the above-mentioned diseases using such antibodies is necessary. [Overview of the project]

[0011] The inventors have identified particularly effective doses of antibodies or binding fragments that specifically bind to citrullinated epitopes on deiminated human histone 2A and / or histone 4 for use in methods of treating or preventing diseases associated with the release of extracellular traps from cells, such as neutrophil extracellular trap (NET)-associated pathology or eosinophil extracellular trap (EET)-associated pathology in subjects requiring such treatment. The doses of the antibodies or binding fragments range from 0.01 mg / kg to 0.6 mg / kg. In one particularly preferred embodiment, the provided dose not only produces the desired effect on NETs and / or EETs but also results in specific cytokine levels that differ from those obtained when higher doses of the antibodies or binding fragments are used. For example, in one embodiment, the dose may result in increased IL-10 release in the subject. In another embodiment, the dose results in a decrease in at least one level of calprotectin, P-selectin, CXCL10, TNF-α, and VCAM in the subject.

[0012] Accordingly, the present invention provides an antibody or its conjugated fragment that specifically binds to citrullinated epitopes on deiminated human histone 2A and / or histone 4, for use in methods of treating or preventing diseases associated with the release of extracellular traps from cells, such as neutrophil extracellular trap (NET)-associated pathology or eosinophil extracellular trap (EET)-associated pathology in subjects requiring such treatment, the antibody or its conjugated fragment being administered in doses of about 0.01 mg / kg to about 0.6 mg / kg.

[0013] The present invention also provides a method for treating neutrophil extracellular trap-associated pathology (NET-associated pathology) in subjects requiring treatment, comprising administering to a subject an antibody or its conjugated fragment that specifically binds to citrullinated epitopes on deiminated human histone 2A and / or histone 4, at a dose of about 0.01 mg / kg to about 0.6 mg / kg.

[0014] The present invention also provides a method for treating eosinophilic extracellular (EET) trap-associated pathology (EET-associated pathology) in subjects requiring treatment, comprising administering to a subject an antibody or its conjugated fragment that specifically binds to citrullinated epitopes on deiminated human histone 2A and / or histone 4, at a dose of about 0.01 mg / kg to about 0.6 mg / kg.

[0015] Sequence listings and brief explanation of terminology Antibody terminology CDR = Complementarity Determination Area. VH = Heavy Chain Variable Domain VL = Light chain variable domain CH = Heavy Chain Steady Domain CL = Light chain constant domain. msVH22.101 = Mouse VH of therapeutic antibody. msVL22.101 = Mouse VL of therapeutic antibody. hVH22.101x = Humanized VH of the therapeutic antibody, where "x" refers to the heavy chain. hVL22.101y = Humanized VL of therapeutic antibody, where 'y' refers to the light chain. hVH22.101(HC)x = Optimized humanized VH for therapeutic antibodies, where "(HC)x" refers to the heavy chain. hVL22.101(LC)y = Optimized humanized VL for therapeutic antibodies, where "(LC)y" refers to the light chain. hMQ22.101x / y = Humanized therapeutic antibody, where "x" represents the heavy chain and "y" represents the light chain. hMQ22.101(HC)x / (LC)y = The optimized humanized therapeutic antibody of the present invention, where "(HC)x" refers to the heavy chain and "(LC)y" refers to the light chain.

[0016] JPEG2026513830000001.jpg221170

[0017] JPEG2026513830000002.jpg178170

Brief Description of the Drawings

[0018] [Figure 1] CIT-013 epitope levels ((Units / ml) in sera from LPS-challenged healthy volunteers treated with placebo. A) Cohort 1 (n = 3) and B) Cohort 2 (n = 6). LPS was dosed at 2 ng / kg 2 hours after placebo treatment. This assay measured only in the placebo group because it relied on the antibody-binding epitopes of CIT-013 that would cross-compete with the CIT-013 present in the sera of volunteers dosed with CIT-013. ULOQ = upper limit of quantification. [Figure 2] Levels of citrullinated histone 3 (ng / ml) in sera from two cohorts of LPS-challenged healthy volunteers treated with either placebo or CIT-013. LPS was dosed at 2 ng / kg 2 hours after placebo or CIT-013 treatment. Cohort 1 was treated with A) placebo (n = 3) or B) 0.3 mg / kg CIT-013 (n = 3). Cohort 2 was treated with C) placebo (n = 6) or D) 0.9 mg / kg CIT-013 (n = 8). E) shows the calculated area under the curve (AUC) in healthy volunteers treated with placebo and CIT-013 in Cohort 2. An unpaired Mann-Whitney test was performed to calculate statistical differences. ***, p = 0.00075. LLOQ is the lower limit of quantification. [Figure 3] Levels of CXCL10 (pg / ml) in plasma from two cohorts of LPS-challenged healthy volunteers treated with either placebo or CIT-013. LPS was dosed at 2 ng / kg 2 hours after placebo or CIT-013 treatment. Cohort 1 was treated with A) placebo (n = 3) or B) 0.3 mg / kg CIT-013 (n = 3). Cohort 2 was treated with C) placebo (n = 6) or D) 0.9 mg / kg CIT-013 (n = 8). The data points circled in a round at 20000 pg / ml indicate that the upper limit of quantification was reached and further sample dilution was impossible. [Figure 4]Levels of TNF-α (pg / ml) in plasma from two cohorts of LPS-challenged healthy volunteers treated with either placebo or CIT-013. LPS was dosed at 2 ng / kg 2 hours after placebo or CIT-013 treatment. Cohort 1 was treated with A) placebo (n = 3) or B) 0.3 mg / kg CIT-013 (n = 3). Cohort 2 was treated with C) placebo (n = 6) or D) 0.9 mg / kg CIT-013 (n = 8). Data points circled in a round shape indicate that the upper limit of quantification was reached and further sample dilution was impossible. [Figure 5] Levels of VCAM-1 (pg / ml) in plasma from two cohorts of LPS-challenged healthy volunteers treated with either placebo or CIT-013. LPS was dosed at 2 ng / kg 2 hours after placebo or CIT-013 treatment. Cohort 1 was treated with A) placebo (n = 3) or B) 0.3 mg / kg CIT-013 (n = 3). Cohort 2 was treated with C) placebo (n = 6) or D) 0.9 mg / kg CIT-013 (n = 8). [Figure 6] Levels of IL-10 (pg / ml) in plasma from two cohorts of LPS-challenged healthy volunteers treated with either placebo or CIT-013. LPS was dosed at 2 ng / kg 2 hours after placebo or CIT-013 treatment. Cohort 1 was treated with A) placebo (n = 3) or B) 0.3 mg / kg CIT-013 (n = 3). Cohort 2 was treated with C) placebo (n = 6) or D) 0.9 mg / kg CIT-013 (n = 8). [Figure 7] Levels of calprotectin (ng / ml) in plasma from two cohorts of LPS-challenged healthy volunteers treated with either placebo or CIT-013. LPS was dosed at 2 ng / kg 2 hours after placebo or CIT-013 treatment. Cohort 1 was treated with A) placebo (n = 3) or B) 0.3 mg / kg CIT-013 (n = 3). Cohort 2 was treated with C) placebo (n = 6) or D) 0.9 mg / kg CIT-013 (n = 8). [Figure 8] Plasma levels of P-selectin (ng / ml) from two cohorts of healthy LPS-loaded volunteers treated with either placebo or CIT-013. LPS was administered at 2 ng / kg two hours after placebo or CIT-013 treatment. Cohort 1 was treated with A) placebo (n=2) or B) 0.3 mg / kg CIT-013 (n=3). Cohort 2 was treated with C) placebo (n=6) or D) 0.9 mg / kg CIT-013 (n=8). [Figure 9] Blood exposure to CIT-013 after intravenous (IV) or subcutaneous (SC) administration. The graph shows the time course of CIT-013 serum concentrations after administration as a single IV dose (five different mg / kg) or a single 100 mg SC dose. [Figure 10] Calibration curves from three different plates are shown in Figure 10a. Figure 10b shows the significant difference in citrullinated NET levels in serum samples from the rheumatoid arthritis (RA) cohort (results shown on the right) compared to serum from the healthy volunteer (HV) cohort (results shown on the left). Significance was not measured using the Mann-Whitney test (unpaired; non-parametric; two-sided p-value and 95% confidence level) (P=0.074).

[0019] Detailed description of the invention It should be understood that the various applications of the disclosed invention may meet specific requirements in the art. Furthermore, it should be understood that the terms used herein are for the sole purpose of describing specific embodiments of the invention and are not intended to be limiting.

[0020] Furthermore, unless otherwise clearly stated, the singular forms "a," "an," and "the" used in this specification and the appended claims include plural references. Therefore, for example, a reference to "an antibody" includes "antibodies," etc. Where the term “comprising” is used herein, embodiments that “consist essentially” or “consist of” what is described are also provided.

[0021] In one embodiment, the term “approximately” in relation to the point values ​​shown herein means that what is provided is within ±10% of the stated point values. In one preferred embodiment, it is within ±5%. In one even more preferred embodiment, it is within ±1%. When used in relation to a numerical range, in one embodiment, the amount of each point value used to indicate the endpoints of the range values ​​is within any of those values.

[0022] All publications, patents, and patent applications cited herein, whether above or below, are incorporated herein by reference in their entirety.

[0023] Dosage and regimen The present invention provides an antibody or its conjugated fragment for use in a method of treating or preventing diseases associated with the release of extracellular traps from cells, such as neutrophil extracellular trap (NET)-related pathology or eosinophil extracellular trap (EET)-related pathology in subjects requiring such treatment, wherein the method comprises administering to a subject at least one dose equivalent to an intravenous administration of about 0.01 mg / kg to about 0.6 mg / kg of the antibody or its conjugated fragment.

[0024] The present invention provides an antibody or its conjugated fragment for use in a method of treating or preventing diseases associated with the release of extracellular traps from cells, such as neutrophil extracellular trap (NET)-associated pathology or eosinophil extracellular trap (EET)-associated pathology in a subject requiring such treatment, wherein the method comprises administering to a subject at least one dose of the antibody or its conjugated fragment ranging from about 0.01 mg / kg to about 0.6 mg / kg. In one embodiment, the at least one dose is about 0.1 mg / kg to about 0.6 mg / kg. In another, it is about 0.1 mg / kg to about 0.5 mg / kg. In yet another, it is about 0.1 mg / kg to about 0.4 mg / kg.

[0025] In a preferred embodiment, at least one dose is from about 0.02 mg / kg to about 0.5 mg / kg. In a more preferred embodiment, at least one dose is from about 0.05 mg / kg to about 0.4 mg / kg. In an even more preferred embodiment, at least one dose is from about 0.1 mg / kg to about 0.3 mg / kg. An example of a preferred dose is 0.02 mg / kg. An example of at least one preferred dose is about 0.05 mg / kg. An example of at least one preferred dose is about 0.1 mg / kg. A further example of at least one preferred dose is about 0.2 mg. A further example of at least one preferred dose is about 0.3 mg / kg. In a preferred embodiment, at least one dose is administered by injection or infusion, more preferably by injection. In a particularly preferred embodiment, it is administered subcutaneously or intravenously. In a preferred embodiment, it is administered subcutaneously. In an even more preferred embodiment, it is administered intravenously.

[0026] In one embodiment, doses 1 to 12 will be administered to the subject. In one embodiment, doses 1 to 10 will be administered. In one embodiment, doses 2 to 8 will be administered. In another embodiment, doses 3 to 7 will be administered. In one embodiment, doses 1, 2, 3, 4, 5, 6, 7, or 8 will be administered. In a more preferred embodiment, doses 5, 6, or 7 will be administered. In a particularly preferred embodiment, dose 6 will be administered. In an alternative embodiment, doses 1, 2, or 3 will be administered. In one embodiment, doses 1 or 2 will be administered. In one embodiment, a single dose of the antibody will be administered. In an alternative embodiment, the antibody will be administered chronically as maintenance therapy. In an alternative embodiment, antibody administration will be continued as maintenance therapy. In an alternative embodiment, antibody administration will be continued as long as deemed necessary. In another embodiment, antibody administration will be continued until the condition of the subject sample improves.

[0027] In embodiments where more than one dose of antibody or conjugated fragment is administered, the administration interval is from one week to six months. In embodiments where more than one dose of antibody or conjugated fragment is administered, the administration interval is from one week to five months in the conjugated fragment embodiment. In embodiments where more than one dose of antibody or conjugated fragment is administered, the administration interval is from one week to four months in the conjugated fragment embodiment. In embodiments where more than one dose of antibody or conjugated fragment is administered, the administration interval is from one week to three months in the conjugated fragment embodiment. In one embodiment, the administration interval is from one week to six weeks. In one embodiment, the dose is administered at intervals of approximately one to four weeks. In one embodiment, the dose is administered at intervals of approximately one month. In a preferred embodiment, the dose is administered at intervals of one to three weeks. In a particularly preferred embodiment, the dose would be administered at intervals of approximately two weeks. In one embodiment, the dose is administered at intervals of approximately one week, two weeks, or one month.

[0028] In one preferred embodiment, the present invention provides an antibody or conjugated fragment that specifically binds to a citrullinated epitope in deiminated human histone 2A and / or histone 4 for use in a method of treating or preventing diseases associated with the release of extracellular traps from cells, such as neutrophil extracellular trap (NET)-related pathology or eosinophil extracellular trap (EET)-related pathology in a subject requiring such treatment, the method comprising administering to a subject one to ten doses of the antibody or conjugated fragment, each dose ranging from about 0.02 mg / kg to about 0.5 mg / kg, and the antibody is administered by injection or infusion. Preferably, the antibody or conjugated fragment is administered subcutaneously or intravenously. Particularly preferably, it is administered subcutaneously. In one preferred embodiment, at least two doses are administered, with an interval of one week to three months between doses. In a particularly preferred embodiment, the interval is one week to three weeks. In a further particularly preferred embodiment, four to eight doses are administered, preferably at intervals of one week to three weeks.

[0029] In a preferred embodiment, the present invention provides an antibody or conjugated fragment that specifically binds to a citrullinated epitope in deiminated human histone 2A and / or histone 4 for use in a method of treating or preventing diseases associated with the release of extracellular traps from cells, such as neutrophil extracellular trap (NET)-associated pathology or eosinophil extracellular trap (EET)-associated pathology in a subject requiring such treatment, the method comprising administering to a subject one to ten doses of the antibody or conjugated fragment, each dose ranging from approximately 0.05 mg / kg to approximately 0.5 mg / kg, and the antibody being administered by injection or infusion. Preferably, the antibody or conjugated fragment is administered subcutaneously or intravenously. Particularly preferably, it is administered subcutaneously. In a preferred embodiment, where at least two doses are administered, the interval between doses is from one week to three months. In a particularly preferred embodiment, the interval is from one week to three weeks. In a further particularly preferred embodiment, four to eight doses are administered, preferably at intervals of one to three weeks.

[0030] In a preferred embodiment, the present invention provides an antibody or conjugated fragment that specifically binds to a citrullinated epitope in deiminated human histone 2A and / or histone 4 for use in a method of treating or preventing diseases associated with the release of extracellular traps from cells, such as neutrophil extracellular trap (NET)-associated pathology or eosinophil extracellular trap (EET)-associated pathology in a subject requiring such treatment, the method comprising administering to a subject one to ten doses of the antibody or conjugated fragment, each dose ranging from approximately 0.1 mg / kg to approximately 0.4 mg / kg, and the antibody being administered by injection or infusion. Preferably, the antibody or conjugated fragment is administered subcutaneously or intravenously. Particularly preferably, it is administered subcutaneously. In a preferred embodiment, where at least two doses are administered, the interval between doses is from one week to three months. In a particularly preferred embodiment, the interval is from one week to three weeks. In a particularly preferred embodiment, four to eight doses are administered, preferably at intervals of one to three weeks.

[0031] In a preferred embodiment, the present invention provides an antibody or conjugated fragment that specifically binds to a citrullinated epitope in deiminated human histone 2A and / or histone 4 for use in a method of treating or preventing diseases associated with the release of extracellular traps from cells, such as neutrophil extracellular trap (NET)-associated pathology or eosinophil extracellular trap (EET)-associated pathology in a subject requiring such treatment, the method comprising administering to a subject 4 to 8 doses of the antibody or conjugated fragment, each dose ranging from approximately 0.02 mg / kg to approximately 0.3 mg / kg, the antibody being administered by injection or infusion, with an interval of 1 to 3 weeks between administrations. In a particularly preferred embodiment, the administrations are spaced 1 to 3 weeks apart.

[0032] In one embodiment, a fixed dose is administered. In one embodiment, a preferred fixed dose is administered subcutaneously at a fixed dose of 2 mg to 100 mg. In one embodiment, a preferred fixed dose is administered subcutaneously at a fixed dose of 2 mg to 90 mg. In one embodiment, a preferred fixed dose is administered subcutaneously at a fixed dose of 2 mg to 80 mg. In one embodiment, a preferred fixed dose is administered subcutaneously at a fixed dose of 2 mg to 70 mg. In one embodiment, a preferred fixed dose is administered subcutaneously at a fixed dose of 2 mg to 60 mg. In one embodiment, a preferred fixed dose is administered subcutaneously at a fixed dose of 2 mg to 50 mg. In a preferred embodiment, the fixed dose is administered subcutaneously at a fixed dose of 2 to 40 mg. In a preferred embodiment, the fixed dose is administered subcutaneously at a fixed dose of 30 mg. In a preferred embodiment, the fixed dose is administered subcutaneously at a fixed dose of 2 to 20 mg. In a preferred embodiment, the fixed dose is administered subcutaneously at a fixed dose of 8 to 30 mg. In one preferred embodiment, the fixed dose is administered subcutaneously in a fixed dose of 8 to 20 mg. In one embodiment, at least two such fixed doses may be administered at any of the intervals described herein. In one embodiment, 2 to 10 fixed doses may be administered at intervals of 1 to 6 weeks. In one preferred embodiment, 4 to 8 fixed doses may be administered. In one preferred embodiment, 4 to 8 fixed doses may be administered at intervals of 1 week to 1 month.

[0033] As shown in Figure 9, subcutaneous administration of 100 mg of CIT-013 results in a serum exposure level of approximately 10,000 ng / ml 72 hours after administration. This exposure level is equivalent to the exposure level reached 72 hours after intravenous (IV) administration of 0.7 mg / kg of CIT-013. The IV curve shows that the exposure in ng / ml is nearly linear. Therefore, if twice the amount of CIT-013 IV is administered, twice as much will be detected in the serum 72 hours later. Thus, it is predicted that subcutaneous administration of 20 mg of CIT-013 will result in a serum exposure level of approximately 2,000 ng / ml 72 hours after administration, which is equivalent to the exposure level seen 72 hours after IV administration of 0.3 mg / kg of CIT-013. Therefore, it is predicted that a subcutaneous administration of 8 mg of CIT-013 will result in a serum exposure level of approximately 800 ng / ml 72 hours after administration, which is equivalent to the exposure level observed 72 hours after an intravenous administration of 0.1 mg / kg of CIT-013.

[0034] Cytokine and markers In a preferred embodiment, the amount of antibody administered results in an increase or decrease in the amount of at least one cytokine or other marker. One of the advantages of the present invention is that it can result in a specific pattern of cytokine release or other markers.

[0035] In one preferred embodiment, the present invention increases the level of IL-10 in a subject. That is, the level of IL-10 after antibody administration is higher than the baseline level of IL-10 in the subject before any antibody is administered. Thus, in one embodiment, administration of an antibody or binding fragment results in stimulation of IL-10. The level of IL-10 may be measured in plasma.

[0036] In one preferred embodiment, the present invention results in the suppression of the release of a specific cytokine or marker, i.e., the level of cytokine or marker release is lower than the baseline level before antibody administration. In one embodiment, administration of the antibody or binding fragment suppresses the level of at least one of the following in the subject: calprotectin, P-selectin, CXCL10, TNF-α, and VCAM. In one embodiment, the levels of at least two cytokines or markers are suppressed. In one embodiment, the levels of at least three cytokines or markers are suppressed. In one embodiment, the levels of at least four cytokines or markers are suppressed. Preferably, all five levels are suppressed.

[0037] In a preferred embodiment, antibody administration results in some increase in IL-10 levels but suppresses levels of at least one of the following in the subject: calprotectin, P-selectin, CXCL10, TNF-α, and VCAM. Preferably, at least two of the following are suppressed. More preferably, at least three of the following are suppressed. More preferably, at least four of the following are suppressed. More preferably, all five of the following are suppressed.

[0038] The levels of IL-10, calprotectin, P-selectin, CXCL10, TNF-α, and VCAM can be measured by any suitable means, including by commercially available kits. In preferred embodiments, the method used is that described in the examples of this application.

[0039] hindrance 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 for use in the treatment or prevention of diseases associated with the release of extracellular traps from cells, such as neutrophil extracellular trap (NET)-related pathology or eosinophil extracellular trap (EET)-related pathology.

[0040] 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 pathologies, such as NET-related pathologies and inflammatory conditions, and / or EET-related pathologies.

[0041] The present invention also encompasses methods for treating a patient, comprising administering to a subject a therapeutically effective amount of an antibody or conjugated fragment or a pharmaceutical composition as defined herein, optionally to treat or prevent citrullination-related pathologies, such as NET-related pathologies and inflammatory conditions, and / or EET-related pathologies.

[0042] The present invention also includes antibodies or their conjugated fragments, as defined herein, or pharmaceutical compositions as defined herein, used in the manufacture of agents for the prevention or treatment of citrullination-related pathologies, such as NET-related pathologies and inflammatory conditions and / or EET-related pathologies.

[0043] The present invention also encompasses pharmaceutical compositions comprising the antibody or its conjugated fragment for treating or preventing citrullination-related pathologies, such as NET-related pathologies and inflammatory conditions, and / or EET-related pathologies.

[0044] Citrullination-related pathologies can be defined as any disease or condition in which citrullination is associated with a 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 affected or disease-associated tissues. This can be achieved by immunological tests such as Western blotting or ELISA, using affected tissue as an antigen and detecting citrullination of that antigen with the help of anti-citrulline antibodies described herein. Alternatively, those skilled in the art may use proteomics applications such as mass spectrometry to compare the levels and types of citrullination in healthy versus diseased tissues from affected patients.

[0045] NET-related pathologies can be considered pathologies associated with citrullination. NET-related pathologies can be defined as diseases or conditions in which NET formation and NETosis are associated with a 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 associated tissues.

[0046] Therefore, the present invention relates to an antibody or its conjugated fragment for use in the treatment or prevention of NET-related pathology.

[0047] Therefore, the present invention relates to a method for treating patients suffering from NET-related pathology who require a therapeutically effective amount of the antibody or its conjugated fragment of the present invention.

[0048] Examples of NET-related pathologies include inflammatory conditions or diseases, inflammatory eye diseases, cardiovascular diseases, respiratory diseases, wound healing, skin diseases, autoimmune diseases, cancer, and organ health after transplantation.

[0049] An "inflammatory condition" or "inflammatory disease" refers to any of several conditions or diseases characterized by vascular changes: edema and neutrophil infiltration (e.g., acute inflammatory response); tissue infiltration by mononuclear cells; tissue destruction by inflammatory cells, connective tissue cells and their cellular products; and attempts at repair by connective tissue replacement (e.g., chronic inflammatory response). Such diseases include, for example, inflammatory arthritis including rheumatoid arthritis and osteoarthritis, SLE, lupus, sepsis, vasculitis, multiple sclerosis, psoriatic arthritis, psoriasis, hidradenitis suppurativa, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, myositis (polymyositis and dermatomyositis), Sjögren's disease, spondyloarthritis, multiple system atrophy, heart failure, atherosclerosis, Parkinson's disease, Lewy body dementia, idiopathic pulmonary fibrosis, dry eye disease, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, as well as lung diseases such as asthma, acute respiratory distress syndrome (ARDS) including but not limited to coronavirus-induced ARDS, COPD and bronchitis, thrombotic diseases, and cardiovascular diseases. Non-granulomatous uveitis may be associated with neutrophil-dominant inflammation, while granulomatous uveitis may be associated with macrophage-dominant inflammation.

[0050] NETs play a role in the pathology of autoimmune diseases, including RA, SLE, and vasculitis. The pathway by which therapeutic antibodies or their binding fragments improve disease is likely mediated through the inhibition of NETosis, promoting the removal of NET residues, NET-ing neutrophils, and other autoantigens, including toxic histones, from tissues and circulation. In many autoimmune diseases, pathology has been shown to improve in PAD knockout models or in wild-type animals treated with PAD inhibitors or DNase, suggesting a strong correlation between the number of NETs in tissues and circulation and disease severity.

[0051] Therefore, inflammatory conditions or diseases and autoimmune diseases can be treated with the antibodies and their conjugated fragments described in the present invention.

[0052] In preferred embodiments, the diseases to be treated include SLE, lupus, sepsis, vasculitis, inflammatory arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, hidradenitis suppurativa, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, myositis (polymyositis and dermatomyositis), Sjögren's disease, antiphospholipid syndrome, Behçet's disease, spondylitis, spondyloarthritis, multiple system atrophy, Parkinson's disease, Lewy body dementia asthma, allergic rhinovirus exacerbated asthma, allergic asthma, cystic fibrosis, fibrosis and idiopathic pulmonary fibrosis, heart failure, atherosclerosis, dry eye disease, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, bronchitis, or wound healing in diabetes, cancer, cancer metastasis, and in vivo or ex vivo. Other NET-related pathologies include NET-related pathologies such as the health of transplanted organs in vivo.

[0053] In preferred embodiments, the diseases to be treated are inflammatory conditions such as SLE, lupus, sepsis, vasculitis, inflammatory arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, hidradenitis suppurativa, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, myositis (polymyositis and dermatomyositis), Sjögren's disease, antiphospholipid syndrome, Behçet's disease, spondylitis, spondyloarthritis, multiple system atrophy, Parkinson's disease, Lewy body dementia asthma, allergic rhinovirus exacerbating asthma, allergic asthma, acute respiratory distress syndrome, cystic fibrosis, fibrosis, idiopathic pulmonary fibrosis, heart failure, atherosclerosis, dry eye disease, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, and bronchitis. In one preferred embodiment, the NET-related disorder is tauopathy. In a particularly preferred embodiment, the disorder is Parkinson's disease.

[0054] The methods disclosed herein may be for the diagnosis, treatment, or prevention of any disease or condition including EET-related pathology. EET-related pathology typically means a pathology that is mediated in whole or in part by the formation of EETs. Such pathologies are typically present in any disease or condition that is mediated in whole or in part, or preferably primarily, by eosinophils. Such diseases or conditions may be described herein as eosinophilic or eosinophil-related. In other words, the methods disclosed herein may be for the diagnosis, treatment, or prevention of eosinophilic diseases or conditions.

[0055] Eosinophilic diseases or conditions can be defined as diseases or conditions in which the number of eosinophils is increased in the affected tissue or organ compared to the same tissue or organ in a healthy individual. Eosinophilic diseases and conditions may include: eosinophilic diseases or conditions of the skin; eosinophilic diseases or conditions of the respiratory tract; eosinophilic diseases or conditions of the gastrointestinal tract; allergic diseases or conditions; or helminthic, fungal, viral or bacterial infections.

[0056] Eosinophilic diseases or conditions of the skin include bullous pemphigoid (PB), atopic dermatitis (AD), and chronic idiopathic urticaria (CSU), allergic contact dermatitis, and eosinophilic cellulitis (also known as Wells syndrome).

[0057] Eosinophilic diseases or conditions of the respiratory tract include eosinophilic asthma, chronic sinusitis with nasal polyps (CRSwNP), allergic sinusitis, allergic rhinisitis, allergic bronchopulmonary aspergillosis (fungal infection), eosinophilic chronic sinusitis, and tropical pulmonary eosinophilia (typically a respiratory helminth infection).

[0058] Eosinophilic diseases or conditions of the gastrointestinal tract include eosinophilic esophagitis (EoE), eosinophilic gastritis (stomach-EG), eosinophilic gastroenteritis (stomach and small intestine-EG), eosinophilic enteritis (small intestine), eosinophilic colitis (colon-EC), and gastrointestinal helminthiatic infections such as ascariasis or trichinellosis.

[0059] Other eosinophilic diseases or conditions include eosinophilic syndrome (HES - affecting the blood and various organs), eosinophilic granulomatosis with polyangiitis (EGPA - affecting various organs, including blood vessels), eosinophilic otitis media (EOM - affecting the middle ear), and drug reactions with eosinophilic and systemic symptoms (DRESS - affecting various organs).

[0060] In one preferred embodiment, the disease to be treated or prevented is arteriosclerosis. In another embodiment, vasculitis is treated.

[0061] The methods disclosed herein may be for the diagnosis, treatment, or prevention of any of the eosinophilic diseases or conditions listed above. Particularly preferred eosinophilic diseases or conditions include those in which the presence of EET is directly confirmed. Such diseases and conditions include, but are not limited to, bullous pemphigoid, atopic dermatitis, allergic contact dermatitis, eosinophilic asthma, chronic sinusitis with nasal polyps (CRSwNP), allergic sinusitis, allergic bronchopulmonary aspergillosis, eosinophilic chronic sinusitis, eosinophilic esophagitis (EoE), eosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA), eosinophilic otitis media (EOM), and drug reaction with eosinophilic and systemic symptoms (DRESS).

[0062] The most preferred eosinophilic diseases or conditions are those in which a direct correlation is observed between eosinophilic inflammatory responses (EETs) and disease incidence and / or severity. Such diseases and conditions include, but are not limited to, eosinophilic asthma, chronic sinusitis with nasal polyps (CRSwNP), eosinophilic chronic sinusitis, and eosinophilic otitis media (EOM).

[0063] The presence and / or role of eosinophils in diseases such as those discussed above is well-established in the art. See, for example: Williams, T. L et al. (2020). “NETs and EETs, a Whole Web of Mess”. Microorganisms, 8(12), 1925 and Mukherjee, M., et al. (2018). Eosinophil Extracellular Traps and Inflammatory Pathologies—Untangling the Web!. Frontiers in Immunology, 9, 2763. The present invention is suitable for the treatment, prevention or diagnosis of any of the diseases described in these documents, which are incorporated by reference.

[0064] The methods disclosed herein may also be for the diagnosis, treatment, or prevention of EET-related pathologies in diseases or conditions that are partially mediated by eosinophils. For example, diseases such as chronic obstructive pulmonary disease (COPD), Crohn's disease, ulcerative colitis, dermatitis herpetiformis, thrombosis, and atherosclerosis may exhibit multiple pathologies caused by multiple cell types and may not be defined as “eosinophilic.” However, they may still exhibit EET-related pathologies and can therefore be diagnosed, treated, or prevented by the methods disclosed herein.

[0065] In one preferred embodiment, the present invention is used to treat lung injury. In particular, the present invention provides a method for treating or preventing lung injury, comprising administering an antibody or a conjugated fragment thereof that specifically binds to citrullinated epitopes in deiminated human histone 2A and / or histone 4 to a subject suffering from or at risk of suffering from the lung injury. In one embodiment, the antibody or conjugated fragment is one of those described herein. In a preferred embodiment, the lung injury may be inflammatory lung injury. In one embodiment, the lung injury is characterized by an influx of inflammatory cells into the lungs compared to a healthy person without the injury. For example, in one embodiment, the condition may be characterized by an influx of leukocytes into the lungs. In one embodiment, the lung injury is characterized by an influx of granulocytes, particularly eosinophils and / or neutrophils into the lungs.

[0066] In one embodiment, the lung injury is characterized by a subject exhibiting a low symptomatic response to corticosteroids. In particular, in one embodiment, the provided approach is used to treat a subject with lung injury exhibiting a low response to dexamethasone. In another embodiment, the subject is treated with both an antibody or its conjugated fragment that specifically binds to citrullinated epitopes on deiminated human histone 2A and / or histone 4, and a corticosteroid. In one embodiment, the subject is treated with both the antibody (or conjugated fragment) and dexamethasone. In one embodiment, combining the two may help enhance the effect of the corticosteroid.

[0067] In one embodiment, a method for treating or preventing lung injury results in a reduction in the presence of NETs. In another embodiment, the method results in a reduction in the presence of EETs. In a preferred embodiment, the method may result in a reduction of both NETs and EETs in the lungs of a subject. In one embodiment, the method may result in a reduction in the formation of NETs and / or EETs.

[0068] These methods may be used to treat any suitable lung disorder, particularly inflammatory lung disorder. In one embodiment, the lung disorder is selected from COPD, bronchitis, emphysema, cystic fibrosis, fibrosis and idiopathic pulmonary fibrosis, as well as asthma. In a preferred embodiment, the condition is asthma. The subject may have severe asthma. In a particularly preferred embodiment, the lung disorder may be allergic asthma. In a preferred embodiment, the lung disorder is allergic asthma with house dust mite allergy. In one embodiment, the lung disorder is asthma characterized by the presence of an increased number of eosinophils and / or neutrophils. In one embodiment, the methods of the present invention may be used to treat a lung condition with an increased number of infiltrating eosinophils. In another embodiment, the methods of the present invention may be used to treat a lung condition with an increased number of infiltrating neutrophils. In another embodiment, the subject has an increased number of infiltrating eosinophils and neutrophils. In one embodiment, the subject may have neutrophilic asthma. In another embodiment, the subject may have eosinophilic asthma. In one embodiment, the subject may have type 2 asthma. In another embodiment, the subject may have non-type 2 asthma.

[0069] In one embodiment, bronchoalveolar lavage (BAL) may be used as a method to assess the presence of inflammatory cells in the lungs. In one embodiment, BAL may be used as a method to measure the total number of white blood cells in the bronchoalveolar lumen. In one embodiment, BAL may be used as a method to measure the number of neutrophils and / or eosinophils in the bronchoalveolar lumen. In one embodiment, the method of the present invention will result in a decrease in the number of neutrophils in the subject's BAL compared to the number before treatment. In another embodiment, treatment will result in a decrease in the number of eosinophils in the subject's BAL compared to the number before treatment or during treatment. In one embodiment, both the number of eosinophils and neutrophils will decrease. In one embodiment, the total number of granulocytes in BAL will decrease as a result of treatment. In one embodiment, the present invention may result in a decrease in perivascular infiltrating neutrophils, perivascular mononuclear cells and / or bronchiolar infiltrating neutrophils. Treatment with the antibodies or their conjugated fragments described herein may also result in a decrease in citrullinated histones, particularly citrullinated histone 3 in BAL, as measured, for example, in BAL.

[0070] Typically, antibodies or compositions containing them are administered to a subject already suffering from a disorder or condition in an amount sufficient to cure, alleviate, or partially block the condition or one or more of its symptoms. Such therapeutic treatment may result in a reduction in the severity of disease symptoms or an increase in the frequency or duration of asymptomatic periods. An amount sufficient to achieve this is defined as a “therapeutic effective dose.” The effective dose for a given purpose will depend 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.

[0071] antibody The present invention relates to an antibody or its conjugated fragment that specifically binds to citrullinated epitopes in deiminated human histone 2A and / or histone 4, and more particularly to the dosage of such an antibody or its conjugated fragment for use in the treatment or prevention of neutrophil extracellular trap-associated pathology (NET-associated pathology). This section provides examples of possible antibodies or their conjugated fragments, which are also provided for use in the uses and methods described herein. For the sake of brevity, however, this section will simply refer to them as antibodies, and both antibodies for the uses described herein and methods including their administration are provided, as are the uses of antibodies in the manufacture of pharmaceuticals.

[0072] 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 peptidylarginine deiminase (PAD); (EC 3.5.3.15). In mammals (humans, mice, and rats), five PAD isotypes (PAD1-PAD6; "PAD4" and "PAD5" are used for the same isotype) have been identified to date, each encoded by a different gene.

[0073] Citrullination of histone 2A and / or histone 4 is associated with NET formation. Downstream pathological effects of NET formation can be wide-ranging. For example, autoantigens may be exposed to the extracellular space, leading to the production of pathological autoantibodies by the subject. NET-derived histones can be toxic to vascular walls and organs, causing vascular damage and organ failure. NETs can lead to the formation of autoantigen / autoantibody immune complexes, for example, in the kidneys of SLE patients, which enhance further inflammation. NETs are also involved in metastasis in cancer progression.

[0074] Any suitable antibody or its binding fragment that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4 may be used in the present invention. Deiminomination of human histones 2A and 4 can be carried out by enzymes such as PADs, e.g., PAD2 and PAD4. In specific embodiments, the antibody or its binding fragment according to the present invention specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, the epitope comprising a peptide selected from the group consisting of SEQ ID NOs: 18, 19, 20, 21 and 22. The antibody or its binding fragment may also bind to an epitope comprising the peptide of SEQ ID NO: 53 or 54.

[0075] As used herein, the terms “antibodies,” “antibody,” or “the binding fragment thereof” refer to a structure, preferably a protein or polypeptide structure, that has the ability to specifically bind to a target molecule, often called an “antigen.” As used herein, an antibody molecule refers to an antibody or its binding fragment. In particularly preferred embodiments, the term “antibody” as used herein generally refers to a complete (full-length) antibody, i.e., an antibody comprising two heavy chains and two light chain elements. Antibodies may include further additional binding domains, such as the molecule DVD-Ig disclosed in WO2007 / 024715, or the so-called (FabFv)2Fc described in WO2011 / 030107. Thus, as used herein, “antibody” includes monovalent, bivalent, trivalent, or tetravalent full-length antibodies. References to “antibody” herein specifically include the use of an antigen-binding fragment unless otherwise evident from the context.

[0076] Antibodies or their binding fragments may be selected from the group consisting of single-chain antibodies, single-chain variable fragments (scFv), variable fragments (Fv), antigen-binding domain fragments (Fabs), recombinant antibodies, monoclonal antibodies, fusion proteins containing the antigen-binding domain of native antibodies or aptamers, single-domain antibodies (sdAbs), also known as VHH antibodies, nanobodies (single-domain antibodies derived from camelid animals), single-domain antibody fragments derived from shark IgNAR called VNAR, bispecific antibodies (diabodies), triabodies, antikalin, aptamers (DNA or RNA), and their active components or fragments.

[0077] A polyvalent antibody may possess multiple specificities, such as bispecificity, or it may have a single specificity.

[0078] In one embodiment, an antigen-binding fragment of the antibody may be used rather than the full-length antibody. 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 or VL or VHH), scFv, monovalent, bivalent, trivalent or tetravalent antibodies, Bis-scFv, bispecific antibodies (diabody), tribody, triabody, tetrabody, and any of the above epitope-binding fragments (see, for example, Holliger P and Hudson PJ, 2005, Nat. Biotechnol., 23, :1126-1136; Adair JR and Lawson ADG, 2005, Drug Design Reviews-Online, 2, 209-217). Methods for creating and producing these antibody fragments are well known in the art (see, for example, Verma R et al, 1998, J.Immunol.Methods, 216, 165-181). The Fab-Fv format was first disclosed in WO2009 / 040562, and its disulfide-stabilized version, Fab-dsFv, was first disclosed in WO2010 / 035012. Other antibody fragments for use in the present invention include Fab and Fab' fragments.

[0079] In one embodiment, the antibody is selected from a full-size antibody, Fab, F(ab')2, single-chain Fv fragment, single-domain VHH, single-domain VH, or single-domain VL.

[0080] 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 are also included in the present invention, including IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE combined with κ or λ light chains. Furthermore, antibodies of all animal origins of various isotypes can also be used in the present invention.

[0081] In this context, the terms “specifically binds to citrulline” or “specifically binds to citrullinated epitopes” mean that 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 circumstances for immunoreactivity with the antibody or its binding fragment described herein, preferably in circumstances similar to those that appear in human or animal bodies, and more preferably in the context of natural polypeptides. The antibody or its binding fragment of the present invention specifically binds to citrullinated epitopes on deiminated human histone 2A and / or histone 4. Binding of the antibody or its binding fragment to citrullinated epitopes on deiminated human histone 2A and / or histone 4 inhibits NET formation. Histone citrullination is associated with NET formation. Inhibition of NET formation may be complete or partial. For example, the antibody or its binding 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 inhibition can be measured by any suitable means, such as in vitro measurement of NETosis (Kraaij T et al., 2016, Autoimmun. Rev. 15, 577-584).

[0082] In the method of the present invention, an antibody or its binding fragment suitable for us specifically binds to citrullinated epitopes on deiminated human histone 2A and / or histone 4. Binding of the antibody or its binding fragment to the citrullinated epitopes on deiminated human histone 2A and / or histone 4 inhibits EET formation. Histone citrullination is associated with EET formation.

[0083] Inhibition of EET formation is complete or partial. For example, antibodies or their binding fragments can reduce EET formation by 10–50%, at least 50%, or at least 70%, 80%, 90%, 95%, or 99%. EET inhibition can be measured by any appropriate means, such as in vitro measurement of EETosis (Fukuchi et al., “How to detect eosinophil ETosis (EETosis) and extracellular traps”; Allergology International, Volume 70, Issue 1, 2021, Pages 19–29).

[0084] 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 a target. Binding affinity can be quantified by measuring the dissociation constant (Kd) of the antibody to its target. Similarly, the binding specificity of an antibody to a target can be defined in terms of the comparison between the dissociation constant (Kd) of the antibody to its target and the dissociation constant of the antibody to another non-target molecule. Typically, the Kd of an antibody to a target will be 2 times, preferably 5 times, more preferably 10 times lower than the Kd to other non-target molecules such as unrelated substances or incidental substances in the environment. More preferably, the Kd will be 50 times lower, even more preferably 100 times lower, and even more preferably 200 times lower.

[0085] The value of this dissociation constant can be measured directly by well-known methods and can be calculated even for complex mixtures by 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 binding assay, as disclosed by Wong I and Lohman TM (1993, Proc. Natl. Acad. Sci. USA, 90, 5428-5432), or by using Octet surface plasmon resonance, for example.

[0086] ELISA is one method for evaluating the binding affinity to deiminated human histone 2A and / or histone 4. Other standard assays for evaluating the binding ability of ligands such as antibodies to targets are known in the art, such as Western blotting, RIA, and flow cytometry. Antibody binding kinetics (e.g., binding affinity) can also be evaluated using surface plasmon resonance (e.g., Biacore). TM It can be evaluated by standard assays known in this field, such as system analysis.

[0087] Preferably, the antibody 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.

[0088] Furthermore, the antibody or its binding fragment may be a fusion protein containing an antigen-binding domain of a native antibody or an aptamer in DNA or RNA form.

[0089] 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 particular epitope. Monoclonal antibodies (mAbs) of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methodologies, such as those disclosed in "Monoclonal Antibodies: a manual of techniques" (Zola H, 1987, CRC Press) and "Monoclonal Hybridoma Antibodies: techniques and applications" (Hurrell JGR, 1982, CRC Press).

[0090] The antibody or its binding fragment of the present invention comprises a binding domain. The binding domain will generally contain six CDRs (three in the case of VHH), three of which are derived from the heavy chain and three from the light chain. In one embodiment, the CDRs are located within a framework and together form a variable region or domain. Thus, in one embodiment, the antibody or binding fragment comprises an antigen-specific binding domain that includes a light chain variable region or domain and a heavy chain variable region or domain.

[0091] The 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). Unless otherwise specified, this numbering system is used herein.

[0092] IMGT residue designation does not always directly correspond to linear numbering of amino acid residues. Actual linear amino acid sequences may contain fewer or more amino acids than the strict IMGT numbering, which corresponds to shortening or insertion of structural components, such as whether the basic variable domain structure is a framework or CDR. The correct IMGT residue numbering can be determined by aligning homologous residues within the antibody sequence with a "standard" IMGT numbering sequence for a given antibody.

[0093] 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.

[0094] The CDRs of the light chain variable domain are located at residues 27-38 (CDR1 of VL), 56-65 (CDR2 of VL), and 105-117 (CDR3 of VL), based on the IMGT numbering system.

[0095] The antibody or its binding fragment of the present invention is described herein by the primary amino acid sequence of its CDR region. The antibody or its binding fragment of the present invention is disclosed herein by the primary amino acid sequences of its heavy chain and light chain.

[0096] In a particularly preferred embodiment, the antibody or binding fragment is given improved properties compared to an antibody or binding fragment containing an unmodified version of the VL CDR1 by specifically using a modified CDR1 of the antibody or its binding fragment. In one embodiment, the unmodified CDR1 of the VL of the antibody used to derive such a modified antibody comprises or consists of the amino acid sequence QSLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37).

[0097] Modified CDR1 of the VL chain of an antibody or its binding fragment preferably comprises or consists of the amino acid sequence QSL-X1-D-X2-D-X3-KTY, where 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). Modified CDR1 of the VL chain of an antibody or its binding fragment exhibits reduced isomerization compared to the unmodified CDR1 of SEQ ID NO: 36 or 37, but maintains the binding properties of the unmodified CDR1.

[0098] The amino acid sequences of the CDRs for VH of the specific antibody or its binding fragment of the present invention are shown in SEQ ID NOs: 1, 2, and 3. The CDRs for VL are shown in SEQ ID NOs: 4 and 5. The amino acid sequences of the VH and VL of specific antibodies or their binding fragments for particularly preferred antibodies are given in SEQ ID NOs: 11 and 13. The CDRs of VH are shown in SEQ ID NOs: 1, 2 and 3. The CDRs of VL are shown in SEQ ID NOs: 6, 4 and 5.

[0099] The amino acid sequences of VH and VL of another preferred antibody or its binding fragment 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.

[0100] The amino acid sequences of VH and VL of another preferred antibody or its binding fragment 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: 8, 4 and 5.

[0101] The amino acid sequences of VH and VL of another preferred antibody or its binding fragment 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.

[0102] The amino acid sequences of VH and VL of another preferred antibody or its binding fragment 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.

[0103] The amino acid sequences of VH and VL of another preferred antibody or binding fragment 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.

[0104] The amino acid sequences of VH and VL of another preferred antibody or its binding fragment 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.

[0105] The amino acid sequences of the VH and VL of another preferred antibody or its binding fragment are given in SEQ ID NOs: 12 and 15. The CDR of the VH chain 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.

[0106] The amino acid sequences of VH and VL of other preferred antibodies or binding fragments 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.

[0107] The amino acid sequences of VH and VL of another preferred antibody or its binding fragment 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.

[0108] In the embodiment, 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.

[0109] In the embodiment, 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.

[0110] The antibody or its binding fragment used in the present invention may contain one or more CDR sequences of any specific antibody described above, except that the amino acid sequence QSL-X1-D-X2-D-X3-KTY, in which X1 is V or L, X2 is T, S, A or N, and X3 is G or A, is always present as containing or consisting of the VL CDR1, the amino acid sequence must not be QSLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37), or it must contain or consist of SEQ ID NOs: 6, 7, 8, 9 or 10.

[0111] The antibody or its binding fragment used in the present invention may include, in addition to VL CDR1, one or more VH CDR sequences of the specific antibody, and alternatively or additionally one or more VL CDR sequences. The antibody or its binding fragment may include one, two or all of the VH CDR sequences of the specific antibody or its binding fragment as described above, and may alternatively or additionally include one, two or all of the VL chain CDR sequences of the specific antibody or its binding fragment, including VL CDR1. The antibody or its binding fragment may include all six CDR sequences of the specific antibody or binding fragment as described above. For example, the antibody used in the present invention may include one of SEQ ID NOs: 6, 7, 8, 9 or 10 and one or more of SEQ ID NOs: 1, 2, 3, 4 and 5.

[0112] In embodiments of the present invention, the modified CDR1 of the VL chain of the antibody or its binding fragment used comprises or consists of the amino acid sequence QSL-Z1-Z2-Z3-Z4-Z5-KTY, where Z1 is V or L, Z2 is D or E, Z3 is T, S, A or N, Z4 is D, E, S or A, and Z5 is G or A, provided that the amino acid sequence is not QSLLDSDGKTY (SEQ ID NO: 36) or QSLVDSDGKTY (SEQ ID NO: 37). The modified CDR1 of the VL chain 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. Modified CDR1 of the VL chain of the antibody or its binding fragment may, in some embodiments, 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 one embodiment, the antibody includes one of SEQ ID NOs: 6, 7, 8, 9, 10, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52, as well as one or more SEQ ID NOs: 1, 2, 3, 4, and 5. In one embodiment, the antibody includes one of SEQ ID NOs: 6, 7, 8, 9, 10, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52, as well as all of SEQ ID NOs: 1, 2, 3, 4, and 5.

[0113] The antibody or its binding fragment used in the present invention may instead include one of these heavy chain variable domains, or a variant of the CDR sequence in 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.

[0114] Mutant antibodies may contain 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions from the specific sequences and fragments discussed above, while maintaining the activity of the antibodies described herein. “Deletion” variants may include, for example, the deletion of 2, 3, 4, or 5 individual amino acids, or one or more small groups of amino acids such as 2, 3, 4, or 5 amino acids. “Small groups of amino acids” can refer to consecutive or non-contiguous but closely related amino acids. “Substitution” variants preferably include those that perform conservative amino acid substitutions, such as substituting one or more amino acids with the same number of amino acids. For example, an amino acid may be substituted with a substitute 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 micro amino acid, another small amino acid, or another large amino acid. Some of the properties of the 20 major amino acids that can be used to select appropriate substituents are as follows:

[0115] JPEG2026513830000003.jpg77164

[0116] Preferred "derivatives" or "mutants" include those in which the amino acids appearing in the sequence are structural analogs of naturally occurring amino acids. The amino acids used in the sequence may also be derivatized or modified, for example, by labeling, as long as the function of the antibody is not significantly adversely affected.

[0117] The derivatives and variants described above may be prepared during antibody synthesis, or by post-production modification, or, if the antibody is recombinant, by known techniques such as site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or ligation of nucleic acids.

[0118] Preferably, the mutant antibody has amino acid identity with the VL and / or VH or fragments of the antibody disclosed herein of more than 60%, or more than 70%, for example, 75%, or 80%, preferably more than 85%, for example, more than 90%, 95%, 96%, 97%, 98%, or 99%. This level of amino acid identity is observed over the entire length of the relevant SEQ ID NO sequence, or over a portion of the sequence, such as over 20, 30, 50, 75, 100, 150, or more amino acids, depending on the size of the full-length polypeptide.

[0119] Preferably, the mutant antibody comprises one or more CDR sequences described herein.

[0120] In relation to amino acid sequences, "sequence identity" refers to sequences that exhibit predetermined values ​​when evaluated using ClustalW (Thompson JD et al., 1994, Nucleic Acid Res., 22, 4673-4680) with the following parameters: Pairwise alignment parameters - Method: slow / accurate, Matrix: PAM, Gap start penalty: 10.00, Gap extension penalty: 0.10; Multiple alignment parameters - Matrix: PAM, Gap start penalty: 10.00, Identity % for delay: 30, Terminal gap penalty: Yes, Gap separation distance: 0, Negative matrix: No, Gap extension penalty: 0.20, Residue-specific gap penalty: Yes, Hydrophilic gap penalty: Yes, Hydrophilic residues: G, P, S, N, D, Q, E, K, R. Sequence identity at specific residues is intended to include simply derivatized identical residues.

[0121] Therefore, antibodies having specific VH and VL amino acid sequences, as well as their variants and fragments that maintain the function or activity of these VH and VL, can be used in the present invention.

[0122] Therefore, antibodies or their binding fragments may be used that contain variants of VH that retain the ability to specifically bind to citrullinated epitopes on 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. VH variants may contain at least 7 amino acid fragments of hVH22.101f or hVH22.101HC9 (SEQ ID NOs: 11 and 12, respectively), and the antibody or its binding fragment may retain the ability to specifically react with citrullinated epitopes on deiminated human histone 2A and / or histone 4; or A variant of hVH22.101f or hVH22.101HC9 (sequence numbers 11 and 12, respectively) having at least 70% amino acid sequence identity with hVH22.101f or hVH22.101HC9 (sequence numbers 11 and 12, respectively), wherein the antibody or its binding fragment retains the ability to specifically react with citrullinated epitopes on deiminated human histone 2A and / or histone 4.

[0123] Polynucleotides, vectors, and host cells The polynucleotides, vectors, and expression vectors encoding antibodies or their binding fragments described herein may be administered in several embodiments, provided, in particular, illustrative guidance on how the antibodies and binding fragments of the present invention may be produced, provided that they result in the administration of the antibodies or their binding fragments in the described doses.

[0124] Polynucleotides 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, either deoxyribonucleotides, ribonucleotides, or their analogues. 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, primers, and the like. Polynucleotides may be provided in isolated or purified forms.

[0125] A nucleic acid sequence that "encodes" a selected polypeptide is a nucleic acid molecule that, under the control of an appropriate regulatory sequence, is transcribed in vivo (in the case of DNA) and translated (in the case of mRNA) to become 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' (carboxyl) terminus. For the purposes of the present invention, such nucleic acid sequences include, but are not limited to, cDNA derived from viral, 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 at 3' of the coding sequence. In one embodiment, a polynucleotide comprises a sequence encoding the VH or VL amino acid sequence described above. The polynucleotide may encode the VH or VL sequence of a specific antibody or its binding fragment disclosed herein.

[0126] Therefore, the antibody or its conjugated fragment of the present invention can be produced from or administered to a patient in the form of a polynucleotide that encodes and can be expressed therein. 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 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. Such polynucleotides or polynucleotide pairs can be expressed together to produce the antibody of the present invention.

[0127] The polynucleotides of the present invention can be synthesized according to methods well known in the art, as illustrated in Sambrook J et al. (1989, Molecular cloning: a laboratory manual; Cold Spring Harbor: New York: Cold Spring Harbor Laboratory Press).

[0128] Nucleic acid molecules may be provided in the form of expression cassettes containing a control sequence operationally linked to an insert sequence, thereby enabling the in vivo expression of the antibody of the present invention. These expression cassettes are typically provided, in turn, within a vector (e.g., a plasmid or recombinant viral vector). Such expression cassettes 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 carries a sufficient amount of genetic information and enables the expression of the polypeptide of the present invention.

[0129] Expression vectors containing such polynucleotide sequences may be used. Such expression vectors are routinely designed in the field of molecular biology and may involve, for example, the use of plasmid DNA and appropriate initiators, promoters, enhancers, and other elements, such as polyadenylation signals, which are necessary and correctly oriented to enable expression. Other suitable vectors will be obvious to those skilled in the art. For further examples in this regard, refer to Sambrook J et al. (1989, Molecular cloning: a laboratory manual; Cold Spring Harbor: New York: Cold Spring Harbor Laboratory Press).

[0130] Those skilled in the art may use the sequences described herein to clone or generate cDNA or genomic sequences, for example, as described in the following examples. Cloning these sequences into a suitable eukaryotic expression vector or derivative such as pcDNA3 (Invitrogen) and transfecting mammalian cells (such as CHO cells) with a suitable combination of light and heavy chain vectors will result in the expression and secretion of antibodies described herein.

[0131] Those skilled in the art can also use the specific binding domain of the antibody sequence to create analogues of the antibodies or their binding fragments described herein, which can be expressed in different contexts, such as polypeptides including fusion proteins. This is well known in the art.

[0132] Cells modified to express the antibodies of the present invention may also be used. Such cells include transient, or preferably stable, higher eukaryotic cell lines such as mammalian cells and insect cells, lower eukaryotic cells such as yeast, or prokaryotic cells such as bacterial cells. Specific examples of cells that can be modified by inserting a vector or expression cassette encoding the antibodies of the present invention include mammalian HEK293, CHO, HeLa, NS0, and COS cells. Preferably, cell lines that are not only stable but also capable of mature glycosylation will be selected.

[0133] Such cell lines can be cultured using routine methods to produce the antibody or its binding fragment of the present invention, or can be used therapeutically or prophylactically to deliver the antibody or its binding fragment of the present invention to a subject.

[0134] Pharmaceutical composition The antibody or its conjugated fragment may be in the form of a pharmaceutical composition comprising the antibody or its conjugated fragment and a pharmaceutically acceptable carrier.

[0135] As used herein, “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic agent and absorption retarder, etc. Preferably, the carrier is suitable for parenteral administration, e.g., intravenous, intraocular, intramuscular, subcutaneous, intradermal or intraperitoneal administration (e.g., by injection or infusion). In certain embodiments, the pharmaceutically acceptable carrier comprises 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-based carriers, surfactants, suspensions, complexing agents such as cyclodextrins, 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, chondroitin, dermatan, heparan, heparin, pentosan, keratan, alginates, cyclodextrin, and salts and derivatives thereof, including esters and sulfates.

[0136] Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in pharmaceutical compositions include water, buffer water, and physiological saline. Other examples of carriers include ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), 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 the use of coating agents such as lecithin, maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyhydric alcohols such as mannitol and sorbitol, or sodium chloride.

[0137] Pharmaceutical compositions may contain pharmaceutically acceptable antioxidants. These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial presence can be ensured by both the aforementioned sterilization procedures and the addition of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable for the compositions to contain isotonic agents such as sugars and sodium chloride. Furthermore, the inclusion of absorption retarders such as aluminum monostearate or gelatin can enable sustained absorption of the injectable pharmaceutical form.

[0138] Therapeutic compositions must generally be sterile and stable under manufacturing and storage conditions. Pharmaceutical compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high-concentration drugs.

[0139] In further embodiments, the pharmaceutical compositions described herein may be administered intravenously, subcutaneously, intraocularly, intramuscularly, intra-articularly, intradermally, intraperitoneally, intraspinally, or by other parenteral routes, such as injection or infusion. Administration may be carried out via rectal, oral, ocular, topical, epidermal, or mucosal routes. Administration may be topical and may include peritumoral, near-tumor, intratumoral, tumor margin, intralesional, perilesional, intracavitary infusion, intravesicle administration, or inhalation. In particularly preferred embodiments, the pharmaceutical composition is administered intravenously or subcutaneously. Therefore, in one particularly preferred embodiment, the route of administration is intravenous. In another particularly preferred embodiment, the route of administration is subcutaneous. Subcutaneous injection is a particularly preferred route. Intravenous administration by infusion or injection is also a particularly preferred route of administration.

[0140] In one preferred embodiment, the antibody or a fragment thereof is provided in a pharmaceutical composition specifically formulated for injection. In one preferred embodiment, the antibody or a fragment thereof is provided in a pharmaceutical composition specifically formulated for subcutaneous injection. In one preferred embodiment, the antibody or a fragment thereof is provided in a pharmaceutical composition specifically formulated for intravenous injection.

[0141] Sterile injectable solutions can be prepared by incorporating one or a combination of the components listed above into a suitable solvent, adding a predetermined amount of an activator (e.g., antibody), and then performing sterile microfiltration. Generally, dispersions are prepared by incorporating an activator into a sterile medium containing a basic dispersion medium and other necessary components listed above. For sterile powders for sterile injectable solution preparation, preferred preparation methods are vacuum drying and freeze-drying (lyophilization) to obtain a powder containing the active ingredient and any additional components from a pre-sterile filtered solution.

[0142] The pharmaceutical composition to be used may be provided in a pre-filled device (e.g., a pre-filled syringe). In one embodiment, the pharmaceutical composition is in the form of an IV bag containing the pharmaceutical composition. In one embodiment, the pharmaceutical composition is contained in a vial.

[0143] As used herein, a dose unit form refers to a physically separated unit suitable for a single dose to a patient. Each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect when combined with a drug carrier. In one preferred embodiment, a pre-filled device, IV bag, or vial is the unit dose form.

[0144] The pharmaceutical composition may contain additional active ingredients in addition to the antibody. As described above, the composition may contain one or more antibodies of the present invention. It may also contain additional therapeutic or prophylactic activators. Such additional agents may be conjugated to the antibody. The scope of the present invention also includes kits comprising antibodies or other compositions of the present invention and instructions for use. The kits may further include one or more additional reagents, such as additional therapeutic or prophylactic agents discussed herein.

[0145] The present invention is further illustrated by the following embodiments, which should not be construed as further limitations. The contents of all drawings, all references, and all patent and published patent applications referenced throughout this specification are incorporated herein by express reference. [Examples]

[0146] Example 1 Introduction The first randomized, double-blind, randomized, placebo-controlled, single-dose escalation study in humans was conducted on CIT-013, a particularly preferred embodiment of an antibody or its binding fragment that specifically binds to citrullinated epitopes on deiminated human histones. This study was conducted in healthy volunteers who received intravenous administration of lipopolysaccharide (LPS), which stimulates an inflammatory response, including NET production from neutrophils. The objectives of the LPS challenge study, in which healthy subjects were administered CIT-013 followed by LPS, were as follows: (i) safety and tolerability; (ii) antibody pharmacokinetics; and (iii) pharmacodynamic effects of antibody administration by characterizing the inflammatory response. The analyses in (i) to (iii) above contributed to identifying the recommended dosage of the antibody effective in inhibiting NET formation and, consequently, treating NET-related pathology. This study was a single-center study.

[0147] Overall research design and plan Table 2 shows the study setup for the group administered 0.3 mg / kg of antibody before LPS loading. Table 3 shows the study setup for the group administered 0.9 mg / kg of antibody before intravenous LPS loading.

[0148] The total study period for each subject was a maximum of 131 days and was divided as follows: • Screening: Up to 42 days prior to administration; • Treatment and trial evaluation: - Day 1 to Day 84 • In-hospital stay: Day 1 to Day 2; • Follow-up visit: 79-89 days after the last dose. The subjects entered the research facility on day 1 and were discharged approximately 36 hours after administration of the test drug.

[0149] Subjects / Group Cohort 1a consisted of one effective participant and two placebo participants, with the effective participant receiving 0.3 mg / kg of antibody intravenously. Cohort 1b consisted of two effective participants and one placebo participant, with the effective participant receiving 0.3 mg / kg of antibody intravenously. Cohort 2 consisted of eight effective participants and six placebo participants, with the effective participants receiving 0.9 mg / kg.

[0150] Eligibility Criteria All subjects in this study had to meet the following criteria at the time of screening: 1. Healthy men or women aged 18 to 55 years (inclusive) at the time of screening. Cohort 2 only includes healthy men. Participants must be in good health, confirmed by a detailed medical history, a full physical examination including vital signs, laboratory measurements, and a 12-lead ECG, with no evidence of clinically significant active disease or unmanageable chronic disease. 2. The participant has the willingness and ability to comply with the requirements of the research protocol and has obtained signed informed consent. 3. At the time of screening, the body mass index (BMI) is comprehensively between 18 and 32 kg / m². 2 And their weight must be between 50 and 150 kg. 4. Male and female volunteers of childbearing potential must practice effective contraception during the study period and be willing and able to continue doing so for at least 90 days after the last dose. 5. The applicant must be able to communicate effectively with researchers in Dutch and be willing to abide by research restrictions.

[0151] Exclusion criteria Subjects who met any of the following criteria at the time of screening or before administration were excluded: 1. Evidence of any active or chronic disease or condition that could interfere with the conduct of the study, or that treatment could interfere with the conduct of the study (detailed medical history, physical examination, vital signs, 12-lead ECG, and laboratory parameters). 2. Abnormal laboratory results (including liver and kidney function tests, complete blood count, chemistry tests, and urinalysis) that the principal investigator deems clinically significant. Minor deviations from the normal range of laboratory values ​​may be tolerated if the principal investigator or a medically qualified representative deems them not clinically significant. If the results are uncertain or questionable, the tests performed at screening before randomization may be repeated to confirm eligibility or to determine that they are clinically irrelevant to healthy subjects. 3. A confirmed or suspected disease or condition related to an immune system disorder, including autoimmune disease, HIV, asplenia, or recurrent severe infections. 4. Use of chronic (more than 14 days) immunosuppressants or immunomodulators within 3 months prior to IMP administration, or single (non-chronic) use within 30 days prior to IMP administration. 5. Having a history of severe allergic reactions. 6. Confirmed severe drug hypersensitivity reactions (including skin reactions or anaphylaxis), or known allergies (inactive hay fever is acceptable). 7. If the patient is positive for hepatitis B surface antigen (HBsAg), hepatitis C antibody (HCV Ab), or human immunodeficiency virus antibody (HIV Ab) at the time of screening, or if the patient has a known infection requiring systemic antibiotic treatment within 3 months prior to the start of the study. 8. Subjects who have had an active, uncontrolled acute or chronic systemic fungal, bacterial, and / or viral infection within the past 30 days. 9. Subjects with evidence or a history of clinically significant hematological, renal, endocrine, pulmonary, gastrointestinal, cardiovascular, hepatic, psychiatric, or neurological disorders. 10. Subjects who test positive for urinary drug screening at the time of screening or before administration. 11. Subjects who tested positive for SARS-CoV-2 in a PCR test within 72 hours prior to administration of CIT-013. 12. History of abuse of addictive substances (alcohol, illegal drugs), or current alcohol consumption, drug abuse, or regular use of sedatives, hypnotics, tranquilizers, or other addictive substances exceeding 14 units per week. 13. Treatment with the investigational drug within 30 days prior to the first dose of CIT-013 or within 5 half-lives (whichever is longer). 14. Use of prescription or over-the-counter (OTC) medications, vitamins, minerals, and dietary supplements from 7 days prior to the first dose of the study drug or 5 half-lives (whichever is longer) until the end of the study (EOS). Herbal supplements and hormone replacement therapy must be discontinued from 30 days prior to the first dose of the study drug until the end of the study (EOS). However, paracetamol doses of less than 4 g per day on all study days except Day 1 of Part B are excluded from this list. Exceptions are permitted only if the principal investigator has documented clear evidence. 15. Vaccination with a live vaccine or attenuated vaccine 90 days prior to the initial trial intervention. 16. The patient had received the SARS-CoV-2 vaccine or influenza vaccine (including the second dose, if applicable) within 14 days prior to the initial administration of the investigational drug. 17. A known hypersensitivity to any component or additive of CIT-013, or a history of related drug and / or food allergies (anaphylaxis, anaphylactic-like reactions). 18. Excessive caffeine intake, defined as intake exceeding 800 mg per day during the period from 7 days before the first administration of the study drug to 24 hours before administration. Subjects should refrain from consuming caffeine-containing products from 24 hours before the start of administration until leaving the study unit. Approximate caffeine content: One cup of coffee contains 100 mg of caffeine; one cup of tea, one glass of cola, or one serving of chocolate (dark: 100 g, milk: 200 g) contains approximately 40 mg of caffeine; one can of Red Bull contains approximately 80 mg of caffeine. 19. Donation (or loss) of 500 mL or more of whole blood or plasma within 12 weeks prior to CIT-013 administration. 20. Within one month prior to CIT-013 administration, you must not have smoked more than 10 cigarettes per week (or an equivalent amount) and / or used nicotine-containing products, and / or have no intention of refraining from such use from screening to end-of-surgery (EOS). 21. Any other known factors, conditions, diseases, or other factors that the principal investigator determines may impair adherence to treatment, conduct of the trial, or interpretation of results, or that may impair the safety of the subject. 22. Strenuous exercise within two weeks prior to screening (e.g., marathon or triathlon). 23. The subject has previously participated in an intravenous LPS loading test.

[0152] Concomitant medications The subjects were also required to adhere to the following regarding concomitant medications: • From 7 days prior to the first dose of the study drug or 5 half-lives (whichever is longer) until the end of the study (EOS), the intake of prescription or over-the-counter (OTC) medications, vitamins, minerals, and dietary supplements is prohibited. Herbal supplements and hormone replacement therapy must be discontinued from 30 days prior to the first dose of the study drug until the end of the study (EOS). However, paracetamol was permitted as an exception during the study period, excluding the day of the first dose, if the dose was less than 4g per day. Clear documentation of the rationale by the principal investigator was required for the exception to be applied. • Chronic (more than 14 days) use of immunosuppressants or immunomodulators within 3 months prior to IMP administration, or single (non-chronic) use within 30 days prior to IMP administration, is not permitted. Based on the adverse event profile observed in a prior single-dose escalation study involving healthy volunteers, which is most likely to be of gastrointestinal origin, we decided to permit the use of pre-administration medications. All medications (prescription and over-the-counter [OTC]) taken within 30 days from the trial screening to the final follow-up visit at the end of the trial will be recorded in the CRF.

[0153] Basis for sample size Regarding sample size, formal power calculations were performed for plasma NETs as the endpoint, and it was shown that each group of 8 subjects had 80% power to detect the LPS-induced NET component (MPO: myeloperoxidase). A two-sample t-test was used, assuming a common standard deviation of 0.0503 and a significance level of 0.05. Intergroup comparisons were performed with 6 subjects in the placebo group as controls.

[0154] Three subjects in LPS cohort 1a received a pilot dose. These subjects were administered in a blinded, randomized manner (one receiving the active drug, two receiving placebo). The first three subjects in this cohort were observed and monitored for at least 48 hours (vital signs, physical examination, electrocardiogram, and laboratory values) to detect acute adverse events. After this period, the remaining three subjects in cohort 1b received the drug. All subjects in cohorts 1a and 1b, excluding those receiving placebo, received 0.3 mg / kg.

[0155] screening Screening visits were conducted up to 42 days prior to baseline / CHDR admission. The screening phase commenced only after obtaining complete written and oral explanations and signed informed consent, in accordance with the CHDR Standard Operating Procedures. A full medical screening was performed to assess the study eligibility of the subjects. A summary of the assessments during this visit is shown in the study flowchart (Tables 2 and 3).

[0156] Re-screening

[0157] If the reason for ineligibility was determined to be temporary (e.g., abnormal laboratory values, insufficient washout period for prohibited substances, positive drug screening), selected participants underwent rescreening. In rescreening, only assessment items that could change within the screening interval were repeated (e.g., medical history, demographic information, and virology were not repeated).

[0158] Treatment and observation period All endpoints and schedule outlines are provided in the trial flowchart (Tables 2 and 3). Subjects visited the CRU on day 1 (i.e., one day before administration of the study drug) and underwent baseline procedures performed one day prior to administration. Subjects began nocturnal fasting at least 8 hours before morning intravenous administration of CIT-013. Water was available freely. Subjects stayed overnight in the CHDR and were discharged on the afternoon of day 2, approximately 36 hours after administration.

[0159] Follow-up survey Follow-up visits were conducted up to 79 and 89 days after the final dose. A summary of the evaluations at these visits is described in the study flowchart (Tables 2 and 3). End of study (EOS) was defined as the final visit date for the last subject. In cases of early termination, follow-up visits were conducted 21–28 days after the final administration of the study drug.

[0160] Dosage selection In cohorts 1a and 1b, subjects who received the drug instead of a placebo were administered 0.3 mg / kg of CIT-013. In cohort 2, subjects who received the drug instead of a placebo were administered 0.9 mg / kg of CIT-013.

[0161] Antibodies and corresponding placebos The study drug CIT-013 is an IgG1k monoclonal antibody composed of two identical light chain polypeptides, each consisting of 219 amino acids, and two identical heavy chain polypeptides, each consisting of 451 amino acids. The study drug or placebo was administered to subjects as detailed in Tables 2 and 3. A lyophilized CIT-013 formulation of 250 mg per vial was prepared and required reconstitution before addition to the intravenous (iv) administration system. The study drug was administered at a constant rate over 2 hours using a Braunn Space P infusion pump after an 8-hour fasting period. One group of subjects received 0.3 mg / kg of the study drug, and the other group received 0.9 mg.

[0162] Participants were allowed to freely access water. The placebo was 0.9% sodium chloride.

[0163] Blinding This study was conducted using a double-blind method. Except as otherwise provided in this section, the principal investigator, clinical trial staff, subjects, sponsors, medical monitors, and monitors were not allowed access to the randomization list during the study period. The investigational drug and its corresponding placebo were indistinguishable and provided in identical packaging. The randomization list was provided only to pharmacists preparing the investigational drug, personnel responsible for the biological analysis of PK and PD samples, and statisticians involved in creating blinded summaries, graphs, and lists to assist in dose determination. The summaries, graphs, and lists provided by statisticians or programmers were created in areas inaccessible to other team members.

[0164] The principal investigator received a sealed set of randomization codes for emergency access, with copies kept by the open-label monitor and open-label medical monitor. In the event of a subject condition management, i.e., a medical emergency or serious adverse event requiring identification of the administered investigational drug, the subject's treatment emergency code could be opened to identify the drug. All such events were recorded in the study file. Treatment emergency codes were not to be deciphered except in the emergencies described above, and the sponsor was to be contacted before opening the emergency code if possible. During the final monitoring visit, unused emergency code labels were confirmed, and the database lock form was noted as "all intact (or not intact, depending on the circumstances)."

[0165] Specific safety and tolerability assessments Vital signs: Systolic blood pressure, diastolic blood pressure, pulse rate, and body temperature were assessed throughout the study period. Pulse rate and blood pressure were measured 5 minutes after the subjects were in the supine position. Automated oscillometric blood pressure and pulse rate were measured using Dash 3000, Dash 4000, Dynamap 400, and Dynamap ProCare 400.

[0166] Weight and Height: Weight (kg) was recorded at screening, day -1, at follow-up visits, or at early termination. Height (cm) was recorded at screening, and Body Mass Index (BMI) was calculated.

[0167] Physical Examination: Physical examinations (i.e., visual inspection, percussion, palpation, and auscultation) were performed during the study period. Clinically relevant findings present before the start of administration of the study drug were recorded along with the subject's medical history. Clinically relevant findings observed after the start of administration of the study drug that met the definition of an adverse event (AE) (new AE or exacerbation of an existing condition) were recorded.

[0168] Electrocardiogram (ECG) testing: During the study period, ECGs were acquired using Marquette 2000 / 5500 and stored in MUSE Cardiology Information. ECGs were taken after the subjects had rested in a supine position for at least 5 minutes. If timing overlapped, the ECG was performed before safety blood sampling. PK sampling was prioritized. The principal investigator evaluated the ECG records as "normal," "abnormal - not clinically significant," or "abnormal - clinically significant," and added descriptions of abnormalities as needed. The ECG parameters evaluated included heart rate, PR interval, QRS interval, QT interval, and QTcF (calculated using the Fredericia method).

[0169] Laboratory parameters Blood and other biological samples were collected for the following clinical tests, stored at room temperature, and analyzed within 4 hours of collection using the methodologies described in Table 1 Part A below. The biomarkers listed in Table 1 Part B were evaluated when an appropriate number of samples were collected. Table 1 Part C shows the types of blood collection tubes and blood volumes required for each test and biomarker evaluation.

[0170] [Table 1-1]

[0171] [Table 1-2]

[0172] [Table 1-3]

[0173] [Table 1-4]

[0174] Cancellation Criteria The criteria for temporarily suspending or adjusting dose escalation are as follows: If an unacceptable tolerability profile is observed, based on the nature, frequency, and severity of the observed adverse events, as jointly determined by the principal investigator, medical director, and sponsor's medical monitor. If two or more subjects experience a serious adverse event (SAE) or severe AE while receiving the investigational drug, and the principal investigator, medical officer, or medical staff of the sponsor determine that there is a causal relationship with IMP.

[0175] When making decisions regarding dose increases, the decision-maker had access to unblinded data at that time. If data validation found that any of the above criteria were met and a correlation with drug administration was found, the dose escalation was to be discontinued. However, this situation never actually occurred.

[0176] Treatment period A single dose of CIT-013 was administered to achieve the research objectives (safety, tolerability, and pharmacokinetics of a single dose). To evaluate the pharmacodynamic effects of CIT-013 in this study, intravenous LPS was administered 2.5 hours after the start of CIT-013 infusion.

[0177] Key evaluation criteria No formal primary endpoint was established for this study. All endpoints are considered exploratory.

[0178] [Table 2-1]

[0179] [Table 2-2]

[0180] [Table 3-1]

[0181] [Table 3-2]

[0182] [Table 3-3]

[0183] result The main results of this study are shown in Figures 1-8. The results shown for each are as follows:

[0184] Figure 1 shows the results of CIT-013 epitope concentrations (Units / ml) in the serum of placebo-treated, LPS-loaded healthy volunteers. Measurements were performed only in the placebo group because the assay relies on antibodies that bind to the CIT-013 epitope and cross-compete with CIT-013 present in the serum of CIT-013-administered volunteers. Panel A) shows the results for each placebo subject in Cohort 1 (n=3). Panel B) shows the results for each placebo subject in Cohort 2 (n=6).

[0185] Figure 2 shows the results for serum citrullinated histone 3 levels (ng / ml) in a cohort of healthy volunteers treated with either placebo or CIT-013. LPS was administered at 2 ng / kg two hours after placebo or CIT-013 treatment. Cohort 1 results are shown in panels A) and B). Panel A) shows the results for subjects (n=3) in Cohort 1 treated with placebo. Panel B) shows the results for subjects (n=3) treated with 0.3 mg / kg CIT-013. Cohort 2 results are shown in panels C) and D). Panel C) shows the results for subjects in Cohort 2 treated with placebo (n=6). Panel D) shows the results for Cohort 2 treated with 0.9 mg / kg CIT-013 (n=8). Panel E) shows the calculated area under the curve for placebo and healthy volunteers treated with CIT-013 in Cohort 2. Unpaired Mann-Whitney tests were performed to calculate statistical differences. *** p=0.00075.

[0186] Figure 3 shows the results of CXCL10 levels (pg / ml) measured in plasma from two cohorts of healthy volunteers treated with either placebo or CIT-013. LPS was administered at 2 ng / kg two hours after placebo or CIT-013 treatment. Cohort 1 was treated with A) placebo (n=3) or B) 0.3 mg / kg CIT-013 (n=3). Cohort 2 was treated with C) placebo (n=6) or D) 0.9 mg / kg CIT-013 (n=8). The circled data point of 20,000 pg / ml indicates that the upper limit of quantification was reached and further sample dilution was not possible.

[0187] Figure 4 shows the results for plasma TNF-α levels (pg / ml) in two cohorts of healthy volunteers treated with either placebo or CIT-013. LPS was administered at 2 ng / kg two hours after placebo or CIT-013 treatment. Cohort 1 was treated with A) placebo (n=3) or B) 0.3 mg / kg CIT-013 (n=3). Cohort 2 was treated with C) placebo (n=6) or D) 0.9 mg / kg CIT-013 (n=8). Data points circled indicate that the upper limit of quantification was reached and further sample dilution was not possible.

[0188] Figure 5 shows the results for plasma VCAM-1 levels (pg / ml) in two cohorts of healthy volunteers treated with either placebo or CIT-013. LPS was administered at 2 ng / kg two hours after placebo or CIT-013 treatment. Cohort 1 was treated with A) placebo (n=3) or B) 0.3 mg / kg CIT-013 (n=3). Cohort 2 was treated with C) placebo (n=6) or D) 0.9 mg / kg CIT-013 (n=8).

[0189] Figure 6 shows the results of plasma IL-10 levels (pg / ml) in two cohorts of healthy volunteers treated with either placebo or CIT-013. LPS was administered at 2 ng / kg two hours after placebo or CIT-013 treatment. Cohort 1 was treated with A) placebo (n=3) or B) 0.3 mg / kg CIT-013 (n=3). Cohort 2 was treated with C) placebo (n=6) or D) 0.9 mg / kg CIT-013 (n=8).

[0190] Figure 7 shows plasma calprotectin levels (ng / ml) in two cohorts of healthy volunteers treated with either placebo or CIT-013. LPS was administered at 2 ng / kg two hours after placebo or CIT-013 treatment. Cohort 1 was treated with A) placebo (n=3) or B) 0.3 mg / kg CIT-013 (n=3). Cohort 2 was treated with C) placebo (n=6) or D) 0.9 mg / kg CIT-013 (n=8).

[0191] Figure 8 shows the results of plasma P-selectin concentrations (ng / ml) in two cohorts of healthy volunteers treated with either placebo or CIT-013. LPS was administered at 2 ng / kg two hours after placebo or CIT-013 treatment. Cohort 1 was treated with A) placebo (n=2) or B) 0.3 mg / kg CIT-013 (n=3). Cohort 2 was treated with C) placebo (n=6) or D) 0.9 mg / kg CIT-013 (n=8).

[0192] Example 2 Given the specific efficacy and distinctive cytokine pattern of 0.3 mg / kg compared to 0.9 mg / kg, which was effective but did not exhibit a different cytokine pattern, lower endpoints were calculated based on target binding calculations. Specifically, the calculations were based on the number of CIT-013 molecules (i.e., target binding) that (completely) bind to citrullinated target epitopes on histone H2A and H4 in the blood compartments of SLE patients and LPS-loaded healthy volunteers (HVs), based on CitH3 levels measured in the serum of active SLE patients at levels equivalent to or slightly lower than those in LPS-loaded HVs. All calculations were based on CitH3 levels measured in the serum of active SLE patients at levels equivalent to or slightly lower than those in LPS-loaded HVs.

[0193] The approach adopted was as follows: • Serum citrullinated H3 levels in SLE patients were determined using ELISA (Cayman). In patients with active or remittent SLE, mean levels of 12 ng / mL and 19 ng / mL were detected, respectively. • Total circulating blood (5 liters) contains 60-95 μg, and assuming an estimated size of histone H3 of 15 kD, this corresponds to 4 nmol-6.33 nmol. The findings served as a starting point for performing target binding calculations for CIT-013.

[0194] In LPS-loaded HV (high-variable vein) testing, the highest CitH3 levels among healthy (HV) volunteers reached approximately 15 ng / mL. This is comparable to the average CitH3 levels in SLE patients. The results are shown in Table 4 below.

[0195] [Table 4]

[0196] Next, we considered the following factors: The protein sizes of the four core histones range from 11.4 kD(H4) to 15.4 kD(H3). The total molecular weight of a nucleosome (8-mer) is approximately 110 kD. Histone H2A has a molecular weight of approximately 14 kD (including when citrullinated). Histone H3 has a molecular weight of approximately 15 kD (including when citrullinated). CIT-013 recognizes citrulline residues on histone H2A and H4, which means that four histone H2A and H4 per nucleosome provide a total of four citrullinated epitopes. • Proportion of histone H3: H2A / H4 = 2:4 (1:2).

[0197] Next, the following was considered in the calculation: 1. The CitH3 content in serum from SLE or LPS-loaded HV is approximately 15 ng / mL, which is equivalent to 75 μg in 5 L of blood. 2. The histone ratio H2A / H4:H3 is 2:1, meaning that approximately 150 μg of CitH2A / CitH4 is present in 5L of blood. 3. Since the molecular weight of the tetramer H2A / or H4 is approximately 15 kD, the molecular weight is 150 μg / 15000 = 10 nmol. 4. Existing epitope molecules: 10 nmol × 6.03E23 = 6,022E15. 5. When CIT-013 binds simultaneously to H2A and H4 citrulline residues (divalent), 6,022E15 molecules need to be blocked. The required amount of CIT-013 is 6,022 molecules (10 nmol) / 2, which is equivalent to 750 μg of the drug. 6. When CIT-013 binds separately to citH2A and citH4 (monovalent), the required amount doubles: 6,022 molecules (10 nmol) is equivalent to 1500 μg of the drug.

[0198] The calculations assumed that all citrulline residues were present and that CIT-013 was accessible. The calculations were based on NETs in plasma; NETs in tissues were not included.

[0199] Calculations show that in SLE patients or those with LPS-loaded HV, a dose of 750 μg (based on divalent binding) to 1500 μg (based on monovalent binding) is already sufficient to bind all citrullinated H2A / H4 histones to the blood compartment. This corresponds to a lower limit of 0.01 mg / kg to 0.02 mg / kg in the effective dose range.

[0200] Example 3: Determination of the presence of the CIT-013' epitope in rheumatoid arthritis serum samples.

[0201] overview The presence of the CIT-013 epitope in the serum of rheumatoid arthritis (RA) patients was compared to the epitope levels in the serum of healthy volunteers. Using our proprietary epitope ELISA, we clearly demonstrated that neutrophil extracellular traps (NETs) containing citrullinated histones H2A and H4 can be detected in RA serum, and that NET levels are higher compared to the serum of healthy volunteers. These findings further suggest the potential of CIT-013 for the treatment of rheumatoid arthritis patients. The main objective of this study was to determine the presence of the CIT-013 epitope in the serum of rheumatoid arthritis patients and to investigate whether its levels are elevated in the RA population compared to the serum of healthy subjects.

[0202] material and method Human serum sample On December 23, 2021, a well-characterized serum cohort consisting of 58 informed-consent patients with rheumatoid arthritis (RA) was received from the -80°C storage facility of the biobank at Radboud University Medical Center in Nijmegen, Netherlands. Upon arrival at Citryll, the samples were aliquoted and stored in a -80°C temperature-controlled freezer until analysis.

[0203] Normal human serum from 35 healthy subjects was obtained by venipuncture into serum separation tubes (SSTs), processed, aliquoted, and stored at -80°C. All healthy subjects provided informed consent and were recruited from multiple companies located in Pivotpark, Oss, Netherlands. Blood processing was performed by Citryll using standard procedures (coagulation at room temperature for 30-45 minutes, centrifugation at 1,300g, and aliquoting of serum). Serum samples from both RA patients and healthy volunteers (HV) were analyzed using the company's proprietary "epitope" ELISA method. This ELISA determines the presence of citrullinated histones H2A and H4 in the NET structure, thereby determining the epitope of CIT-013. The reagents essential for performing the ELISA are listed in Table 5 below.

[0204] [Table 5]

[0205] Data and statistical analysis Data obtained from epitope ELISA showed elevated epitope levels in the RA cohort compared to the HV cohort. To demonstrate significance, the Mann-Whitney test (unpaired; non-parametric; two-sided test, p-value and 95% confidence level) was performed using GraphPad Prism software (GraphPad Prism, San Diego, USA). A p-value of less than 0.05 was considered to indicate a significant difference between the RA and HV cohorts. A p-value of 0.074 was observed, indicating no significant difference.

[0206] NET detection The method used to detect circulating NETs in serum samples from both RA patients and healthy subjects was a proprietary "epitope" (i.e., citrullinated histone H2A and H4) ELISA (Figure 10). Serum samples, along with a dilution series of activated NETs as a standard, were pipetted into wells of coated microtiter plates, similar to quality control. The coating was a recombinant multiclonal antibody specific to citrullinated histone H3. Any NETs containing citrullinated histone H3 were bound to the immobilized antibody, and after washing away unbound material, mouse anti-citrullinated histone antibody (ACHA) specific to citrullinated histone H2A and H4 was added to the wells. After washing to remove any unbound mouse ACHA, enzyme-conjugated polyclonal goat anti-mouse IgG antibody was added to the wells. After a final wash to remove any unbound antibody-enzyme reagent, substrate solution (TMB) was added to the wells, and color development was proportional to the amount of citrullinated NETs present in the wells. The color development was stopped using a strong acid, and the optical density (wavelengths 450 and 620 nm) was measured using a Tecan ELISA reader.

[0207] All calibrators and quality control samples were freshly prepared on the day of analysis. Serum samples were diluted 5-fold on the day of analysis. Each calibrator, quality control, and serum sample was measured in duplicate (i.e., 2 wells). The coefficient of variation (%CV) of each duplicate measurement must be ≤30.0% (calculated as CV% = 100 × SD of duplicate measurement / mean of duplicate measurement).

[0208] Samples were analyzed using a 96-well absorbance reader (Infinite F50 Plus; Tecan Ltd, Mannedorf, Switzerland). Data processing was performed using GraphPad Prism (version 9.1.0) software provided by GraphPad Prism Software (San Diego, USA). Sample concentrations were calculated using a 4-parameter logistic (4-PL) nonlinear regression model; no weighting coefficients were used.

[0209] The LLOQ for epitopes (i.e., citrullinated NETs) in 100% serum of both RA and HV was 0.625 U / mL, and the ULOQ was 120 U / mL.

[0210] Results and Discussion Serum samples from both RA patients and healthy volunteers were analyzed on three different plates in a single analytical run using epitope ELISA. As shown in Figure 10a, the calibration curves were comparable, with accuracy (%CV) and recovery rate (%RE) ranging from 2.9–20.7% and 92.3–107.9%, respectively (Table 2). For quality control, the %CV was between 1.3–9.8% and the %RE was between 132–277%. Although the QC recovery rate was too high, the NET data obtained from the RA and HV serum samples were relatively comparable, and the calibration curves for all three plates were very comparable; therefore, we decided to report the obtained NET data (Figure 1a).

[0211] The back-calculated values ​​for all analyzed serum samples were determined and plotted against a calibration curve (Figure 10b). Here, we showed that epitope levels in the serum of the RA cohort were higher than those in the HV cohort. Although there was a size imbalance between the groups, it was clear that citrullinated NET levels were higher in RA patients compared to healthy subjects.

[0212] [Table 6]

[0213] The presence of citrullinated NETs in the serum of RA patients opens up the possibility of treating rheumatoid arthritis with CIT-013, the first therapeutic agent in its class. The observation that NET content in RA patients is elevated compared to that in HV is consistent with Bach et al. (2020) Arthritis & Rheumatology; 72: pp47-56. Their study demonstrated significantly elevated NET levels in RA patients from three different cohorts (two plasma, one serum) compared to healthy controls, regardless of the matrix used (p < 0.001 for all three cohorts). While Bach et al. determined NET content in plasma and serum matrices by myeloperoxidase:DNA complex analysis, our study used our proprietary epitope ELISA to determine specific citrullinated NETs containing the CIT-013 epitope.

[0214] conclusion Serum from RA patients showed higher levels of citrullinated NETs and epitopes compared to serum from HV patients. Since NETs contribute to the development and pathology of RA disease, these findings support the development of CIT-013 for the treatment of RA patients. Based on this study, it may be possible to set an epitope level threshold, for example, >10 U / mL, for recruiting the RA population in the CIT-013 clinical trial.

[0215] Appendix: List of Abbreviations %CV: Percentage of coefficient of variation %RE Recovery Rate DNA (Deoxyribonucleic Acid) ELISA (Enzyme-mediated Immunoassay) HV Healthy Volunteers ACHA anti-citrullinated histone antibody NET neutrophil extracellular trap PAD4 Peptide Arginine Deiminase 4 RA (Rheumatoid Arthritis) RT room temperature SST serum separation tube TMB 3,3′,5,5′-Tetramethylbenzidine U / mL units / milliliter

[0216] Array List Sequence ID: 1- msVH22.101 and hVH22.101(HC)x CDR1 GYTFTNYG Sequence IDs: 2- msVH22.101 and hVH22.101(HC)x CDR2 INTYSGEA Sequence ID: 3- msVH22.101 and hVH22.101(HC)x CDR3 LRGYTYQSFDEGGDY Sequence IDs: 4- msVL22.101 and hVL22.101(LC)y CDR2 LVS Sequence ID: 5- CDR3 of msVL22.101 and hVL22.101(LC)y WQGTHFPYT Sequence ID: 6- hVL22.101LC17 CDR1 QSLLDTDGKTY Sequence ID: 7- hVL22.101LC21 CDR1 QSLLDSDAKTY CDR1 of Sequence No.: 8 - hVL22.101LC27 QSLLDTDAKTY CDR1 of Sequence No.: 9 - hVL22.101LC41 QSLLDADGKTY CDR1 of Sequence No.: 10 - hVL22.101LC42 QSLLDNDGKTY Sequence No.: 11 - hVH22.101f RIQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS Sequence No.: 12 - hVH22.101HC9 RIQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYVDDFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS Sequence No.: 13 - hVL22.101LC17 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDTDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Sequence No.: 14 - hVL22.101LC21 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDAKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Sequence No.: 15 - hVL22.101LC27 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDTDAKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Accession number: 16 - hVL22.101LC41 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDADGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Accession number: 17 - hVL22.101LC42 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDNDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Accession number: 18 - SEQ ID NO:1 from WO2016092082 (used in Example 1 / 7), derived from histone 2A SGXGKQGGKARA X is citrulline Accession number: 19 - SEQ ID NO:2 from WO2016092082 (used in Example 7), derived from histone 4 SGXGKGGKGLGKGGAKRHRKVLR X is citrulline Accession number: 20 - Truncated SEQ ID NO:2 from WO2016092082 (used in Example 7), derived from histone 4 SGXGKGGKGLGK X is citrulline Accession number: 21 - Peptide No. 4 (human histone 2A) (SEQ ID NO:24 of WO2011070172) QFPVGXVHRLLR X is citrulline Sequence ID: 22- Peptide number 6 (Human Histone 2A) (Sequence ID 26 of WO2011070172) VHRLLXKGNYSE X is citrulline Sequence ID: 23- Human heavy chain constant domain of IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Sequence ID: 24- Human kappa chain constant domain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID: 25- msVH22.101 RIQLVQSGPELKKPGEAVKISCKASGYTFTNYGMHWMKQTPGKDFRWMGWINTYSGEATYVDDFKGRFAFSLGTSASTAYLQINNLKNDDTATYFCLRGYTYQSFDEGGDYWGQGTALTVSS Sequence ID: 26- hVH22.101j QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS Sequence ID: 27- hVH22.101HC7 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS Sequence ID: 28- hVH22.101HC8 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYVDDFQGRVTITADESTSTAYMELSSLRSEDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS Sequence ID: 29- hVH22.101HC10 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMHWVRQAPGQGLEWMGWINTYSGEATYVDDFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLRGYTYQSFDEGGDYWGQGTLVTVSS Sequence ID: 30- msVL22.101 DVVMTQTPLTLSVTTGQPASISCKSSQSLLDSDGKTYLNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPYTFGGGTNLEIK Sequence ID: 31- hVL22.101e DVVMTQSPLSLPVTLGQPASISCRSSQSLVDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Accession number: 32 - hVL22.101g DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Accession number: 33 - hVL22.101h DVVMTQSPLSLPVTLGQPASISCRSSQSLVASDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Accession number: 34 - hVL22.101i DVVMTQSPLSLPVTLGQPASISCRSSQSLVESDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Accession number: 35 - hVL22.101j DVVMTQSPLSLPVTLGQPASISCRSSQSLVSSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK Accession number: 36 - CDR1 of msVL22.101 and hVL22.101g QSLLDSDGKTY Accession number: 37 - CDR1 of hVL22.101e QSLVDSDGKTY Sequence ID: 38-hVL22.101h CDR1 QSLVASDGKTY Sequence ID: 39-hVL22.101i CDR1 QSLVESDGKTY Sequence ID: 40- hVL22.101j CDR1 QSLVSSDGKTY Sequence ID: 41- hVL22.101LC16 CDR1 QSLLESDGKTY Sequence ID: 42- hVL22.101LC19 CDR1 QSLLDSEGKTY Sequence ID: 43- hVL22.101LC20 CDR1 QSLLDSSGKTY Sequence ID: 44- hVL22.101LC22 CDR1 QSLLESEGKTY Sequence ID: 45- hVL22.101LC23 CDR1 QSLLESSGKTY Sequence ID: 46- hVL22.101LC24 CDR1 QSLLESDAKTY Sequence ID: 47- hVL22.101LC25 CDR1 QSLLDTEGKTY Sequence ID: 48- hVL22.101LC26 CDR1 QSLLDTSGKTY Sequence ID: 49- hVL22.101LC37 CDR1 QSLLDSAGKTY Sequence ID: 50- hVL22.101LC38 CDR1 QSLLESAGKTY Sequence ID: 51-hVL22.101LC39 CDR1 QSLLDAEGKTY Sequence ID: 52- hVL22.101LC40 CDR1 QSLLDNEGKTY Sequence ID: 53- msFibβ XG (Sequence ID 37 of WO2011070172) EPTDSLDAXGHRPVDRR X is citrulline Sequence ID: 54- msVim XS / XL (Sequence ID 38 of WO2011070172) YVTXSSAVXLXSSVP X is citrulline Sequence ID: 55 - CDR2 peripheral region of msVL22.101 and hVL22.101(LC)y LVSKLDS Sequence ID: 56- Heavy chain steady region of hCH22.101f ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. An antibody or its conjugated fragment that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, for use in a method of treating or preventing a disease associated with the release of extracellular traps from cells, such as neutrophil extracellular trap (NET)-associated pathology or eosinophil extracellular trap (EET)-associated pathology, wherein the method comprises administering at least one dose of the antibody or its conjugated fragment at a dose of about 0.01 mg / kg to about 0.6 mg / kg.

2. The antibody or its bound fragment: (a) Approximately 0.02 mg / kg to approximately 0.5 mg / kg; (b) Approximately 0.05 mg / kg to approximately 0.4 mg / kg; or (c) Approximately 0.1 mg / kg to approximately 0.3 mg / kg An antibody or its conjugated fragment for use according to claim 1, administered in the dose of [specify dose].

3. The antibody or its conjugated fragment for use according to claim 1, wherein the antibody or its conjugated fragment is administered in a dose of about 0.3 mg / kg.

4. (a) The antibody or its conjugated fragment is administered intravenously or subcutaneously to the subject; and / or (b) Administration of the antibody or its conjugated fragment increases the level of IL-10 in the subject; and / or (c) Administration of the antibody or its binding fragment suppresses the level of at least one of the following in the subject: calprotectin, P-selectin, CXCL10, TNF-α, and VCAM. An antibody or its conjugated fragment for use according to any one of the preceding claims.

5. (a) 1 to 10 doses of the antibody are administered; (b) Two to eight doses of the antibody are administered; or (c) 4 to 6 doses of the antibody or binding fragment are administered, and optionally, multiple doses: (i) From one week to three months; (ii) One to three weeks; or (iii) Approximately 2 weeks It is administered at intervals of, An antibody or its conjugated fragment for use according to any one of the preceding claims.

6. The antibody or its bound fragment: a) VL CDR1, wherein the CDR has the amino acid sequence QSL-X １ -D-X ２ -D-X ３ -Includes or consists of KTY, X １ is V or L, X ２ is T, S, A or N, and X ３ is G or A, provided that the amino acid sequence is not QSLLDSDGKTY (SEQ ID NO: 36) or QSLLVDSDGKTY (SEQ ID NO: 37); and b) At least one CDR selected from sequence numbers 1 to 5 An antibody or its conjugated fragment for use according to any one of the preceding claims, including

7. The antibody or its bound fragment: a) VL CDR 1 of Sequence ID No. 9 (QSLLDADGKTY); and b) VH CDR 1 of SEQ ID NO: 1 (GYTFTNYG), VH CDR 2 of SEQ ID NO: 2 (INTYSGEA), VH CDR 3 of SEQ ID NO: 3 (LRGYTYQS FDEGGDY), VL CDR 2 of SEQ ID NO: 4 (LVS), and VL CDR 3 of SEQ ID NO: 5 (WQGTHFPYT) Includes, Optionally, the antibody or its bound fragment may be: i) VL CDR1 of sequence number 9; ii) VL CDR2 of SEQ ID NO: 4 and VL CDR3 of SEQ ID NO: 5; and iii) Amino acid sequence of the heavy chain variable domain of SEQ ID NO: 11 or 12 An antibody or its conjugated fragment for use according to claim 6, comprising:

8. The aforementioned antibody or binding fragment is: a) The heavy chain variable domain amino acid sequence described in SEQ ID NO: 11 and the light chain variable region amino acid sequence described in SEQ ID NO: 16; or b) The heavy chain variable domain amino acid sequence described in SEQ ID NO: 12 and the light chain variable region amino acid sequence described in SEQ ID NO: 16 An antibody or its conjugated fragment for use according to claim 6 or claim 7, comprising:

9. The aforementioned NET-related pathologies include tauopathy, systemic lupus erythematosus (SLE), lupus, sepsis, vasculitis, inflammatory arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, hidradenitis suppurativa, Alzheimer's disease, autoimmune hepatitis, juvenile idiopathic arthritis, myositis (polymyositis and dermatomyositis), Sjögren's disease, antiphospholipid syndrome, Behçet's disease, spondylitis, spondyloarthritis, multiple system atrophy, Parkinson's disease, and Lewy body dementia (Lewy body dementia). Other NET-related pathologies such as asthma, allergic rhinovirus exacerbated asthma, allergic asthma, acute respiratory distress syndrome, cystic fibrosis, fibrosis and idiopathic pulmonary fibrosis, heart failure, atherosclerosis, dry eye disease, uveitis, non-granulomatous uveitis, granulomatous uveitis, dermatitis, atopic dermatitis, COPD, bronchitis, thrombotic disease, cardiovascular disease, or wound healing in diabetes, cancer, cancer metastasis, or wound healing in diabetes, cancer, cancer metastasis, and the health of transplanted organs in vivo or ex vivo. An antibody or its conjugated fragment for use according to any one of the preceding claims.

10. An antibody or its conjugated fragment for use according to any one of the preceding claims, wherein the NET-associated pathology is Parkinson's disease.

11. The aforementioned EET-related pathologies include: eosinophilic diseases or conditions of the skin; eosinophilic diseases or conditions of the respiratory system; eosinophilic diseases or conditions of the gastrointestinal tract; allergic diseases or conditions; or infections of helminths, fungi, viruses, or bacteria; or bullous pemphigoid atopic dermatitis, allergic contact dermatitis, eosinophilic asthma, chronic sinusitis with nasal polyps (CRSwNP), allergic sinusitis, allergic bronchopulmonary aspergillosis, eosinophilic chronic sinusitis, eosinophilic esophagitis (EoE), eosinophilic syndrome (HES), eosinophilic polyangiitis (EGPA), eosinophilic otitis media (EOM), and drug reactions with eosinophilic and systemic symptoms. An antibody or its conjugated fragment for use according to any one of claims 1 to 8, which is an eosinophilic disease or condition such as eosinophilic diseases or conditions such as dysplasia (DRESS), arteriosclerosis, and vasculitis.

12. A method for treating neutrophil extracellular trap-associated pathology (NET-associated pathology) in a subject requiring such treatment, comprising administering to the subject at least one dose of an antibody or a conjugated fragment thereof that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, wherein each dose of the antibody or conjugated fragment is approximately 0.01 mg / kg to approximately 0.6 mg / kg.

13. (a) The dose of the antibody or its conjugated fragment to be administered is as defined in claim 2 or 3; (b) The administration route of the antibody or its binding fragment is as defined in claim 4; (c) The administration of the antibody or its conjugated fragment increases the level of IL-10 in the subject; (d) The administration of the antibody or its conjugated fragment suppresses the level of at least one of the following in the subject: calprotectin, P-selectin, CXCL10, TNF-α, and VCAM; (e) The sequence of the antibody is defined in any one of claims 6 to 8; and / or (f) The NET-related pathology is defined in claim 9 or claim 10. The method according to claim 12.

14. A method for treating eosinophil extracellular trap (EET)-associated pathology in a subject requiring such treatment, comprising administering to the subject at least one dose of an antibody or a conjugated fragment thereof that specifically binds to a citrullinated epitope on deiminated human histone 2A and / or histone 4, wherein each dose of the antibody or conjugated fragment is approximately 0.01 mg / kg to approximately 0.6 mg / kg.

15. (a) The dose of the antibody or its conjugated fragment to be administered is as defined in claim 2 or 3; (b) The administration route of the antibody or its binding fragment is as defined in claim 4; (c) The administration of the antibody or its conjugated fragment increases the level of IL-10 in the subject; (d) The administration of the antibody or its conjugated fragment suppresses the level of at least one of the following in the subject: calprotectin, P-selectin, CXCL10, TNF-α, and VCAM; (e) The sequence of the antibody is defined in any one of claims 6 to 8; and / or (f) The EET-related pathology is defined in claim 11. The method according to claim 14.

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