Cell-free nucleosome-level triage method

Abstract

This invention provides a method for identifying patients at risk of developing NETosis-related adverse reactions to infections, using cell-free nucleosome levels. [Solution] A method for monitoring the progression of a disease in a subject suffering from an infectious disease, (i) Contacting a body fluid sample obtained from the subject with a binder to detect or measure the level of cell-free nucleosomes or their components; (ii) Repeating step (i) on one or more occasions; and (iii) The method provides comprising monitoring the progression of an infection in a subject using any changes at the level of the cell-free nucleosome or its components.

Description

[Technical Field]

[0001] (Field of Invention) This invention is particularly relevant to patients at high risk of developing NETosis-related adverse reactions to infectious diseases. Cell-free cells as biomarkers in bodily fluid samples from patients with infectious diseases to identify individuals. The present invention relates to the use of nucleosomes. The present invention relates to therapeutic antibodies for the treatment of NETosis-related conditions and This also relates to the use of anti-nucleosome antibodies. [Background technology]

[0002] (Background of the invention) Influenza spreads worldwide in annual epidemics, resulting in approximately 3 to 5 million cases. It causes severe illness and approximately 290,000 to 650,000 deaths. Very recently, a new infectious disease called COVID-19 has emerged. The outbreak and rapid progression of D-19 led to a pandemic. Several infectious diseases, Acute respiratory syndrome (ARS), which can progress to a life-threatening stage requiring medical intervention. This can lead to acute respiratory distress syndrome (ARDS) or severe acute respiratory syndrome (SARS). This can also lead to an epidemic of infectious diseases. The pandemic has placed a significant burden on international healthcare services, making hospital interventions more urgent. A method of triaging patients to identify those most likely to need it is healthcare Providers prioritize patients, save lives, and more effectively meet the higher demand for medical services. It is extremely important in helping to manage things.

[0003] COVID-19, influenza, and other infectious diseases can be severe and potentially fatal. NETosis-related complications can progress to disability. Such complications include infection. One possible complication of the disease is sepsis, a life-threatening organ failure. Methods for treating severe NETosis, methods for identifying individuals at high risk of NETosis-related complications, and Methods for monitoring the progression of such complications requiring treatment, and the efficacy of treatment. Methods for monitoring such diseases, and methods for monitoring the progression of such diseases, are currently unavailable. Adding.

[0004] Previously, in the literature by Holdenrieder et al., Int. J. Cancer (2001) 95: 114-120, benign and malignant diseases It is described that the level of nucleosomes in serum samples from patients with the disease may be detected. . Cyclo-free content of histone modifications, histone variants, DNA modifications, and adducts. The epigenetic composition of cellular nucleosomes is also a blood-based biomarker for cancer. This has been studied in WO 2005 / 019826, WO 2013 / 030577, WO 2013 / 030579, and WO Please refer to issue 2013 / 084002.

[0005] Effective treatment of NETosis-related conditions, and treatment of NETosis-related complications with poor prognosis during infection. A simple and cost-effective method for identifying and prioritizing individuals at high risk of developing the disease, as well as the disease itself. There remains a need in this field to provide methods for monitoring treatment and disease progression. [Brief explanation of the drawing]

[0006] (Brief explanation of the drawing) [Figure 1]Results of an immunoassay on neutrophil extracellular trap (NET)-derived nucleosomes in EDTA-treated plasma and heparin-treated plasma samples collected from two healthy volunteers. The EDTA sample contained low levels of NET-derived nucleosome material. In contrast, heparin induced NET formation, and the heparin-treated plasma sample contained high levels of induced NET-derived nucleosomes. [Figure 2] Bioanalyzer electrophoresis results for NET-derived nucleosomes in EDTA plasma and heparin plasma samples collected from two healthy volunteers. The EDTA sample contains both low levels of mononucleosome material and NET-derived nucleosome material. In contrast, heparin induces NET formation, and the heparin plasma sample contains low levels of mononucleosomes (peak at approximately 60 seconds) but induces high levels of induced NET-derived nucleosomes (broad peak at approximately 110 seconds). Narrow peaks at approximately 43 seconds and 110 seconds represent DNA samples added for reference purposes. [Figure 3] Levels of nucleosomes containing histone isoform H3.1 measured in 50 patients hospitalized with symptoms of COVID-19 infection, including 34 symptomatic patients who tested positive for COVID-19 by PCR and 16 symptomatic patients who tested negative by PCR, as well as in 50 normal subjects who did not show symptoms of the disease. [Figure 4] Levels of nucleosomes containing histone isoform H3.1, measured in 15 patients with PCR-confirmed COVID-19 infection, including: patients attending scheduled outpatient clinics or being examined in the hospital emergency room (ER); 3 patients hospitalized in general wards; 2 patients hospitalized in the intensive care unit (ICU) (requiring respiratory support and surviving); and 4 patients hospitalized in the ICU (requiring respiratory support and dying). [Figure 5]Levels of nucleosomes containing histone modification H3R8Cit measured in 15 patients with PCR-confirmed COVID-19 infection, including five samples recovered from patients who were coming to the reserved outpatient clinic or undergoing examination in the emergency room (ER) of the hospital; three patients admitted to the general ward; two patients admitted to the intensive care unit (ICU) (requiring respiratory assistance and survived); and four patients admitted to the ICU (requiring respiratory assistance and died). [Figure 6] Results of the experiment described in Example 12 showing the average levels of nucleosomes containing histone isoform H3.1 measured in 16 pigs induced with sepsis who received plasmapheresis. In nine pigs, plasma was passed through a cartridge containing a NET binder (with treatment, black bars), and in seven pigs, plasma was passed through a control cartridge without a NETs binder (control, white bars). [Figure 7] Results of the experiment described in Example 12 and shown in Figure 6, except for the levels in individual test subjects. [Figure 8] H3.1-nucleosome levels measured in human subjects diagnosed with sepsis and healthy human subjects. SUMMARY OF THE INVENTION

[0007] (Summary of the Invention) According to a first aspect, a method for monitoring the progression of a disease in a subject suffering from an infectious disease, comprising: (i) contacting a body fluid sample obtained from the subject with a binding agent to detect or measure the level of cell-free nucleosomes or components thereof; (ii) repeating step (i) on one or more occasions; and (iii) using any change in the level of the cell-free nucleosomes or components thereof to monitor the progression of the infectious disease in the subject (iv) repeating step (i) on one or more occasions; and (v) using any change in the level of the cell-free nucleosomes or components thereof to monitor the progression of the infectious disease in the subject is provided. : A method is provided that includes the above steps.

[0008] According to a further aspect, a method of assigning the risk of developing or progressing medical complications in a subject suffering from an infectious disease, comprising: (i) contacting a body fluid sample obtained from the subject with a binding agent to detect or measure the level of cell-free nucleosomes or components thereof; and (ii) using the detected level of cell-free nucleosomes to assign the likelihood that a medical complication will develop or progress in the subject is provided. (ii) using the detected level of cell-free nucleosomes to assign the likelihood that a medical complication will develop or progress in the subject is provided. : is provided.

[0009] According to a further aspect, a method of assigning the risk of adverse outcome to a subject suffering from an infectious disease, comprising: (i) contacting a body fluid sample obtained from the subject with a binding agent to detect or measure the level of cell-free nucleosomes or components thereof; and (ii) using the detected level of cell-free nucleosomes to assign the likelihood of an adverse outcome to the subject where subjects identified as having a high likelihood of adverse outcome are assigned to medical intervention (ii) using the detected level of cell-free nucleosomes to assign the likelihood of an adverse outcome to the subject is provided. : comprising where subjects identified as having a high likelihood of adverse outcome are assigned to medical intervention is provided. <​​​​​​​​​​​​​​​​​In a preferred embodiment, the infectious disease is respiratory influenza or coronavirus infection. It is a disease, and medical complications include ARS, ARDS, SARS, or pneumonia. Therefore, it is a real condition. The method of application detects individuals requiring medical treatment for pneumonia, ARS, ARDS, or SARS. It is a method, (i) The bodily fluid sample obtained from the subject is brought into contact with a binder to form cell-free nucleosomes or To detect or measure the level of the components; and (ii) The level of cell-free nucleosomes in the subject is a medical condition for pneumonia, ARS, ARDS, or SARS. To use as an indicator that medical treatment is needed. A method is provided that includes:

[0012] In a preferred embodiment, the infectious disease is respiratory influenza or coronavirus infection. The disease is characterized by medical complications such as ARS, ARDS, SARS, or pneumonia.

[0013] In another preferred embodiment, the infection is sepsis. Therefore, in one embodiment A method for detecting individuals who require medical treatment for sepsis or septic shock. There is, (i) The bodily fluid sample obtained from the subject is brought into contact with a binder to form cell-free nucleosomes or To detect or measure the level of the components; and (ii) The level of the cell-free nucleosomes in the medical field of sepsis or septic shock. To use as an indicator that appropriate treatment is needed. A method is provided that includes:

[0014] A further aspect of the present invention is a method for monitoring infectious diseases in a subject, , (i) The bodily fluid sample obtained from the subject is brought into contact with a binder to form cell-free nucleosomes or To detect or measure the level of a component; (ii) Testing the level of cell-free nucleosomes or their components in the body fluid obtained from the subject. Repeating the output or measurement on one or more occasions; (iii) Using any change at the level of the cell-free nucleosome or its components, the Monitoring the progression of infectious diseases in elephants A method is provided that includes: [Modes for carrying out the invention]

[0015] (Detailed explanation) Nucleosomes are released into circulation when chromatin fragments during cell death. Infectious diseases, such as viral infections, involve various mechanisms (cell binding and entry, endoscopy). Cell death is initiated through TLR3 activation and gene expression, thereby affecting the circulation of blood cells. It increases the number of ring nucleosomes (Danthi et al., Annu. Rev. Virol. (2016) 3: 533- 53). Furthermore, infections can induce NETosis, and this NETosis leads to histone H Post-translational histone modifications such as acetylation or hypercitrullination of 3 and H4 (Wang Y et al., J. Cell Biol. (2009) 184(2): 205-213) states that as the initial response to infection, cells circulate simultaneously. It promotes the decondensation of released chromatin. However, extracellular nucleosomes and Extracellular traps (NETs) of cytoplasmic cells, if not removed promptly, can cause severe complications. For example, nucleosome binding to the glomerular membrane is associated with renal injury in lupus. (Kalaaji et al., Kidney Int. (2007) 71(7): 665-672), on the other hand, NETs are viruses. It has been shown to enhance lung injury during pulmonary pneumonia (Ashar et al., Am. J. Pathol. (2018)). 188(1): 135-148). In fact, host-targeted NET toxicity causes respiratory distress, obstruction of narrow airways, and endothelial and It is associated with epithelial cell damage, inflammatory responses, thrombus formation, and other medical conditions (Marcos et al.). References: Nat. Med. (2010) 16: 1018-23; Hoeksema et al., Future Microbiol. (2016) 1 1: 441-53).

[0016] Most individuals infected with influenza or coronavirus experience only mild symptoms. However, in elderly people over 60 years of age, as well as those with diabetes, chronic lung disease, and especially chronic heart disease, Several population subgroups, including those with underlying medical conditions such as diseases, have been affected by ARS, SARS, and lung disease. You are exposed to the risk of severe effects, including fire and death. Influenza or coronavirus The exact mechanism by which Rus infection leads to complications, including pneumonia, is unknown, but it is likely due to excessive NETs are viral infections that can contribute to pneumonia and, in the worst cases, acute lung damage that can lead to death. It is thought to be caused by an excessive immune response to the dye.

[0017] This invention utilizes an increase in the level of cell-free nucleosomes, including NETs, ​​to treat infectious diseases. To predict the severity and outcome of the disease.

[0018] Therefore, according to one aspect, the risk of adverse outcomes is assigned to subjects suffering from infectious diseases. A method that (i) The bodily fluid sample obtained from the subject is brought into contact with a binder to form cell-free nucleosomes or To detect or measure the level of the components; and (ii) Using the detected cell-free nucleosome levels, the potential for adverse outcomes in the subject Assign A method is provided that includes: the method for which a person has been identified as having a high potential for adverse outcomes. Elephants can be used to be assigned to medical interventions.

[0019] One embodiment involves assigning the risk of adverse outcomes to individuals suffering from an infectious disease. And, (i) The body fluid sample obtained from the subject is brought into contact with a binder to form a neutrophil extracellular trapping substance or to detect or measure the level of its components; and (ii) Using the levels of detected neutrophil extracellular trapping material, the potential for adverse outcomes Assigning to elephants A method is provided that includes:

[0020] Nucleosomes are the basic units of chromatin structure and consist of eight highly conserved core histograms. From a protein complex of tons (composed of pairs of histones H2A, H2B, H3, and H4) Yes. Approximately 146 base pairs of DNA are wrapped around this complex. Another histone, H1 or H5 acts as a linker and is involved in chromatin condensation. DNA is often "string-like" In a structure described as resembling "connected beads," a continuous network of nucleosomes surrounds a series of nucleosomes. This wrapping process forms the basic structure of open chromatin, or euchromatin. In compressed chromatin, i.e., heterochromatin, this string is coiled and s It forms a supercoil and a closed, complex structure (Herranz and Esteller's literature, Methods) Mol. Biol. (2007) 361: 25-62).

[0021] The reference to "nucleosomes" is used when they are detected in bodily fluid samples, meaning "cell-free nucleosomes." It can refer to "mu". The term cell-free nucleosome is used more than once throughout this document. It is understood that it is intended to contain any cell-free chromatin fragments, including nucleosomes. It will probably happen.

[0022] It is understood that cell-free nucleosomes can be detected by binding to their constituent elements. The term “its constituent elements” as used herein refers to a part of a nucleosome. It refers to a part, meaning that the entire nucleosome does not need to be detected. Cell-free nucleosome The components of the system are histone proteins (i.e., histone H1, H2A, H2B, H3, or H4), post-translational histone modification, histone variant or isoform, nucleos A protein bound to a nucleosome (i.e., a nucleosome-protein adduct), nucleos DNA fragments associated with nucleosomes, and / or modified nucleotides associated with nucleosomes: It can be selected from the group. For example, its components may be histone (isoform) H3.1 or It may be stone H1 or DNA.

[0023] The method and use of the present invention can measure the level of (cell-free) nucleosomes themselves. The reference to "the rhosome itself" implies that nucleosomes contain some kind of epigenetic features. Regardless of whether they are present or not, all nucleosole present in the sample This refers to the nucleosome level or concentration. Detection at the total nucleosome level typically involves detecting all nucleosomes. This involves the detection of histone proteins common to osomes, such as histone H4. Therefore, nucleation The reosome itself detects core histone proteins, such as histone H4. It can be measured. As described herein, histone proteins are eukaryotic proteins. Known as nucleosomes, which are used to package DNA in living cells. It forms a structural unit.

[0024] Normal cell turnover in adults is due to the daily cell division of a vast number of cells. It involves cell generation and a similar number of deaths, mainly through apoptosis. Matin is broken down into mononucleosomes and oligonucleosomes released from the cell. Under normal conditions, the level of circulating nucleosomes observed in healthy subjects has been reported to be low. It has been reported that elevated levels are associated with many cancers, autoimmune diseases, inflammatory conditions, strokes, and myocardial infarction. This is observed in subjects with various conditions, including infarction (Holdenreider and Stieber's literature, Crit. Rev. Clin. Lab. Sci. (2009) 46(1): 1-24).

[0025] Previous nucleosome ELISA methods were primarily used as a method for detecting apoptosis. It was used in cell culture (Salgame et al., Nucleic Acids Res. (1997) 25(3): 680-68) 1; Holdenrieder et al. (2001), see above; van Nieuwenhuijze et al., Ann. Rheum. Dis. (2003) 62: 10-14), also used for the measurement of circulating cell-free nucleosomes in serum and plasma. Holdenrieder et al. (2001). Serum and plasma released into circulation by dying cells. Cell-free nucleosome levels are used to assess their potential use as biomarkers. Therefore, it is measured by the ELISA method in many different cancer studies.

[0026] Cell-free nucleosomes include mononucleosomes, oligonucleosomes, and larger nucleosomes. It may be a component of romatine fragments, a component of NET, or a mixture thereof.

[0027] Mononucleosomes and oligonucleosomes are analyzed using enzyme-linked immunosorbent assay (ELISA). It can be detected by several methods (for example, Salgame et al.'s References (1997); Holdenrieder et al. (2001); van Nieuwenhuijze et al. (2003). These The assay typically involves anti-histone antibodies (e.g., anti-H2B, anti-H3, or anti-H1, H2A, H2B). Using H3 and H4 as capture antibodies, anti-DNA or anti-H2A-H2B-DNA complex antibodies are used as detection antibodies. To use.

[0028] Circulating nucleosomes are not homogeneous groups of protein-nucleic acid complexes. Rather, they are, These are heterogeneous chromatin fragments resulting from the digestion of chromatin during cell death, and are specific to certain types of chromatin. Stone isoform (or variant), post-translational histone modification, nucleotide or modified nucleotide It includes a vast array of epigenetic structures, including creotides and protein adducts. Elevated nucleosome levels include certain histone isoforms (or variants). Nucleosomes, nucleosomes containing specific post-translational histone modifications, specific nucleo- Nucleosomes containing nucleotides or modified nucleotides, and nucleosomes containing specific protein adducts Some circulating nuclei containing specific epigenetic signals, including rhosomes. It will be obvious to those skilled in the art that this is related to an increase in osome subsets. Assays of chromatin fragments are known in the art (e.g., by reference to this specification). It is incorporated into WO 2005 / 019826, WO 2013 / 030579, WO 2013 / 030578, WO 2013 / (See issue 084002).

[0029] Some proteins are attached to NETs directly or indirectly to nucleosomes. These proteins include, but are not limited to, myeloperoxids. Ze (MPO), neutrophil elastase (NE), lactotransferrin, azulocidine, cathepsi ən G, leukocyte proteinase 3, lysozyme C, neutrophil defensin 1, neutrophil defensin Syn3, myeloid cell nuclear differentiation antigen, S100 calcium-binding protein A8, S100 calcium-binding Actin A9, S100, calcium-binding protein A12, actin β, actin γ, α-actin Plastin-2, cytokeratin-10, catalase, α-enolase, and transketra -se can be cited (Urban et al., PLOS Pathogens. (2009) 10: e1000639). It is found in NETs. Any nucleosome-protein adduct used in the method of the present invention is above the NETs level It is a useful adduct for detecting nitrates. C-reactive protein (CRP) is also a nucleo in NETs. Nucleosome-CRP adducts can be added to nucleosomes, and therefore, the method of the present invention This is a useful addition for detecting an increase in NETs levels.

[0030] In a preferred embodiment of the present invention, the adduct used is an MPO-nucleosome adduct. Alternatively, it is an NE-nucleosome adduct.

[0031] In one embodiment, the components of the cell-free nucleosome are the cells of the cell-free nucleosome. It includes bigenetic features.

[0032] The biomarker used in the method of the present invention is the level of the cell-free nucleosome itself. It may be an epigenetic feature of cells and / or cell-free nucleosomes. The terms “netic signal structure” and “epigenetic features” are defined herein. It will be understood that these are used interchangeably in the context of the detection of nuclei. This refers to specific characteristics of an osome. In one embodiment, the epigenetic characteristics of a nucleosome... Key features include post-translational histone modifications, histone isoforms, modified nucleotides, and / or a protein bound to a nucleosome in a nucleosome-protein adduct: Selected from the group.

[0033] In one embodiment, the epigenetic features of a nucleosome are defined as one or more histograms Includes variants or isoforms. Epigenetic of cell-free nucleosomes The characteristic is histone isoforms, for example, coanucleosome histone isoforms In particular, it may be a histone H3 isoform. "Histone variant" and "histo The term "nucleosoisoform" may be used interchangeably in this specification. The structure of a splice consists of different genes or splicing products and different amino acid sequences. It may also vary depending on the inclusion of other histone isoforms or variants. Many histone isoforms are known in the art. Histone variants are, It can be classified into several families, which are further subdivided into individual types. The nucleotide sequences of the variants are publicly known, for example, at the National Human Genome Research Institute (NHGRI). Histone database (Marino-Ramirez et al.'s paper, "Histone Database: Histone and The Histone Database: an integrated resource for histone fold-containing proteins ed resource for histones and histone fold-containing proteins.)” Database Vol.20 11. (and http: / / genome.nhgri.nih.gov / histones / complete.shtml), GenBank (NIH Genetic Distribution) (Column) Database, EMBL Nucleotide Sequence Database, and DNA Databank of Japan (DDB) It is publicly available in J). For example, variants of histone H2 include H2A1, H2A2, This includes mH2A1, mH2A2, H2AX, and H2AZ. In another example, the histone isophore of H3. The term "Mu" includes H3.1, H3.2, H3.3, and H3t.

[0034] In one embodiment, the histone isoform is H3.1.

[0035] Nucleosome structure can vary depending on post-translational modifications (PTMs) of histone proteins. Histone protein PTM typically occurs in the tail of core histones, and is common. Modifications include acetylation, methylation, or ubiquitination of lysine residues, as well as arginine residues. This includes methylation or citrullination of the group, phosphorylation of serine residues, and many others. The histone modifications are known in the art, and their number increases as new modifications are identified. Increasing (Zhao and Garcia (2015) Cold Spring Harb Perspect Biol, 7: a0250) 64). Therefore, in one embodiment, the epigenetic characteristics of cell-free nucleosomes The mark may be a post-translational modification (PTM) of histone. Histone PTMs are coanucreosomes, for example. It may be H3, H2A, H2B, or H4, in particular H3, H2A, or H2B histone PTM. In particular, Histone PTM is histone H3 PTM. Examples of such PTM are described in WO 2005 / 019826. It is being done.

[0036] For example, post-translational modifications include acetylation, monomethylation, dimethylation, or trimethylation. Methylation, phosphorylation, ribosylation, citrullination, ubiquitination, and hydrolysis are possible. This may include xylation, glycosylation, nitrosylation, glutamation, and / or isomerization. See Ausio's literature (2001) Biochem Cell Bio 79: 693). In one embodiment, histone P TM is selected from citrullination or ribosylation. In a further embodiment, hist PTM is H3 citrulline (H3cit) or H4 citrulline (H4cit). Further embodiments In this case, the histone PTM is H3cit.

[0037] In one embodiment, histone PTM is a ribosylation also known as ADP-ribosylation. The posttranslational nucleotides that occupy the promoters of macrophage inflammatory response markers Stone ADP-ribosylation is stimulated by exposure to lipopolysaccharides, which leads to increased transcription, and anti It may possess viral characteristics. Furthermore, all members of the coronavirus family are tampa Enzymatic removal of covalently attached ADP-ribose from the target substance leads to posttranslational ADP-ribose Contains a highly conserved macrodomain within non-structural protein 3 (nsp3), which regulates sylation. It contains a mutated macrodomain with reduced nsp3 de-ADP-ribosylation activity. Recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) strains have low infectivity and early enhancement. Interferon (IFN), interferon-stimulating gene (ISG), and pro-inflammatory sites It induces the Cain response. Therefore, circulating ADP-ribosylated nucleotides released from macrophages Modification at the creosome level is considered useful in the method of the present invention.

[0038] It is also possible to detect groups or classes of related histone post-translational modifications (rather than a single modification). It is possible. While not limited to this, a typical example is the direction to bind to nucleosomes. It binds to one antibody or other selective binder and a group of target histone modifications. Two-site immunization using one antibody or another selective binder directed to perform a specific action. This includes examples of antibodies directed to bind to groups of histone modifications. However, this is not limited to, but for the purpose of explanation, anti-panacetylated antibodies (for example, pan-acetylated antibodies) Examples include cetyl H4 antibody (H4panAc), anti-citrullinated antibody, or anti-ubiquitin antibody.

[0039] In one embodiment, the epigenetic features of a nucleosome are one or more DNA modifications. Includes. Epidurally mediated by nucleosome histone isoforms and PTM composition. In addition to genetic signaling, nucleosomes use their nucleotides and modified nuclei They also differ in the composition of rheotides. Some nucleosomes are different from others. Many 5-methylcytosine residues (or 5-hydroxymethylcytosine residues or other nuclei) It may include ostide or modified nucleotides. In one embodiment, the DNA modification is 5-methyl Lucitosine or 5-hydroxymethylcytosine is selected.

[0040] In one embodiment, the epigenetic characteristics of a nucleosome are one or more proteins Includes nucleotide-nucleosome adducts or complexes. Further types of circulating nucleosomes The subset is a nucleosome protein adduct. Chromatin is one of its components. It has long been known that it contains numerous non-histone proteins bound to DNA and / or histones. These chromatin-related proteins are of a wide variety of types, and are diverse. It has the functions of a transcription factor, transcription enhancer, transcription repressor, histone regulator, and DNA damage repair. Includes numerous proteins, including recombination proteins. Nucleosomes and other non-histone chromatin proteins. These chromatin fragments, which may contain DNA and other non-histone chromatin proteins, This is described in the field of technology.

[0041] In one embodiment, it is attached to a nucleosome (and therefore, as a biomarker) The proteins that can be used include transcription factors, high-mobility proteins, or chromatin-regulating enzymes. Elements: Selected from. The reference to "transcription factors" means that they bind to DNA and promote transcription. In other words, by activating (i.e., repressing) or repressing (i.e., repressor), This refers to proteins that regulate gene expression. Transcription factors are proteins that regulate the DNA adjacent to the gene they regulate. It contains one or more DNA-binding domains (DBDs) that bind to specific sequences. All circulating nucleosomes and parts, types, or subgroups of nucleosomes are, This may be useful in the present invention.

[0042] Multiple epigenetic features of cell-free nucleosomes are used in the method and use of the present invention. It will be understood that it can be detected by combining multiple biomarkers. - It can be used as. Therefore, in one embodiment, use is combined bio Includes multiple epigenetic features of cell-free nucleosomes as markers. Genetic characteristics are of the same type (e.g., PTM, histone isoform, nucleo). Histones (or protein adducts) or different types (e.g., in combination with histone isoforms) (Combined PTM) is possible. For example, detecting post-translational histone modifications and histone variants. It is possible to do so (i.e., to detect multiple types of epigenetic features). Or Or, further, to detect multiple types of post-translational histone modifications, or multiple types of histone modifications It detects isoforms. In one embodiment, it is used for the diagnosis, detection, treatment, and selection of infectious diseases. Post-translational biomarkers as combined biomarkers in a sample for selection, prediction, or monitoring. Includes stone modifications and histone isoforms. In one embodiment, a combination biomer The markers are H3.1 and H3cit. In an alternative example, the combined biomarker is H 3.1 and H4cit.

[0043] The term "biomarker" refers to a differential biological or It means a biologically derived indicator. A biomarker is a diagnostic method, such as a clinical screening tool. In addition to prognosis assessment, and monitoring of treatment outcomes, it is possible to respond to specific therapeutic measures. It can be used in identifying patients with the highest potential, screening for drugs, and in development. Biomarkers and their use are used to identify new drug therapies and new targets for drug therapies. It is useful for discovery.

[0044] Biomarkers are used as companion diagnostics for selecting patients who are suitable for treatment with specific therapies. It is also useful as a discontinuation product. The inventors have identified a method for controlling the circulating nucleosome level, or specific epidensities. Testing of nucleosome levels containing genetic signals or structures is performed on NETs. Or, as stated herein, if it is a useful companion product for the treatment of NETosis-related diseases. It's there and showing.

[0045] The present invention relates to a method for assigning patients who are at risk of adverse outcomes. This includes death and / or emergency medical care, such as hospitalization (i.e., inpatient treatment) and / or outpatient treatment. This includes acute events requiring medical intervention. For many patients, infections require medical intervention. It is overcome by the person's own immune system without needing to be done. However, some In patients with this condition, the infection is not overcome by the immune system, and the severity progresses or The patient's own immune response to the infection may increase, or it may lead to adverse outcomes. For example, Adverse outcomes include acute coronary or cardiac events (e.g., myocardial infarction and / or stroke), acute Multiple organ or single organ failure (e.g., renal failure, hepatic failure, and / or heart failure), wasting Onset of acute illness, and / or acute respiratory illness (e.g., pneumonia, hypoventilation / bradypnea, acute respiratory illness). Distress syndrome (ARDS), severe acute respiratory syndrome (SARS), bronchiolitis and / or bronchitis) It may include. Therefore, in one embodiment, the method described herein is acute respiratory Assign patients or subjects at risk of developing organ diseases. Further ec. Acute respiratory disease is pneumonia. In a further embodiment, acute respiratory disease is hypocongestive. It is a slow respiration. In a further embodiment, the acute respiratory disease is acute respiratory distress syndrome. It is a symptom of ARDS (ardiopulmonary retardation syndrome) and / or Severe Acute Respiratory Syndrome (SARS).

[0046] Assigning patients at risk of adverse outcomes involves allocating immediate or short-term risks. Can we predict or assign medium-term risks? The test indicates that the patient has had a medical examination within 30 days of the onset of symptoms or a confirmed diagnosis, for example, within 2 weeks or 14 days. This includes cases where adverse outcomes may occur within a few days, within a week or seven days, or within five days. Such immediate or short-term risks may be related to the patient's ability to perform the methods described herein. Within 30 days, for example, within 2 weeks or 14 days, within 1 week or 7 days, or within 5 days This may include cases where adverse outcomes may develop within the treatment period. An example of short-term risk is the need for hospitalization. The key risk is the development of NETs-related complications from COVID-19 infection. Mid-term risks include: The patient has been alive for 30 days since the onset of symptoms, definitive diagnosis, and / or the implementation of the method described herein. This includes cases where adverse outcomes may develop after exceeding a certain threshold. An example of a medium-term risk is the so-called One example is the long-term onset of COVID-19, where the effects of COVID-19 infection can last for several months. Therefore, In one embodiment, the method described herein is performed within two weeks from the onset of symptoms or definitive diagnosis. Alternatively, assign patients or subjects at risk of developing an adverse outcome within 14 days. In the embodiments described herein, the method is used within one week from the onset of symptoms or definitive diagnosis. This assigns a risk of developing an adverse outcome within 7 days. In a further embodiment, The methods described herein are for preventing adverse outcomes within 5 days of the onset of symptoms or definitive diagnosis. Assign the risk.

[0047] Therefore, in another aspect of the present invention, a subject having an infectious disease requiring hospitalization is specifically A method of determination, (i) The bodily fluid sample obtained from the subject is brought into contact with a binder to form cell-free nucleosomes or To detect or measure the level of the components; and (ii) Using the detected cell-free nucleosome levels, the subject should be hospitalized for treatment. Deciding whether or not hospitalization is necessary. A method is provided that includes:

[0048] Using the method of the present invention, it is possible to identify patients who do not require hospitalization, i.e., detection Using the cell-free nucleosome levels obtained, the subject should be admitted to a hospital for treatment. It will be understood that it is also possible to determine whether or not. It helps identify patients who can be discharged early, even if they are already hospitalized. It is likely.

[0049] The methods and uses described herein apply to body fluid samples, particularly blood, serum, or plasma samples. The test can be performed. Preferably, a plasma sample is used. The plasma sample contains one or more anticoagulants. Agents, for example, ethylenediaminetetraacetic acid (EDTA), heparin, or sodium citrate, in particular It can be collected in a collection tube containing EDTA.

[0050] (infectious disease) The method of the present invention is particularly useful in managing infectious disease outbreaks. Infectious diseases are various diseases. It can be caused by pathogens and environmental factors. In one embodiment, the infection is caused by a virus. These are bacterial, fungal, or microbial infections. Bacterial infections include mycobacteria, pneumococcus, And influenza infections, such as those caused by Streptococcus pneumoniae and Escherichia coli. (Escherichia coli), Mycobacterium tuberculosis, Haemophilus influenzae (Haemoph) Caused by Staphylococcus aureus (Illus aromae) and Staphylococcus aureus. This may include infectious diseases (e.g., pneumonia). In a further embodiment, the infectious disease may include viral infections. It is an infectious disease. Viral infections include respiratory syncytial virus (RSV), influenza A, and influenza B. Infections caused by influenza and coronaviruses (e.g., COVID-19) It may include.

[0051] Infectious diseases can be defined by the tissues affected by the disease. For example, disease It can affect the heart, brain, kidneys, liver, pancreas, lungs, and / or blood, and infections can occur. Bacteria, viruses, fungi, etc., that are generally known to affect tissues or organs such as Alternatively, it may be a microbial infection. In one embodiment, the infection is a respiratory tract infection. According to the embodiment, the infection affects the lungs, upper and / or lower respiratory tract.

[0052] Other tissues that can be affected by the disease include peripheral tissues such as the limbs, hands, and feet, and infections. The symptoms may be bacterial infections (e.g., gangrene). In one embodiment, infectious diseases and / or diseases The disease can affect multiple tissues or organs simultaneously. For example, an infection can affect the limbs, hands, or It can be a bacterial infection of the foot, and the disease can also affect the blood (for example, sepsis). In one embodiment, the infection is sepsis. In another embodiment, the disease is heart failure or coronary artery disease. Pulse failure may occur, and other tissues or organs affected by the disease include the kidneys and renal system, as well as / or may include the brain (e.g., stroke). In further examples, the disease may affect the lungs. The infection may be a respiratory tract infection, and other affected tissues or organs may include the heart. This may include the coronary artery system and / or the brain (e.g., heart failure, myocardial infarction, and / or stroke).

[0053] In one embodiment, circulating nucleosome levels are used to determine the prognosis of a disease. It is measured in samples taken from subjects suffering from the disease. In another embodiment, Ring nucleosome levels are used to monitor disease progression and / or therapeutic efficacy. To evaluate this, among a large number of samples taken at intervals from subjects infected with the infectious disease... It is measured at [location / location].

[0054] In a further embodiment, circulating nucleosome levels are used, in particular, to evaluate the prognosis of a disease. To do this, measurements are taken in samples taken from subjects suffering from sepsis or septic shock. Multiple samples are taken at intervals from subjects suffering from sepsis or septic shock. Further measurements on a number of samples are needed to monitor disease progression and / or treatment. This can be done to evaluate the effectiveness of [the law].

[0055] In one embodiment, respiratory tract infections include influenza, pneumonia, and severe acute respiratory syndrome. Group (SARS): Selected from. SARS is caused by SARS coronavirus (SARS-CoV), etc. It is a respiratory infection caused by the coronavirus, and related coronaviruses are known (e.g., COVID-19). 9 (also known as SARS-CoV-2, and previously known as 2019-nCoV)). It was It causes fever, flu-like symptoms, cough, and fatigue, and can lead to pneumonia (e.g., direct viral infection). It is known that this can lead to russic pneumonia or secondary bacterial pneumonia.

[0056] The emergence of COVID-19 and its rapid progression into a pandemic situation has impacted international healthcare services. This places a heavy burden on them. The predicted infectivity ranges from 70 to 80 percent of the country's population. While some people experience mild symptoms, a double-digit percentage of those infected suffer serious consequences. It seems that this is sometimes the case.

[0057] By identifying COVID-19 positive individuals at high risk of severe reactions or complications, including pneumonia, Until triage becomes possible and herd immunity is established, emergency beds and ventilators will be provided. The allocation of urgent medical resources, including equipment, will proceed smoothly, and the community will be able to handle future large-scale outbreaks. This will protect against the effects of the drug. Therefore, in a preferred embodiment, medical treatment is not required. A method for identifying a person infected with influenza or coronavirus infection, (i) The bodily fluid sample obtained from the subject is brought into contact with a binder to form cell-free nucleosomes or To detect or measure the level of the components; and (ii) Using the detected cell-free nucleosome levels, determine whether the subject requires medical treatment. To decide whether or not to A method is provided that includes:

[0058] (Diagnosis and monitoring methods) In a further aspect, a method for monitoring the severity of an infectious disease in a subject, , (i) The bodily fluid sample obtained from the subject is brought into contact with a binder to form cell-free nucleosomes or To detect or measure the level of a component; (ii) Testing the level of cell-free nucleosomes or their components in the body fluid obtained from the subject. Repeating the output or measurement on one or more occasions; (iii) Using any change at the level of the cell-free nucleosome or its components, the Monitoring the progression of infectious diseases in elephants A method is provided that includes:

[0059] In further circumstances, a person may have an infectious disease or be suspected of having an infectious disease, This involves monitoring the progression of infections in subjects who tend to have a poor prognosis for infections. A method for doing so, (i) The sample obtained from the subject is brought into contact with a binder to determine the level of the cell-free nucleosomes. A process for detecting or measuring; and (ii) The level of detected cell-free nucleosomes in earlier samples taken from the subject In comparison, the process of monitoring the progression of the infection. A method is provided that includes:

[0060] In a further aspect, monitoring the progression of the disease in subjects suffering from an infectious disease. A method, (i) The bodily fluid sample obtained from the subject is brought into contact with a binder to form cell-free nucleosomes or To detect or measure the level of a component; (ii) Repeating step (i) on one or more occasions; and (iii) Using any change at the level of the cell-free nucleosome or its components, the Monitoring the progression of infectious diseases in elephants A method is provided that includes:

[0061] Even if the subject is determined to be free from infectious diseases or to have a mild infectious disease, the present invention Furthermore, it is used for the purpose of monitoring disease progression in order to detect future medical complications. This can be done. For example, this method can be used with samples from subjects that have been determined to have a mild infection. In this case, the measurement of the biomarker level is repeated at a different time point, and if the biomarker level changes... It is possible to determine whether or not it has transformed.

[0062] Detection and / or quantification are performed directly on purified or concentrated nucleosome samples. Alternatively, this can be done indirectly with an extract from the sample or a dilution thereof. Quantifying the amount of biomarkers present in a sample is necessary to determine the amount of biomarkers present in the sample. This may include determining the concentration of -. Detection, monitoring according to the present invention as described herein The use and methods of diagnosis are to assess the onset and progression of the disease in order to confirm its presence. This is done to monitor the progression of the disease, or to evaluate its improvement or regression. Useful for detection, monitoring, and diagnosis. The methods and uses are clinical. For the evaluation of treatment, prognosis, therapy selection, and assessment of treatment benefits, i.e., drug screening It is also useful in methods for training and development.

[0063] In one embodiment, the disease is a pathological clinical complication of high levels of NETs or NETosis. It is a disease that involves multiple illnesses.

[0064] Detection or measurement can be performed using immunoassay, immunochemistry, mass spectrometry, chromatography, or chromatography. This may include matin immunoprecipitation or biosensor methods. In particular, detection and / or measurement may involve nucleation. This may include a two-site immunoassay method of the reosome region. Such a method involves two anti-nucleosomal immunosynoassays. Somesome binding agent or one anti-nucleosome binding agent to provide anti-histone modification or anti-histone barrier It utilizes a combination of an anti-DNA modification or anti-addition protein detection binder. Nucleosomes or epigenetic components incorporated into nucleosomes in situ It is preferable for measuring features. Also, detection and / or measurement can be performed, for example, on labeled or fixed surfaces. Anti-nucleosome, anti-histone modification, anti-histone variant / isoform, anti-DNA This may include a two-site immunoassay utilizing a combination of modifications or anti-addition protein binders. .

[0065] The present inventors hereby describe a substance present in an intact nucleosome, but isolated. It binds to epitopes that are not present on (free) histones or DNA nucleosome components. Along with a labeled anti-nucleosome antibody directed to target the histone H3.1 protein Immobilized antihistamines directed to bind to epitopes around amino acids 30-33 Using a two-site immunoassay for H3.1-nucleosomes utilizing H3.1 antibodies, cleavage was performed. This type of epidendrocyte captured cleaved and uncleaved nucleosomes. The top requires that the native three-dimensional arrangement of the target nucleosome be intact. Therefore, in this specification, it can be referred to as a "three-dimensional structural nucleosome epitope."

[0066] The H3R8Cit nucleosome measurement described herein is a structural nucleosome epiphysis. Along with the same labeled anti-nucleosome antibody directed to bind to the tope, hist It is oriented to bind to citrullinated nucleosomes of H3 arginine 8. The procedure was performed using a two-site immunoassay utilizing immobilized antibodies.

[0067] In one embodiment, the detection or measurement method involves a body fluid sample being subjected to cell-free nucleosomes or The component is brought into contact with a solid phase containing a binder, and the binding to the binder is detected. This includes releasing.

[0068] In one embodiment, the detection or measurement method is: (i) the sample is subjected to the epidendrome of cell-free nucleosomes. (ii) Contacting with a first binder that binds to the genetic features; (ii) the first binder in step (i) The sample bound by the binder is then brought into contact with a second binder that binds to cell-free nucleosomes. (iii) to detect or quantify the binding of a second binder in the sample.

[0069] In another embodiment, the detection or measurement method is: (i) attaching a sample to cell-free nucleosomes (ii) to bring into contact with a first binder; (ii) to be bonded by the first binder in step (i) The sample is then brought into contact with a second binder that binds to the epigenetic features of cell-free nucleosomes. (iii) to cause; and to detect or quantify the binding of the second binder in the sample: nothing.

[0070] The detection or measurement of biomarker levels is performed using one or more reagents, such as a suitable binder. This can be implemented. For example, one or more binders may be one or more interleukins and any A desired biomarker, for example, a nucleosome or its constituent part, combined with the desired biomarker, for example. , epigenetic characteristics of nucleosomes, nucleosomes or their constituent parts It may contain ligands or binders specific to the structure / shape mimic.

[0071] The terms “antibody,” “binder,” or “ligand” as used herein are limited to… It contains any binder that can bind to a specific molecule or entity, rather than a specific one. It is intended that any suitable binder may be used in the method of the present invention. It will be obvious to those skilled in the art that this is possible. The term "nucleosome" is used in fluid media. Mononucleosomes, oligonucleosomes, NETs, ​​and any tans can be analyzed. It is also clear that the protein is intended to contain DNA chromatin fragments.

[0072] Methods for detecting biomarkers are known in the art. Reagents are used in conjunction with the desired target. One or more ligands or binders capable of specific binding, e.g., natural compounds or chemically synthesized compounds. It may contain compounds. The ligand or binder is a peptide capable of specific binding to the desired target. Antibodies, antibodies, or fragments thereof, or synthetic ligands such as plastic antibodies, or It may contain a ptamer or oligonucleotide. The antibody is a monoclonal antibody or its It can be a fragment. When using an antibody fragment, it can be a biomarker (in this invention). (According to) retain the ability to bind to a biomarker so that it can be detected This will be understood. The ligand / binder is a detectable marker, e.g., luminescence, firefly. It can be labeled with light, enzymes, or radioactive markers; or, further, according to the present invention The ligand is an affinity tag, for example, biotin, avidin, streptavidin, or His( For example, it can be labeled with a hexa-His tag. Alternatively, ligand binding can be performed using an unlabeled technique. This can be determined using techniques such as label-free technology from ForteBio.

[0073] As used herein, the terms “detect” or “diagnose” refer to the identification of a disease state. This includes confirmation and / or feature analysis. The detection, monitoring, and diagnosis methods according to the present invention. The law aims to confirm the presence of a disease by evaluating its onset and progression, thereby mitigating the development of the disease. Useful for monitoring or evaluating improvement or regression of a disease. Detection The methods of monitoring and diagnosis include evaluation of clinical screening, prognosis determination, and selection of therapy. Selection, methods for evaluating therapeutic benefits, i.e., methods for drug screening and development. It is also useful for this purpose.

[0074] The method of the present invention may involve standardization of marker levels. For example, specific epigenetics Cellless nucleosome levels containing distinctive characteristics, the nucleosome itself (or several Standardized to the levels of several other types of nucleosomes or parameters, This level can be expressed as the ratio of nucleosomes containing the characteristic. For example, The level of citrullinated nucleosomes is determined by the ratio of citrullinated nucleosomes and To express it.

[0075] In one embodiment, the method described herein is repeated on multiple occasions. This offers the advantage of enabling monitoring of detection results over a certain period of time. Such preparations are beneficial for monitoring or evaluating the effectiveness of treatment for the disease condition. The present invention provides monitoring methods for onset, progression, stabilization, and improvement. Relapse and / or remission can be monitored.

[0076] In the monitoring method, the test sample can be collected on two or more occasions. This method allows for the collection of test samples. The level of biomarkers present in the material is compared to one or more controls and / or, for example, at the start of treatment. Previously taken from the same subject and / or earlier in the treatment of the same subject. This method may further include comparing it with one or more previously tested samples. This may include detecting changes in the properties or quantities of biomarkers in a test sample collected. .

[0077] The levels in the test sample were compared to those in past test samples taken early from the same subject. Changes in Iomarker levels indicate the beneficial effect of the therapy on the disorder or suspected disorder. For example, it may show stabilization or improvement. Furthermore, once treatment is completed, the disease The method of the present invention can be repeated periodically to monitor for disease recurrence.

[0078] Using methods to monitor the efficacy of therapy, in human subjects and non-human animals (e.g., animals) It is possible to monitor the therapeutic effectiveness of existing and new therapies in a model. These monitoring methods can be used to screen for new drug substances and combinations of substances. It can be incorporated into.

[0079] In a further embodiment, monitoring of more rapid changes by immediate-acting therapy is more It can be implemented at short intervals of time or days.

[0080] A diagnostic or monitoring kit (or panel) is provided for carrying out the method of the present invention. Such kits are preferably used for the detection and / or detection of biomarkers according to the present invention. One or more ligands for quantification, and / or biosensors and / Alternatively, the array may be included, optionally, along with the kit's user manual.

[0081] A further aspect of the present invention is a kit for detecting the presence of an infectious disease, which is described herein. A device capable of detecting and / or quantifying one or more of the defined biomarkers. This is a kit that includes an io-sensor. When used herein, the term "biosensor" is used. The term refers to anything that can detect the presence of a biomarker. Examples of biosensors are described herein. Biosensors are biomarkers and It may contain ligand binders or ligands described herein that are capable of specific binding to the ligand. Such biosensors are used in the detection and / or quantification of the biomarkers of the present invention. It is useful.

[0082] Preferably, a biosensor for detecting one or more biomarkers recognizes biomolecules. This is used to detect the presence of a biomarker in a sample or to quantify a biomarker in a sample. Combine with appropriate means of conversion. Biosensors are used, for example, in wards, outpatient departments (out For "alternative site" diagnostic testing in subjects' department, operating rooms, homes, fields, and workplaces. It can be adapted. A biosensor for detecting one or more biomarkers of the present invention The sensors include acoustic sensors, plasmon resonance sensors, holographic sensors, and bio-sensors. There are ear interferometry (BLI) sensors and micro-engineering sensors. Lint detection elements, thin-film transistor technology, magnetoacoustic resonator devices, and other Novel acoustic-electric systems are being utilized in biosensors for the detection of one or more biomarkers. It can be used.

[0083] Biomarkers for detecting the presence of disease are novel in that they delay or halt the progression of the disorder. It is an essential target for the discovery of targets and drug molecules. The level of biomarkers is Since it indicates harm and drug response, biomarkers are used in vitro and / or in vitro. Useful for identifying novel therapeutic compounds in boassays. The biomass described herein The manufacturer uses this method to screen compounds that modulate the activity of biomarkers. It can be used.

[0084] Therefore, in a further embodiment of the present invention, directing the biomarker according to the present invention A peptide, antibody or its fragment, or aptamer or oligonucleotide that has been processed Possible use of the described binder or ligand; or generation of a biomarker. A biosensor according to the present invention for identifying substances that can promote and / or inhibit Use of a siphon, array, or kit is provided.

[0085] The immunoassays described herein include those using the biomarkers defined herein. Any person who uses one or more antibodies or other specific binders directed to bind. One example of an immunoassay is a two-site immunoassay utilizing enzyme detection or Immunoassays (e.g., ELISA), fluorescently labeled immunoassays, time-resolved fluorescently labeled immunoassays Measurement assays, chemiluminescence immunoassays, immunoturbidimetric assays, particulate labeling immunoassays SEA, and immunoradioanalysis assays, as well as single-site immunoassays, reagent-limited immunoassays Competitive immunoassay methods including labeled antigens and labeled antibodies, radioactive, enzyme-based, fluorescent, time-resolved fluorescence Examples include single-antibody immunoassays using various labeling types, including microparticle labeling. All of these immunoassay methods are well known in the art, for example, Salgame et al. See reference (1997) and the literature by van Nieuwenhuijze et al. (2003).

[0086] Identification, detection, and / or quantification are performed using biological samples derived from the target, or purified biological samples or This is good for identifying the presence and / or amount of specific proteins in extracts or their dilutions. This can be carried out by any suitable method. In particular, quantification can be performed in one or more samples. This can be carried out by measuring the concentration of the target. In the method of the present invention, the test is performed. The biological samples obtained include the biological samples defined above in this specification. It can be prepared by conventional methods, for example, by diluting or concentrating as appropriate, and then stored. (This invention) It is particularly used in plasma samples that can be obtained from the subject.

[0087] Identification, detection, and / or quantification of biomarkers may involve the biomarker or a fragment thereof, e.g. This can be done by detecting fragments having C-terminal or N-terminal cleavage. Preferably, the length is greater than 4 amino acids, for example, lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, or 20 amino acids. The same as or related to the histone tail sequence. It should be noted that peptides with this sequence are particularly useful histone protein fragments. .

[0088] For example, detection and / or quantification can be performed using immunoassays, immunochromatography, SELDI (-TOF), and MA. LDI (-TOF), 1-D gel-based analysis, 2-D gel-based analysis, mass spectrometry (MS) Reverse-phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC, and other LC methods This can be carried out by one or more methods selected from the group consisting of LC MS-based techniques. Appropriate LC-MS techniques include ICAT® (Applied Biosystems, CA, USA) or iTRA. Q(registered trademark) (Applied Biosystems, CA, USA) is an example. Liquid chromatography (e.g.) For example, high-pressure liquid chromatography (HPLC) or low-pressure liquid chromatography (LPLC), thin Layer chromatography and NMR (nuclear magnetic resonance) spectroscopy can also be used.

[0089] The method for detecting and / or quantifying one or more biomarkers according to the present invention is performed using a benchtop device. It may be possible to perform the experiment in an environment outside the laboratory, such as a doctor's office or beside the subject's bed. Can be incorporated into disposable diagnostic or monitoring platforms. It is possible to do so. Suitable biosensors for carrying out the method of the present invention are optical or One example is a "credit" card equipped with an acoustic reader. Biosensors collect data It is configured to allow the collected data to be electronically transmitted to a physician for interpretation, however Therefore, it is possible to form the foundation of e-medicine. , bedside or point-of-care immunotherapy for measuring biomarkers according to the present invention The use of an epidemic assay method is provided. In one embodiment, a bedside immunoassay method is provided. , point-of-care immunoassay devices (e.g., Abbott i-STAT or LightDeck Diagno) Includes a point-of-care immunoassay device. In one embodiment, a bedside The immunoassay method includes lateral flow testing. In a preferred embodiment, biomass A maker is a nucleosome or a nucleosome containing epigenetic features. ru.

[0090] Identifying biomarkers for disease status enables the integration of diagnostic procedures and treatment regimens. Iomarkers are used to assess treatment response, response failure, undesirable side effect profile, and medication compliance. It provides a means to indicate the degree of alliance and the achievement of appropriate serum drug levels. Using the KAR system, warnings of adverse drug responses can be provided. The response can be evaluated to determine medication. By fine-tuning the dosage and minimizing the number of prescribed medications, delays in achieving effective therapy are reduced. Because it can prevent adverse drug reactions and avoid them, biomarkers are useful in personalized therapy. It is useful in development. Therefore, monitoring the biomarker of the present invention Therefore, the care for the subject is determined by the subject's disability and pharmacological profile. It can be precisely matched to the size, and therefore, using biomarkers, Adjusting the optimal dose, predicting a positive therapeutic response, and targeting patients at high risk of severe side effects. It is possible to identify this.

[0091] Biomarker-based testing provides a first-line evaluation for "new" targets and surpasses current methods. It provides an objective means for accurate and rapid diagnosis that is not achievable by other means.

[0092] Methods for monitoring biomarkers, biosensors, and point-of-care tests Lateral flow tests and kits can also be used to determine whether the recurrence is due to the worsening of the disorder. It is important as a target monitoring tool to enable the therapist to make decisions. If the treatment is deemed insufficient, the treatment may be reinstated or increased; if appropriate. Therefore, therapies can be modified. Because biomarkers are sensitive to the state of the disorder, This provides an indicator of the effects of drug therapy.

[0093] References to "subject" or "patient" are used interchangeably in this specification. Subject refers to... The subject may be a human or an animal. In one embodiment, the subject is a human. The subjects are (non-human) animals. The panels and methods described herein are in vivo This can be done using either Ro or Exvivo.

[0094] Detection and / or quantification can be compared to the cutoff level. The cutoff value is Analyze the results from multiple patients and controls and determine suitable values for classifying subjects as having or not having the disease, which can be determined in advance. For example, for a disease where the biomarker level is higher in patients with the disease, if the detected level is higher than the cut-off, the patient is indicated as having the disease. Or, for a disease where the biomarker level is lower in patients with the disease, if the detected level is lower than the cut-off, the patient is indicated as having the disease. The advantages of using a simple cut-off value include the ease with which a clinician can understand the test and the elimination of the need for software or other assistance in the interpretation of test results. The cut-off level can be determined using methods in the art. It can be determined in advance by analyzing the results from multiple patients and controls and determining suitable values for classifying subjects as having or not having the disease. For example, for a disease where the biomarker level is higher in patients with the disease, if the detected level is higher than the cut-off, the patient is indicated as having the disease. Or, for a disease where the biomarker level is lower in patients with the disease, if the detected level is lower than the cut-off, the patient is indicated as having the disease. The advantages of using a simple cut-off value include the ease with which a clinician can understand the test and the elimination of the need for software or other assistance in the interpretation of test results. The cut-off level can be determined using methods in the art. It can be determined in advance by analyzing the results from multiple patients and controls and determining suitable values for classifying subjects as having or not having the disease. For example, for a disease where the biomarker level is higher in patients with the disease, if the detected level is higher than the cut-off, the patient is indicated as having the disease. Or, for a disease where the biomarker level is lower in patients with the disease, if the detected level is lower than the cut-off, the patient is indicated as having the disease. The advantages of using a simple cut-off value include the ease with which a clinician can understand the test and the elimination of the need for software or other assistance in the interpretation of test results. The cut-off level can be determined using methods in the art. It can be determined in advance by analyzing the results from multiple patients and controls and determining suitable values for classifying subjects as having or not having the disease. For example, for a disease where the biomarker level is higher in patients with the disease, if the detected level is higher than the cut-off, the patient is indicated as having the disease. Or, for a disease where the biomarker level is lower in patients with the disease, if the detected level is lower than the cut-off, the patient is indicated as having the disease. The advantages of using a simple cut-off value include the ease with which a clinician can understand the test and the elimination of the need for software or other assistance in the interpretation of test results. The cut-off level can be determined using methods in the art.

[0095] Detection and / or quantification can also be compared to controls. For example, it will be apparent to those skilled in the art that subjects known to not have the disease may be included or control subjects, which may be subjects having different diseases (e.g., for differential diagnosis studies), can be selected according to various criteria. The "control" may include healthy subjects, non-affected subjects, and / or subjects without infectious diseases. The control may also be a subject having an infectious disease showing asymptomatic or mild symptoms, e.g., a subject infected with a respiratory virus showing asymptomatic or mild symptoms. Mild symptoms can include manageable symptoms that do not require hospital intervention and / or intensive medical treatment. In one embodiment, subjects with a positive test result by the method of the present invention are viral In one embodiment, subjects with a positive test result by the method of the present invention are viral In one embodiment, subjects with a positive test result by the method of the present invention are viral In one embodiment, subjects with a positive test result by the method of the present invention are viral

[0096] In one embodiment, subjects with a positive test result by the method of the present invention are viral You may be infected with a disease and may develop further medical complications, or Further medical complications followed. In contrast, the control group also contracted a viral disease. It is possible that they do not suffer from medical complications, or do not suffer from medical complications in succession. No. Comparison with controls is well known in the field of diagnosis. The range of values ​​observed in the control group is: The values ​​observed for the subject of the test can be compared as normal, healthy, or within the reference range. It can be used. For example, if the reference range is less than 10 units, a test value of 5 units is considered normal. Alternatively, it may be considered that no treatment is necessary, but if the value is 11 units, it is abnormal and requires treatment. It is considered to indicate that

[0097] Therefore, in one embodiment, this method involves cell-free nucleosomes in the target body fluid sample. or further includes comparing the level of its components with one or more controls. For example, this method This involves comparing the cell-free nucleosome levels present in the sample obtained from the target with those of a normal target. This may include comparing the levels of cell-free nucleosomes present in the obtained sample with those of cell-free nucleosomes. Teru could be considered a healthy individual.

[0098] In one embodiment, the cell-free nucleosome or its component level was compared to a control. And it is rising.

[0099] In no case is it necessary to measure a control level for comparative purposes. This will be understood. For example, regarding healthy / non-infected controls, once the "normal range" is Once established, it can be used as the standard for all subsequent tests. The normal range is Obtaining samples from multiple control subjects that do not have infectious diseases and the level of biomarkers It can be established by testing. Subsequently, if an infectious disease is suspected... The results of the subjects (i.e., biomarker levels) were examined, and these were compared to their respective normal ranges. It is possible to determine whether it is on the inside or outside. The use of "normal range" is for the disease. This is standard practice for detection.

[0100] In one embodiment, this method determines at least one clinical parameter of a patient. This further includes the following. This parameter can be used in the interpretation of the results. Clinical As a meter, any relevant clinical information, such as, but not limited to, body temperature, This can include gender, weight, body mass index (BMI), smoking status, and eating habits. Therefore, in one embodiment, the clinical parameters are body temperature, age, sex, and body mass index. (BMI): Selected from the group consisting of the following.

[0101] In one embodiment, the method of the present invention has a high risk of causing severe reactions to infectious diseases. This is done to identify individuals with a condition, and therefore those who require medical intervention. Such medical interventions may include one or more of the therapies described herein. .

[0102] According to another aspect of the present invention, the risk of adverse outcomes is assigned to subjects suffering from infectious diseases. It is a method that (i) The bodily fluid sample obtained from the subject is brought into contact with a binder to form cell-free nucleosomes or To detect or measure the level of the components; and (ii) Using the detected cell-free nucleosome levels, the potential for the adverse outcome is assessed. Assigning The use of a binder in the manufacture of a kit for use in a method, which comprises

[0103] According to a further aspect of the invention, a method of detecting a subject who requires medical treatment for pneumonia, acute respiratory syndrome (ARS), or severe acute respiratory syndrome (SARS), comprising: (i) contacting a body fluid sample obtained from the subject with a binder to detect or measure the level of cell-free nucleosomes or a component thereof; and (ii) using the level of the cell-free nucleosomes as an indicator that the subject requires medical treatment for pneumonia, ARS, or SARS.

[0104] According to a further aspect of the invention, a method of detecting a subject who requires medical treatment for sepsis or septic shock, comprising: (i) contacting a body fluid sample obtained from the subject with a binder to detect or measure the level of cell-free nucleosomes or a component thereof; and (ii) using the level of the cell-free nucleosomes as an indicator that the subject requires medical treatment for sepsis or septic shock. The use of a binder in the manufacture of a kit for use in a method, which

[0105] According to a further aspect of the invention, a method of detecting a subject who requires medical treatment for pneumonia, acute respiratory syndrome (ARS), or severe acute respiratory syndrome (SARS), comprising: (i) contacting a body fluid sample obtained from the subject with a binder to (ii) The level of the cell-free nucleosomes in the subject is medical for pneumonia, ARS, or SARS. To use as an indicator that appropriate treatment is needed. A method is provided that includes:

[0106] According to a further aspect of the present invention, medical treatment for sepsis or septic shock is not necessary. A method for detecting the target object, (i) The bodily fluid sample obtained from the subject is brought into contact with a binder to form cell-free nucleosomes or To detect or measure the level of the components; and (ii) The level of the cell-free nucleosomes is such that the subject is in response to sepsis or septic shock. To be used as an indicator that medical treatment is needed. A method is provided that includes:

[0107] (Further biomarkers) The level of cell-free nucleosomes can be detected or measured as one of the measurement panels. The panel is a different epigenetic component of the nucleosome described above in this specification. It may include netic features (e.g., histone isoforms and PTM). Medical intervention Useful biomarkers in panel testing for the detection of severe respiratory infections. While not limited to these, the cytokine portion (especially interleukins), C-antibody Responsive proteins, myeloperoxidase, D-dimer, factor VII activated protease ( Examples include FSAP, fibrinogen, and fibrin / fibrinogen degradation products. In one embodiment, the panel comprises a C-reactive protein. In one embodiment, the panel is It contains one or more cytokines, for example, one or more interleukins.

[0108] Interleukins (ILs) act as signaling molecules and are normally secreted by white blood cells. Interleukins are a group of cytokines that stimulate immune responses and inflammation. Interleukins play an important role. Interleukins were first identified in the 1970s, and many more have been discovered. Each time an interleukin species is discovered, it has been named with a number. Examples of interleukins and IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-1 2. IL-13, IL-14, and IL-15 are examples, but are not limited to these.

[0109] In one embodiment, one or more interleukins are interleukin-6 (IL-6) and Selected from the group consisting of: enterleukin-12 (IL-12).

[0110] Interleukins can be IL-6. Interleukin-6 (IL-6) is found in a wide variety of living organisms. It is a functional cytokine. It is a potent inducer of fever and acute phase responses. The sequence of human IL-6 is publicly known in the art and is recorded under UniProt accession number P05231. It is stated that in one particular embodiment, the interleukin may be IL-6.

[0111] Alternatively, or furthermore, the interleukin may be IL-12. Interleukin-12(I IL-12A is a T cell stimulating factor because it stimulates the proliferation and function of T cells. It is a heterodimer cytokine composed of IL-12A and IL-12B. The sequence of human IL-12A is used in this technology. It is publicly known in the field and is described in UniProt accession number P29459, and is related to human IL-12B. The sequence is also publicly known and is listed under UniProt accession number P29460. Depending on the application, the interleukin may be IL-12.

[0112] In one embodiment, the panel is a cell-free nucleosome or its epigenetic Features and interleukins are included. In another embodiment, the panel is a cell-free nucleo It contains the epigenetic characteristics of the cell and two interleukins. For example, cell-free cells Creosome measurement is combined with the measurement of multiple interleukins such as IL-6 and IL-12. This can be done. In a further embodiment, the epigenetic effects of cell-free nucleosomes The distinctive features include histone isoforms such as H3.1 and post-translational modified histones such as H3cit. They are selected. In another embodiment, the measurement panel is H3.1, H3cit, H4cit, And IL-6.

[0113] In one embodiment, the panel contains C-reactive protein (CRP). CRP is found in plasma. It is a pentameric protein, and whether or not it is attached to a nucleosome is irrelevant to CRP. (i) The level is, for example, in response to inflammation in bacterial, viral, fungal, and microbial infections. It increases in the plasma. CRP levels increase after IL-6 secretion by macrophages and T cells. Its physiological role is to activate the complement system via C1q, and to serve as a marker for dead or dying cells. This involves binding to lysophosphatidylcholine expressed on the surface. This also applies to some skins. It binds to phosphocholine on the surface of bacteria, enhancing phagocytosis. Measuring CRP levels indicates the progression of the disease. It is useful in determining the effectiveness of treatment, and elevated CRP levels indicate diabetes, hypertension, This has been shown in patients with an increased risk of cardiovascular disease. Increased CRP levels are associated with renal failure. It is also seen in patients with inflammatory bowel disease (IBD, including Crohn's disease and ulcerative colitis), and generally When elevated CRP levels correlate with coronary heart disease, but are not directly related to heart disease, It is not a specific prognostic marker. CRP increases during inflammation, so it may be relevant in SARS or COVID-19. Viral infections, such as those caused by viruses (e.g., COVID-19), also cause an increase in CRP levels in the plasma. It is possible.

[0114] In one embodiment, the panel contains myeloperoxidase (MPO). MPO is neutrophil It is expressed in granulocytes and produces hypohalite, thereby performing its antimicrobial activity. It is stored in azurophilic granules and released into the extracellular space upon degranulation. MPO levels are found in the myocardium. It is known to be a useful predictor of infarction, and its accuracy in predicting the risk of myocardial infarction in patients. To increase the effect, it is combined with CRP measurement. In one embodiment, the panel is It contains neutrophil elastase (NE).

[0115] The biomarkers of the present invention can be used to derive a model or algorithm. Methods for deriving the m are well known in the art, and suitable software packages are available. A typical software tool for this purpose is SPSS (Social Sciences Management System). Examples include the "R" software package. These software packages are used for clinical data It provides linear and nonlinear data modeling.

[0116] Any combination of the biomarkers disclosed herein can be used to detect or predict complications of infectious diseases. It can be used in panels and algorithms for further markers. It will be apparent to those skilled in the art that these markers can be added to the panel.

[0117] According to an aspect of the present invention, a panel for detecting or predicting complications of infectious diseases in patients The use of the test, wherein the panel test is used to determine nucleosomes or so in a sample obtained from a patient The use is provided, including reagents for detecting the components and the measurement of one or more interleukins. In one embodiment, the complication is a NETs-related complication. In one embodiment, Complications include ARDS, ARS, SARS, or embolism or thrombotic complications.

[0118] (Treatment method) In a further embodiment, a method for treating an infectious disease in a subject, comprising the following steps: (i) To detect or measure the level of cell-free nucleosomes in a sample obtained from the subject. and; (ii) The level measured in step (i) indicates the presence and / or severity of the infection in the subject. To use as a means of / or to indicate medical complications; and (iii) If the subject is determined to have a severe infection or medical complication in process (ii), Administering therapy A method is provided that includes this.

[0119] In a further aspect, it is a method for treating infectious diseases in those who need it. When compared to the level of cell-free nucleosomes in the sample obtained from the control, The samples obtained from the target were identified to contain varying levels of cell-free nucleosomes. A method is provided which includes the step of administering a therapy (e.g., a therapeutic agent) to an elephant. The therapy is limited It is not a definitive rule, but drugs (for example, anti-inflammatory drugs, anticoagulants or anti-blood clotting drugs, therapeutic drugs) Anti-NETs antibody drugs, DNase drugs, NETosis inhibitors, antibacterial drugs, or antiviral drugs), aphe One or more treatments for the disease, including LASES therapy, mechanical ventilation, fluid support, or other treatments. The following are some of the most suitable treatments.

[0120] In one embodiment, the treatment is antibiotic therapy (e.g., penicillin, cephalosporin) Tetracycline, aminoglycoside, macrolide, clindamycin, sulfonate Mid, trimethoprim, metronidazole, tinidazole, quinolone, and / or nitro Frantoin), antimicrobial therapy (e.g., ethambutol, isoniazid, pyrazinamide, Rifampicin, aminoglycosides (amikacin, kanamycin), polypeptides (capre Omycin, Biomycin, Enbiomycin), Fluoroquinolone (Ciprofloxacin) (Levofloxacin, Moxifloxacin), Thiamide (Ethionamide, Prothionamide) (D), cycloserine (croserine), terizidone, rifabutin, macrolide (clarith) Romycin, linezolid, thioacetazone, thioridazine, arginine, vitamin D and / or R207910), antiviral COVID treatment (e.g., remdesivir), antiviral influenza The treatment (e.g., amantadine, umifenovir, moloxidine, rimantadine, umif Enovir, zanamivir, and neuraminidase inhibitors, cap-dependent endonuclease Inhibitors, adamantane, peramivir, zanamivir, oseltamivir phosphate, and var (Xavir marboxil), as well as antivirals for other viral diseases that can result in high levels of NETosis Viral treatment, as well as antifungal treatment (e.g., clotrimazole, econazole, miconazole) (of which telbinafine, fluconazole, ketoconazole, and amphotericin are included) Select one or more from the following.

[0121] In one embodiment, the treatment is an anti-inflammatory drug. Many steroidal and non-steroidal anti-inflammatory drugs. Steroidal anti-inflammatory drugs are known in this art. Some examples of steroidal anti-inflammatory drugs include: While not limited to these, dexamethasone, hydrocortisone, cortisone, betamethasone Examples include prednisone, prednisolone, triamcinolone, and methylprednisolone. It can be said that, although not limited to, some examples of nonsteroidal anti-inflammatory drugs are, Aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen n, indomethacin, CD24Fc (CD24 protein bound to the Fc region of immunoglobulin G), and EXO-CD24 (CD24-exosome) is another example.

[0122] In one embodiment, the treatment involves DNase therapy to digest excess NETs, ​​or NETosi Inhibitors of s, for example, anthracycline drugs. In a further embodiment, ant Lacyclin drugs include epirubicin, daunorubicin, doxorubicin, and idarubicin. Selected from:

[0123] In one embodiment, the treatment is directed to bond to NETs or component parts of NETs. Therapeutic antibody drugs, but not limited to, nucleosomes or optional nucleosomes These are therapeutic antibodies directed to bind to the constituent parts of a molecule. For example, histone Isoform H3.1, citrullinated histone, myeloperoxidase, neutrophil elastin Oriented to bind to nucleosomes containing -ase or C-reactive proteins. One example is a therapeutic antibody.

[0124] In one embodiment, the treatment involves adsorbing and / or removing nucleic acids from circulation or from the body. A scavenger, for example, is the DNA scavenger polyamidoamine.

[0125] No treatments that inhibit NETosis have been used, or to the best of our knowledge, in humans... It has not been tested for use to date. There are several reasons for this. Firstly, as mentioned above. Thus, NETosis is an important component of the immune system. It is a vital component against infectious pathogens. In patients who may not yet have developed an antibody response, and in the NETosis process, phagocytosis Furthermore, it is especially important in patients, as it can be the primary mode of combating and preventing the spread of infectious diseases. Furthermore, this is even more important in the case of NETosis inhibitor therapy, because Rather than removing the products of NETosis (which can be replaced), the immune process itself This is because it renders it ineffective and prevents further production of NETs. Secondly, intravenously administered DN The half-life of -ase has been reported to be 3-4 hours, but the half-life of anthracycline is... Anthracyclines remain in circulation for more than 40 hours, and for at least several days. Disabling the immune system for several days in patients with infectious diseases is sufficient for a significant worsening of the disease. It will be time. Thirdly, anthracyclines kill cancer cells by It is a cytotoxic drug commonly used to treat cancer. It is one of the important mechanisms of action of the immune system. A drug that inactivates one, is long-lasting, and is cytotoxic, is needed for a disease related to NETosis. It should not be administered to patients with diseases that do not require such treatment or diseases that do not require such treatment. It is clear that drugs that inhibit NETosis are used for patients with normal or low levels of NETs. The treatment of such patients is inappropriate and potentially dangerous. Therefore, the administration of such therapies The patient having a high NETs level who requires treatment using the method described herein. This requires careful selection.

[0126] Therefore, in one aspect of the present invention, a drug and a cell-free nutrient for the treatment of NETosis-related diseases A combination product is offered that includes a companion diagnostic test for the detection or measurement of creosomes. In some embodiments, the combination product includes multiple drugs (e.g., NETosis inhibitors). (and cardioprotective agents and / or antibiotics) and / or multiple companion tests (e.g., no This may include (examination of cellular nucleosomes and examination of cytokine portions). In one embodiment, The combination product is a companion for the detection or measurement of cell-free nucleosomes and DNase drugs. Includes on-the-job diagnostic testing.

[0127] In one embodiment, the combination product is a drug that digests NETs, ​​for example, a longer nucleo NETs composed of somal chains are shorter oligonucleotide chains or mononucleotides. DNase drugs that digest into smaller fragments and cell-free nucleosomes for detection or measurement. This includes a companion diagnostic test.

[0128] In one embodiment, the combined product is a nucleic acid scavenger and a cell-free nucleosome. Includes companion diagnostic tests for detection or measurement.

[0129] In one embodiment, the combination product comprises NETs, ​​component parts of NETs, ​​MPOs, NEs, or CRPs. For the detection or measurement of cell-free nucleosomes with therapeutic antibody drugs directed to bind. This includes companion diagnostic testing.

[0130] In one embodiment, the combination product is a drug that inhibits or interferes with NETs formation by neutrophil cells. This includes physical therapy and companion diagnostic tests for the detection or measurement of cell-free nucleosomes. Drugs that inhibit or interfere with NETs formation are currently used to treat NETosis-related diseases. It has not been done. Anthracycline drugs are effective in treating sepsis induced in mice. A certain finding was reported by Figueiredo et al., Immunity (2013) 39: 874-884. Lethal Large amounts of sepsis were caused by cecal ligation and puncture, which resulted in death in 100% of mice within 72 hours. Induced in mice. When measured using various circulating inflammation biomarkers, anthracite Treatment with the drug epirubicin resulted in a 75% survival rate and reduced inflammation. However, However, it is not possible to clarify the mechanism of action of anthracyclines in achieving this effect. Figueiredo says nothing about NETs or NETosis. Later, Khan et al.'s literature, C Ancers (2019) 11: 1328 describes the efficacy of anthracycline drugs on neutrophil cells in cell culture. Khan investigated the fruit in vitro and reported that it inhibits NETosis. Ikurin and other DNA chelating agents suppress unwanted NETosis in NETs-related diseases. It was concluded that it could be considered a potential treatment for [the condition]. However, Anthrasa Since the main toxic side effect of Ikurin drugs is cardiotoxicity, Khan also suppresses NETosis. It does not affect the ability of anthracyclines, and suppresses the side effects of anthracyclines. Anthra is a cardioprotective agent used to limit the number of cases of heart disease, and is used in combination with dexrazoxane. He proposed that cyclin drugs could be administered.

[0131] Anthracycline drugs are extremely effective chemotoxic agents for the treatment of cancer. However, However, it can cause alopecia, skin rash, nausea and vomiting, discomfort, fever, peripheral neurotoxicity, and secondary complications. It has several side effects, including sexual leukemia and cardiotoxicity. Cardiotoxicity occurs during treatment or recovery. It can potentially lead to fatal congestive heart failure (CHF) several months to several years after treatment, It is dose-limiting. For example, it is based on a composite index of signs, symptoms, and left ventricular ejection fraction reduction. The probability of developing myocardial dysfunction is increased by the number of doxorubicin 300 mg / m 2 1-2% of the total cumulative dose, 400 mg / m² 2 At a dose of 3-5%, 450 mg / m² 2 5-8%, and 500 mg / m² 2 in The estimated risk of developing CHF is 6-20%. The risk of developing CHF is associated with a total cumulative dose of doxorubicin of 400 mg / m². 2 If increased beyond a certain point, it increases rapidly. Typical doses used in chemotherapy are for treatment. 60-75 mg / m² per unit 2 That is the case.

[0132] Anthracyclines have been observed to be useful in treating a mouse model of sepsis. The dosage is 0.6 μg / g (Figueiredo et al.), which is approximately 25 mg / m² for an 80 kg human male. 2 The inventors have clarified that it corresponds to. Therefore, a single intravenous dose is less than 1 / 10 of the dose that causes 1-2% of myocardial toxicity. The concentration of anthracycline drugs required for almost complete inhibition of NETosis of neutrophil cells in vitro is 5 μM (Khan et al.'s paper), which corresponds to approximately 3 μg / ml. A study of the pharmacokinetic properties of doxorubicin shows that a single 25 mg / m bolus intravenous dose produces a serum concentration of 10 μg / ml serum. Furthermore 2 The half-life of circulating doxorubicin is approximately 41 hours, and the serum level remains above 3 μg / ml for about 4 days. Therefore, a single 25 mg / m bolus dose can inhibit NETosis for several days and can be used as a treatment for sepsis or other NETosis-related diseases. A single bolus dose level or repeated cumulative dose level of less than 25 mg / 2 m may also be effective. m 2 Nanoparticle anthracycline drug formulations provide a targeted NETosis inhibitor approach

[0133] in which the active drug is released only in activated neutrophils, thus avoiding toxic side effects while maintaining the NETosis ability of the remaining neutrophils to respond to further infections (Zhang et al., Science Advances 2019;5: eaax7964). Therefore, in one embodiment, a combination product is provided that includes a nanoparticle anthracycline drug formulation for the treatment of NETosis-related diseases and a companion diagnostic test for the detection or measurement of cell-free nucleosomes.

[0134]

[0134] Plasmapheresis provides a circulatory treatment for NETosis that avoids drug toxicity side effects. Therapies involving the removal of NETs and NET degradation products have been reported (WO2019053243). In one embodiment, plasmapheresis therapy for the treatment of NETosis-related diseases and Combination products including companion diagnostic tests for the detection or measurement of cell-free nucleosomes It will be provided.

[0135] Anti-inflammatory drug therapies based on the CD24 protein have been reported for individuals suffering from COVID-19. In one case, 29 out of 30 moderate / severe COVID cases were cured within a few days. It has been reported that this is the case (Times of Israel 5 Feb 2021 and ClinicalTrials.gov Identi fier: NCT04747574), and CD24-exosomes accompanied by CD24 delivery in exosomes Yes, there is another such treatment, CD24Fc, which binds to the crystallizable region (Fc) of human IgG1 fragments. It contains the non-polymorphic region of the combined CD24 (ClinicalTrials.gov Identifier: NCT04317040). In one embodiment, plasmapheresis therapy for the treatment of NETosis-related diseases and Combination products including companion diagnostic tests for the detection or measurement of cell-free nucleosomes It will be provided.

[0136] NETs or NETosis-related diseases include sepsis, pneumonia, COVID-19, and influenza. Infectious diseases, and, but not limited to, pneumonia, SARS or ARDS of any cause, NETo for thrombotic or microthrombotic conditions, many inflammatory disease conditions, and other diseases including amputation. Sis-related complications, as well as thrombotic complications of diabetes and cancer, and many other Other diseases that involve pathologically elevated NETs production include other diseases, including cerebrospinal fluid disorders.

[0137] This method, (i) The level of cell-free nucleosomes in the sample obtained from the subject (optionally, 1 or more in Measure (in combination with tarleukin levels); (ii) Based on a higher level of cell-free nucleosomes compared to the control, the subject is treated Identifying that the patient has a NETosis-related disease (e.g., an infection) that requires medical treatment; and (iii) Administering the treatment to the subject It may include a colon (:).

[0138] In a preferred embodiment, the treatment involves DNase therapy to digest excess NETs, ​​anti-Nuclear Creosome or anti-NETs therapeutic antibody therapy, apheresis to remove excess NETs Alternatively, plasmapheresis therapy, or an inhibitor of NETosis as described herein. .

[0139] In one embodiment, a person has a medical complication that requires treatment, or the medical complication A method for identifying individuals who are at risk of developing an infectious disease, (i) The level of cell-free nucleosomes in the sample obtained from the subject (optionally, 1 or more in The process of measuring (in combination with the level of tarleukin); (ii) Based on a higher level of cell-free nucleosomes compared to the control, the subject is treated The process of identifying a person who has an infectious disease requiring medical treatment; and (iii) The process of administering the treatment to the subject. A method is provided that includes:

[0140] In one embodiment, the infection is sepsis or septic shock.

[0141] In a preferred embodiment, the patient has a medical complication requiring treatment, or the medical condition This is a method for identifying individuals infected with respiratory viruses that are at risk of developing complications. That is, (i) The level of cell-free nucleosomes in the sample obtained from the subject (optionally, 1 or more in The process of measuring (in combination with the level of tarleukin); (ii) Based on a higher level of cell-free nucleosomes compared to the control, the subject is medically The process of identifying the risk of developing medical complications; and (iii) The process of administering the treatment to the subject. A method is provided that includes:

[0142] In a preferred embodiment, the respiratory infection is influenza or coronavirus. The medical complication is pneumonia. Suitable treatments are, but are not limited to, extracorporeal. Respiratory support using oxygen is necessary for patients who are physically unable to breathe adequately without assistance. Respiration using a medical ventilator designed to provide artificial respiration of air into and out of the lungs. The provision of supplements, and / or oxygen and / or antiviral, antifungal, or anti-inflammatory drugs. It can be listed.

[0143] According to another aspect of the present invention, a method for treating an infectious disease, wherein the infection is detected by a panel test. This includes identifying patients who require treatment for a disease and providing such treatment, The panel test includes reagents for detecting the measurement of nucleosomes or their components. A method is provided. Patients with infections have higher cell-free nucleosole levels compared to controls. It is thought to have a level of "M".

[0144] (therapeutic antibody) Therapeutic antibodies are administered intravenously to neutralize the damaged or disease-causing parts of the target. This can be done. Therapeutic antibodies, as well as other similar or induced therapeutic bacteria such as Fab and Fv fragments. Inder is typically human in terms of the properties of the amino acid sequence of its heavy and light chains. or humanized. Therapeutic antibodies and methods for their development and production are in the art. This is well known. Therefore, in a further aspect of the present invention, severe hyperimmunity, including pneumonia Anti-nucleosome antibodies are provided for the treatment of the reaction.

[0145] Therefore, in a further embodiment, a respiratory virus with medical complications is present. A method of treating the subject, (i) The level of cell-free nucleosomes in the sample obtained from the subject (optionally, 1 or more in The process of measuring (in combination with the level of tarleukin); (ii) Based on a higher level of cell-free nucleosomes compared to the control, the subject is medically The process of identifying the presence of medical complications; and (iii) The step of administering a therapeutic anti-nucleosome antibody to the subject. A method is provided that includes:

[0146] Prevention or inhibition of NETosis by treatment with NETosis inhibitors is inappropriate for those with high levels of NETosis. The circulating and / or tissue-mediated NETs and nucleosomes of subjects suffering from diseases accompanied by Ts Lowering the bell. This is because the patient is suffering from a NETosis-related disease condition such as sepsis or stroke. It has been shown to lead to improved clinical outcomes in the subjects (Figueiredo et al., Immunity (2020) 13) 39: 874-884 and Zhang et al., Science Advances (2019) 5: eaax7964). Similarly, N NETs and nucleoses of circulating and / or tissue origin in subjects suffering from ETosis-related disease conditions. Removing the complication leads to improved clinical outcomes.

[0147] This is a guide for the measurement of H3.1-nucleosomes, citrullinated nucleosomes, MPO, and NE. The immunoassays described in this document have high binding affinity to nucleosomes and NETs and are particularly effective. Heterozygous monoclonal antibodies are used. These antibodies target NETs, ​​NETs metabolites, and nucleotides. They bind strongly and specifically to osomes. Therefore, these antibodies can be bound to NETs in vivo. It is used as a therapeutic antibody to neutralize NETs and, for example, to remove them from the body through phagocytosis. It can facilitate clearance (Weiskopf and Weissman, Mabs (2015) 7:303-10) ).

[0148] CRP is known to be physically associated with NETs that can further induce NETosis. It is an acute-phase protein. Therefore, antibodies against CRP neutralize and remove NETs. This can also inhibit the induction of NETosis by neutralizing and clearing CRP.

[0149] Therefore, in one embodiment of the present invention, a method for treating NETosis-related diseases, wherein Nuc Rheosomes or their components, DNA, myeloperoxidase, neutrophil elastase or a method comprising administering a therapeutic antibody directed to bind to a C-reactive protein. The therapeutic antibody is provided to neutralize NETs derived from a diseased subject or to neutralize the NETs. This can promote clearance. The method of administering therapeutic antibodies is well known in the art. ru.

[0150] In another aspect of the present invention, excessive or inadequate NETosis (i.e., NETosis-related disease) Anti-nucleosome, anti-DNA, anti-myeloperoxin for use in the treatment of conditions accompanied by Sidase, anti-neutrophil elastase, or anti-C-reactive protein therapeutic antibodies are provided.

[0151] Used by the inventors for the nucleosome assay described herein The anti-histone H3.1 antibody, anti-nucleosome antibody, and anti-citrullinated H3 antibody are extremely powerful. It has been selected as a highly effective and specific antibody, and therefore is particularly useful as a therapeutic antibody. be.

[0152] In particular, anti-histone H3.1 antibodies can be extremely specific. Nucleosomes are histones The tail is removed physically and irreversibly by regulatory proteolysis or cleavage. Furthermore, histone degradation has been shown to be involved in the formation of NETs (Papayan See the literature by nopoulos et al. (2010) J. Cell Biol. 191(3): 677-691. Regarding histone H3. It has been reported that cleavage occurs around amino acid position 21 (Yi and Kim (2018) B) MB Reports, 51(5): 211-218). The amino acid sequence of histone H3.1 at positions 27-36 is: [ka] The amino acid sequence at positions 29-35 is generally a post-translational modified amino acid (e.g., lig It does not contain (amino acids, serine, or arginine). Therefore, this epitope (i.e., amino acids) Antibodies directed to bind to acid positions 29-35) are used in the post-translational modification of nucleosomes. Depending on the circumstances, it may not be affected or may be minimally affected, and is independent of the PTM structure. It binds to all or most of the nucleosomes containing ston H3.1. Therefore, this specification Solid-phase capture selected for use by the inventors for immunoassays described in this book. Antibodies detect both intact and cleaved nucleosomes, regardless of their PTM status. Regardless, within the core of histones near amino acid positions 30-33, so that they can be captured by antibodies. It was an anti-histone H3.1 antibody directed to bind to an epitope located in that region. This maximizes the capture of H3.1-nucleosomes, and the effectiveness of this approach is shown in Figure 6. It is clear that the amino acid sequence of histone H3.1 is publicly known in the art, and UniProt It is listed under accession number P68431.

[0153] Therefore, in one embodiment of the present invention, the therapeutic antibody is located above amino acid position 21. To bind to the core histone epitope of histone H3.1 in the amino acid epitope. It is directed to. In a preferred embodiment, the anti-histone H3.1 therapeutic antibody is histo Within the core of H3, or near amino acid positions 29-35, particularly amino acid position 30 It is oriented to bind to epitopes located at or near ~33.

[0154] The labeled antibody used herein by the inventors for immunoassays is DN It is present in intact nucleosomes containing a histone octamer core that has formed a complex with A. Antinucleosomes oriented to bind to nucleosome epitopes in a three-dimensional structure It was an antibody. This antibody is a free histone octamer complex, free histone (i.e., DNA) It does not contain, does not bind to (or weakly binds to), free DNA, or free histones. In this case as well, This antibody is relatively unaffected by the histone PTM composition of the nucleosome to which it binds. Sometimes.

[0155] Therefore, in one embodiment of the present invention, the therapeutic antibody forms a complex with DNA. The three-dimensional structure of nucleosomes present in intact nucleosomes containing a ton octamer core It is oriented to bond to a pitope.

[0156] In one embodiment of the present invention, the therapeutic antibody is, for example, one or more histone tails removed. By binding to epitopes present in the cleaved nucleosomes, cleavage occurs. It is directed to selectively bind to the nucleosome. In this embodiment And the epitope, due to the presence of a complete histone tail, is within the intact nucleosome. It may be hidden beforehand (and therefore prevent antibody binding). Therefore, the cleaved Nu Epitopes bound by antibodies selective for creosomes remain intact (i.e., It may be an inaccessible epitope within a nucleosome (whether complete or uncleaved). In one embodiment, the cleaved nucleosome has its histone tail removed. Contains ston H3, H2A, and / or H4 proteins.

[0157] In one embodiment, a mixture of two or more therapeutic antibodies can be administered to the target. For example However, although not limited to, nucleosome epithelium present in intact nucleosomes One antibody directed to bind to a loop, and the core histone epithelium of histone H3.1 A mixture of another antibody directed to bind to a specific target.

[0158] The antibody used by the inventors for the immunoassay was a mouse monoclonal antibody. These mouse monoclonal antibodies are useful therapeutic antibodies in mice. It is likely that (since monoclonal antibodies are foreign proteins) in humans or other animals Its use results in an antigenic immune response and the production of anti-mouse immunoglobulin antibodies. To avoid answering, non-human monoclonal antibodies may be humanized. Humanized antibodies are... The protein composition of the non-human antibody has been modified to be similar to that of naturally occurring human antibodies. It is an antibody and is well known in this art. The antibody is specific to the antibody and the antibody epithelium Variable domains including complementarity-determining regions (CDRs) that determine linkage specificity and binding strength, and species-specific It consists of a constant domain and a target domain. One method of humanization is to encode the variable CDR of a non-human antibody. This involves the fusion of the DNA that encodes the human constant domain with the DNA that encodes the human constant domain. Using this method, human pairs It is not antigenic in elephants, and possesses the binding specificity and binding affinity of the original non-human monoclonal antibody. Most of them can produce DNA vectors that encode human antibodies. Humanized antibodies The resulting DNA vector can then be used for large-scale production.

[0159] Therefore, in one embodiment, the therapeutic antibody is a humanized antibody.

[0160] The embodiments described herein can be applied to all aspects of the present invention, i.e., The embodiments described for use may be equally applicable to the claimed methods, etc. This will be understood.

[0161] The present invention will now be illustrated with reference to the following non-limiting embodiments. [Examples]

[0162] (Example 1) The inventors have found that the addition of heparin alters the NETs form of leukocytes in fresh healthy whole blood samples. This demonstrated the induction of the NETs substance produced in plasma, followed by detection of the NETs substance produced in plasma (Lelliott et al., I International Immunology (2019) pii: dxz084). The inventors have identified low levels of circulating NET material. Whole blood samples from two healthy volunteers, which are expected to be affected, were collected in an EDTA plasma blood collection tube and Heparinized plasma was collected in blood collection tubes. Two EDTA plasma blood collection tubes were immediately inserted. The cells and plasma fractions are separated by centrifugation, and the large chromatin (containing NET material) is mixed. With minimal input, the plasma was transferred to a freezing tube and frozen. Two heparinized plasma blood samples were collected. The collection tube was gently rotated and incubated at room temperature for 1 hour. Next, the tube was centrifuged, the plasma was transferred to a freezing tube, and frozen.

[0163] The sample was subjected to a double-cycle ELISA procedure to obtain a nucleo containing histone isoform H3.1. The H3.1-nucleosome was assayed. Briefly, 20 μl of the sample was subjected to an assay. Microtiter wells containing magnetic particles pre-coated with histone H3.1 antibody. It was added to the sample. The sample was incubated, the magnetic particles were isolated and washed. Horseradish peroxide To bind to the three-dimensional nucleosome epitope conjugated by xidase. Directed anti-nucleosome antibodies were added to magnetic particles. The particles were incubated, The samples were then isolated and washed. The bound anti-nucleosome antibody was measured using a colorimetric substrate reaction. The results were determined. The results are shown in Figure 1, which shows that the NET material level was high in the heparin tube. However, this indicates that the level was lower in the EDTA tube. This is the level of circulating NET material. It has been clearly demonstrated that an increase in [the substance] can be detected by a simple, low-cost immunoassay test. Yes, they are.

[0164] (Example 2) DNA was extracted from the two heparin and plasma samples described in Example 1, and then into a chip base. This is applied to a capillary electrophoresis system (Agilent Bioanalyzer) to separate DNA fragments by size. The analysis revealed that DNA fragments approximately 150 bp in size, corresponding to mononucleosomes, were retained for approximately 60 seconds. There is a gap. As shown in Figure 2, the observed level of mononucleosome-associated DNA. The number of DNA fragments corresponding to NET material was low (as expected for healthy volunteers). The size has a longer retention time of approximately 110 seconds. As shown in Figure 2, the NET material Bell's levels were low in EDTA plasma (as expected for healthy volunteers), but NET formation was low. The results were higher in heparinized plasma tubes stimulated by exposure to valine. In Example 1, the increase in nucleosome levels observed in heparinized plasma indicated that mononucleosomal This confirms that it originates from NETs, ​​not rheosomes.

[0165] (Example 3) EDTA plasma samples were collected from 50 control subjects with mild symptoms and from individuals with respiratory complications. All coronavirus infection test results, including those for 50 people requiring respiratory support, are now available. The circulating NET material will be collected from 100 subjects who tested positive. The NET material will be collected using the method described in Example 1. Measurement is performed using a crosome immunoassay. 50 control subjects had low NET nucleos It was found to have range levels, and these levels were used to determine the range of comparison. It was found that the 50 people tested had higher NET nucleosome levels. These high levels indicate that, in addition to viral infections, the subjects have respiratory complications requiring treatment. It is used as an indicator of possession.

[0166] (Example 4) The experiment performed in Example 3 will be repeated, but the immunoassay performed will be citrullinated Creosomes were measured. Fifty control subjects had low levels of citrullinated nucleosomes. It was found that these levels are used to determine the control range. 50 people The subjects tested were found to have higher levels of citrullinated nucleosomes, and these High levels indicate that, in addition to viral infections, the subject has respiratory complications requiring treatment. It is used as an indicator to show that...

[0167] (Example 5) The experiment performed in Example 3 will be repeated, but the immunoassay performed will be myeloperoxic. Sidase-nucleosome adduct levels were measured. 50 control subjects had low myeloperidol levels. It was found that oxidase-nucleosome levels were present, and these levels were within the control range. Used to determine the range. 50 test subjects had higher myeloperoxidase levels. It was found that these levels are present at the nucleosome level, and these high levels are associated with viral infections. In addition, it can be used as an indicator that the subject has respiratory complications requiring treatment. It will be done.

[0168] (Example 6) The experiment performed in Example 3 will be repeated, but the immunoassay performed will be neutrophil elastinase. -Nucleosome adducts were measured. 50 control subjects had low neutrophil elastase levels. It was found that these levels were present at the creosome level, and these levels were used to determine the control range. Used for: 50 subjects tested showed higher neutrophil elastase-nucleosome levels. It was found that these high levels, in addition to viral infections, indicate that the subjects require treatment. It is used as an indicator that a patient has respiratory complications that require intervention.

[0169] (Example 7) The experiment conducted in Example 5 was repeated, but the levels of CRP and IL6 were also measured in the EDTA plasma sample. Measurement is performed in or in serum samples from the same subject. Clinical sensitivity and for identifying the subject of the test. To maximize specificity, the resulting logistic regression analysis is used to open up the algorithm. To emit.

[0170] (Example 8) EDTA plasma samples were collected from 50 healthy individuals in 2019, before the COVID-19 pandemic, and from individuals infected with COVID-19. The data was collected from 50 people who were hospitalized for suspected symptoms of COVID-19 infection. Of the 50 people who underwent the examination, 34 tested positive for COVID-19 using polymerase chain reaction (PCR) testing. Sixteen people tested positive for COVID-19 using PCR testing. The test result was negative.

[0171] Patients with symptoms include both those who have experienced severe illness and those who have experienced milder illness. The selection was made to include: 5 subjects admitted to the ICU, of which 2 subjects were... Forty individuals who received artificial respiration for 9 and 10 days respectively were hospitalized. Even if a PCR test result is negative, those who are hospitalized will still receive treatment for symptoms of respiratory infection. I received it.

[0172] H3.1-nucleosome levels were measured in plasma samples. The results are shown in Figure 3. All R-positive COVID-19 patients have elevated levels of plasma H3.1-nucleosomes, and based on this... Therefore, 100% of PCR-positive COVID-19 patients had a specificity of 94% (3 false positives out of 50 control subjects). ) and distinguishable from normal controls with an AUC of 98.7%.

[0173] PCR-positive COVID-19 patients were divided into two groups based on their H3.1-nucleosome levels. They could be divided into two groups. The first group, which included 15 of the 34 subjects, had a blood sugar level of less than 600 ng / ml. They had H3.1-nucleosome levels. The second group included 19 out of 34 subjects. The sample had H3.1-nucleosome levels higher than the upper limit of the assay (>700 ng / ml). He was.

[0174] A similar pattern was observed in six of those individuals, who had H3.1-nucleosome levels below 600 ng / ml. Ten people had H3.1-nucleosome levels >700 ng / ml and were experiencing COVID-19 symptoms. However, observations were made in 16 patients who tested negative by PCR.

[0175] (Example 9) Whether circulating H3.1-nucleosome levels predict the severity of COVID-19 disease. To determine this, EDTA plasma samples are used in a PCR COVID-19 virus test, and the positive result of the COVID-19 virus test is determined by the results of the COVID-19 virus test. The samples were collected from 14 individuals diagnosed with D-19 infection. Of these, five samples were from outpatients. The data was collected from individuals who had appointments at the hospital or were being examined in the hospital's emergency room (ER), and consisted of three categories. The samples were collected from patients hospitalized in regular addiction treatment wards (i.e., not in the intensive care unit). The six samples were collected from patients receiving ventilators and extracorporeal membrane oxygenation (patients who had undergone cannula treatment). Advanced respiratory support, including (where lung function is replaced by supplying blood through an artificial lung) Those with extremely severe conditions are transferred to the intensive care unit (ICU) of a tertiary hospital center for other clinical support. The data was collected from patients with the disease. The mortality rate among these subjects was 4 deaths out of 6.

[0176] Circulating H3.1-nucleosome levels were measured in plasma samples. The results are shown in Figure 4. The difference between patients examined in the outpatient / ER and patients admitted to a regular ward due to COVID-19 infection is clear. There was an increase in the kana level. The area under the curve (AUC) for this distinction was 100%. The patient was admitted to the ICU. The level was also clear when comparing those who had COVID-19 with those who were hospitalized in a regular ward. There was an increase. The area under the curve for this distinction was also 100%. Furthermore, the four patients who died... It was found to have four of the highest circulating H3.1-nucleosome levels. Prediction of mortality The AUC was also 100%.

[0177] These results indicate that circulating H3.1 nucleosome levels predict disease severity and mortality. This demonstrates that it can be used for prognosis determination. Therefore, the method of the present invention is used In the examination of respiratory infections, the level of clinical assistance and / or the nature of the treatment required. It is required for patients or subjects at various stages of the clinical process, including the later stages that determine quality. It is possible to predict the level of care required.

[0178] Similarly, these results, using circulating H3.1-nucleosome levels, indicate that the levels are elevated. Patients are monitored by continuous sampling to determine whether they are healthy or declining. This indicates that clinical decisions regarding future treatment and clinical support systems can be communicated. For example, a lower level means that intensive respiratory support is no longer essential. The decision and / or the decision that the treatment used was effective may be communicated.

[0179] Conversely, rising levels indicate a decision and / or use that intensive respiratory support is needed. This means that the current treatment is not effective and additional or alternative treatments should be considered. The decision can be communicated. Therefore, the method of the present invention can be used, for example, NETs are removed by NETosis inhibitors, apheresis, or plasmapheresis. Treatment to reduce inflammation, or anti-inflammatory drug treatment, such as CD24 therapy (e.g., EXO-CD24 or CD24Fc). Patients requiring treatment for NETosis-related conditions, including [specific condition], can be selected. Therefore, the present invention provides a companion for drugs and treatments for diseases accompanied by NETosis. It can be used as a diagnostic product.

[0180] Similarly, this relates to the efficacy of the investigational drug for treating respiratory disease conditions using the method of the present invention. It also shows that it may be suitable for use in the evaluation of circulating H3. .1-Nucleosome levels can be measured alone or in combination with other parameters. It can be used as a surrogate endpoint in clinical trials.

[0181] (Example 10) To determine whether the method of the present invention is effective for other nucleosome regions, The same subject as described in Example 9 is the citrulline residue at the arginine residue at position 8 of histone H3. At the level of circulating nucleosomes (H3R8Cit-nucleosomes) containing histone modifications We also tested this. Since NETs chromatin is known to be citrullinated, this The nucleosome region was selected. The results are shown in Figure 5, and the method of the present invention is effective in citrulline Using nucleosome denaturation assays, and more generally, any circulating nucleosome portion Alternatively, it is effective using measurements of NETs parts that include MPO, NE, or other NETs component parts. This supports the point.

[0182] These results suggest that circulating H3R8Cit nucleosome levels predict disease severity and mortality. This indicates that it is a viable option and can be used for prognosis determination. Similarly, H3R8Cit-nucleo Using some-level techniques, the care required by patients or subjects at various stages of the clinical process is assessed. Predicting the level and the H3.1-nucleosome level as described in Example 9 above The patient can be monitored through continuous sampling.

[0183] (Example 11) Blood samples were collected from subjects suffering from NETosis-related diseases, and the excess production of NETs by the subjects was investigated. Nucleosomes described herein as indicators of live or overproduction The assay is performed. If the measured NETs level is below the threshold cutoff value, the patient is A NETosis inhibitor therapy, such as intracycline therapy, is not administered. Measured NETs level If the threshold cutoff value is exceeded, the patient will receive NETosis inhibitor therapy such as anthracycline therapy. The drug is administered. Further blood samples are taken at appropriate intervals (e.g., every 4 hours or daily). The patient monitors the decline in circulating NETs levels and determines the effectiveness of the treatment. When used in law, the present invention method is a NETosis inhibitor therapy and a NETosis therapy (e.g., antivirus) Antibacterial drugs or anticoagulants, anti-inflammatory drugs, anticoagulants or blood coagulation inhibitors, NETosis inhibitors, DN Aase drugs, anti-nucleosome therapeutic antibody drugs, anti-MPO therapeutic antibody drugs, and anti-NE therapeutic antibody drugs, or Apheresis or plasmapheresis treatment is required to remove NETs from the circulation. Companion diagnostics for patient selection and monitoring of patients with pathological NETs levels We provide combination products that include the following.

[0184] (Example 12) Sepsis was treated with an infection caused by E. coli administered via intravenous infusion over a 3-hour period. Induction was performed in 16 more pigs (0-3 hours in Figures 6 and 7). Sepsis in pigs was described in WO2019053243. As described, the NETs were removed from the bloodstream by plasmapheresis. In short, whole blood is taken from the pig's body through a tube into a plasmapheresis device. Remove the NETs and separate the whole blood into a cell fraction and a plasma fraction. The plasma is then processed into a plasma containing the NETs binder. The plasma is passed through a smapheresis cartridge to remove NETs, ​​and then the plasma is divided into blood cells. The cells were reassembled and returned to the pig's body. The plasmapheresis procedure was performed for 5 hours. The procedure was performed (2-7 hours in Figures 6 and 7). Plasma ferresis cartridge used on 9 pigs. It contains NETs binder (treated pig), and the cartridge used on the other 7 pigs was The control pig did not contain any of the specified ingredients.

[0185] Eight plasma samples were collected from each pig at one-hour intervals from 0 to 7 hours after the onset of infection. The method of (i) is effective as a monitor during the infection period and (ii) is effective as a treatment. To determine if it was effective as a monitor, we measured circulating nucleosomes.

[0186] Furthermore, the degree of plasma removal by the NETs binder in the cartridge can be controlled by the method of the present invention. To determine if it is possible to monitor the plasma sample in a cartridge, Upstream (to sample the plasma that enters the NETs binder cartridge) and cart Downstream of the ridge (to sample plasma flowing out of the NETs binder cartridge) Samples were collected from plasmapheresis devices in both locations. Five upstream and five downstream samples were infected. Samples were taken every hour from 3 to 7 hours after the start of the experiment (Figures 6 and 7, 3 to 7 hours).

[0187] Plasma samples containing histone isoform H3.1 (H3.1-nucleosome) levels We performed an assay on creosomes. The assay measurements were performed using an automated immunoassay instrument. The test was performed by an immunoassay. Briefly, the calibration sample or sample (50 μl) was subjected to an acridinase. Along with ester-labeled anti-nucleosome antibody (50 μl) and assay buffer (100 μl) The samples were incubated at 37°C for 1800 seconds. Magnetic beads coated with anti-histone H3.1 antibody were then used. Add 20 μl of the solution and incubate the mixture for another 900 seconds. Then, remove the magnetic beads. Isolate, wash three times, and then apply magnetically coupled acridinium ester to the luminescence output over 7000 milliseconds. Therefore, it was decided.

[0188] The mean results for circulating H3.1-nucleosome levels in control and treated pigs are shown in Figure 6a. It has been shown that control pigs (infected and sepsis induced, but not treated) subsequently The patient developed sepsis over several hours, which was attributed to the observed circulating H3.1 nucleosome levels. This was reflected in the increase. The increase in average H3.1-nucleosome levels was evident after 1 hour (after the start of infection). It was slow, and accelerated after 3 hours, which is consistent with the time course of the NETosis process. H3.1 nucleosome levels continued to rise, reaching 361 ng / ml in 7 hours. Mean circulating H3.1 - Similar initial elevations in nucleosome levels were observed in treated pigs from 0–2 hours. Initiating plasmapheresis treatment slows down the increase in nucleosome levels. The average level observed after 7 hours was 150 ng / ml. This is the average level observed in control pigs. The level was considerably lower than the average level, which demonstrated the effectiveness of plasmapheresis. H3.1 Nucleosome levels are effective in monitoring the course and severity of septic diseases. It has been shown to be a treatment guideline and an effective monitor of the in vivo NETosis process. It will be done.

[0189] During the operation, measurements were taken in the sample collected from the plasmapheresis device upstream of the cartridge. The mean results for the determined plasma H3.1-nucleosome levels are shown in Figure 6b. The results are shown in Figure 6a for the measured mean circulating H3.1-nucleosome levels. The result is the same as what was predicted.

[0190] During the operation, measurements were taken in a sample taken from within the plasmapheresis device downstream of the cartridge. The mean results for the determined plasma H3.1-nucleosome levels are shown in Figure 6c. (Control pigs) Regarding this, the results in Figure 6c are similar to those in Figure 6b (and 6a), and the plasma was used as a binder for NETs. Passing the sample through a cartridge that does not contain the substance significantly affects the observed H3.1-nucleosome levels. This indicates that the level of NETs in plasma did not affect the binder of NETs. The expected result was that the passage of cartridges that did not contain the substance did not have a significant impact. The results are consistent. For the treated pigs, the results in Figure 6c are all low. This is because the plasma NETs When passed through a binder-containing cartridge, almost all or all NETs are removed from the plasma. This is consistent with the expected result. Furthermore, these results are in the cartridge The NETs binder is not saturated with NETs in 7 hours, and the NETs present in the plasma entering the device... This indicates that it remained bound to all or most of the molecules. Therefore, H3.1-nucleo Measuring the level of the foam determines when the bonding material in the cartridge becomes saturated, and therefore, It is no longer useful as a tool for removing NETs and should be replaced with a fresh cartridge. It is useful for deciding whether or not to do something.

[0191] Therefore, the combined results of Figures 6b and 6c show that the measurement of H3.1-nucleosome levels is N This study demonstrates its usefulness as a monitor and guideline for the treatment of ETosis and sepsis.

[0192] The circulating H3.1 nucleosome levels were measured in samples taken from all 16 pigs. The results are shown individually in Figure 7a. Average H3.1-nucleosole observed in control pigs at 7 hours. The glycemic level was 361 ng / ml, which was above 120 ng / ml in all control pigs (range 1). (23-743 ng / ml). In contrast, the mean H3.1-nucleosome level observed in treated pigs at 7 hours. The level was 150 ng / ml, which is below 120 ng / ml in most (7 out of 9) treated pigs. (Range 27-111 ng / ml). These results demonstrate the effectiveness of plasmapheresis treatment. These results indicate that H3.1 nucleosome levels are related to the course and severity of septic disease. It is an effective monitoring and treatment guideline, and also an effective monitoring of excessive NETosis in vivo. It also indicates that it is a monitor and treatment guideline. Furthermore, the results in Figure 7a show that circulating H3.1-nucleate Using osome-level measurements, individuals with elevated NETs levels are classified as having NETs or NETosis. This indicates that it can be identified as a suitable candidate for treatments that reduce the level. ru.

[0193] Seven control pigs showed elevated nucleosome levels and clinical stress indicators. The levels also rose, and the seven treated pigs with lower nucleosome levels required more intensive medical support. It required assistance. Furthermore, it had H3.1-nucleosome levels exceeding 120 ng / ml. The two treated pigs observed also showed elevated indicators of clinical stress, requiring more intensive medical care. Support was needed. Therefore, the method of the present invention monitors the efficacy of NETosis treatment. This is a good method for that purpose.

[0194] During the operation, measurements were taken in the sample collected from the plasmapheresis device upstream of the cartridge. The results for the determined plasma H3.1-nucleosome levels are shown individually in Figure 7b for all 16 pigs. As shown above regarding Figure 6, the results shown in Figure 7b are the same as those shown in Figure 7a. The results are similar. The mean H3.1-nucleosome level observed in control pigs at 7 hours was 368n The levels were g / ml (range 121-629 ng / ml). In contrast, the H3.1-nucleotides observed in treated pigs at 7 hours were... Rheosome levels were lower, with the average result (for all 9 treated pigs) being 143 ng / ml. For the seven responder pigs, the range of results at 7 hours was 34-127 ng / ml.

[0195] During the operation, measurements were taken in a sample taken from within the plasmapheresis device downstream of the cartridge. The results for the determined plasma H3.1-nucleosome levels are shown individually in Figure 7c for all 16 pigs. The mean level of H3.1 nucleosomes observed in control pigs over 7 hours is shown. The blood glucose level was 378 ng / ml (range 147-617 ng / ml). In contrast, the carton in treated pigs was 378 ng / ml after 7 hours. The mean level of H3.1-nucleosomes observed in plasma downstream of the ridge was 2.4 ng / ml (range 0.7). The levels were ~6.5 ng / ml, and all 9 treated pigs were below 7 ng / ml at all time points.

[0196] The combined results of Figure 7b and Figure 7c show that the measurement of H3.1-nucleosome levels is related to NETosis and This study demonstrates its usefulness as a monitor and guideline for the treatment of sepsis.

[0197] When combined, the results shown in Figure 7 indicate that the plasmapheresis treatment system was used for all 9 animals. We successfully removed NETs from the pig circulation, and 7 out of 9 pigs underwent the treatment. Although they responded sufficiently, this indicates that two were non-responders. Somone results correlate very well with clinical observations in pigs.

[0198] Furthermore, based on the observed nucleosome results, the seven treated responder pigs were selected. Treated non-responder pigs and untreated pigs can be clearly distinguished.

[0199] (Example 13) Plasma samples were obtained from 20 human subjects diagnosed with sepsis and 10 healthy human subjects. The sample was subjected to histone isoflavone assay using the automated immunoassay apparatus described in Example 12. Assay on the level of nucleosomes containing foam H3.1 (H3.1-nucleosomes) Elevated levels were observed in sepsis samples compared to healthy controls. This indicates that this disease is associated with This is likely due to the effects of NETosis at various stages (Figure 8).

Claims

1. A method for monitoring the progression of disease in subjects suffering from an infectious disease, (i) The bodily fluid sample obtained from the subject is brought into contact with a binder to form cell-free nucleosomes or To detect or measure the level of a component; (ii) Repeating step (i) on one or more occasions; and (iii) Using any change at the level of the cell-free nucleosome or its components, the Monitoring the progression of infectious diseases in elephants The method including:

2. A method of assigning the risk of adverse outcomes to individuals suffering from an infectious disease, (i) The bodily fluid sample obtained from the subject is brought into contact with a binder to form cell-free nucleosomes or To detect or measure the level of the components; and (ii) Using the detected cell-free nucleosome levels, the potential for adverse outcomes in the subject Assign : includes, In this stage, subjects identified as having a high potential for adverse outcomes are assigned to medical interventions. The aforementioned method.

3. Claim 1 or Claim 2, wherein the infectious disease is a viral, bacterial, fungal, or microbial infection. Method of description.

4. The method according to any one of claims 1 to 3, wherein the infectious disease is a respiratory tract infection.

5. The aforementioned respiratory infection is selected from influenza, pneumonia, and severe acute respiratory syndrome (SARS): The method described in claim 4, which is selected.

6. The person according to claim 1 or claim 2, wherein the subject is suffering from sepsis or septic shock. Law.

7. The body fluid sample is a blood, serum, or plasma sample, according to any one of claims 1 to 6. method.

8. The cell-free nucleosomes are part of or derived from neutrophil extracellular traps. The method according to any one of claims 1 to 7.

9. The components of the cell-free nucleosome are the epigenetic components of the cell-free nucleosome. A method according to any one of claims 1 to 8, comprising the features described above.

10. The aforementioned epigenetic features are histone isoforms, for example, coanucreos The histone isoform of a histone, particularly the histone H3 isoform, as described in claim 9. The method.

11. The method according to claim 10, wherein the histone isoform is H3.

1.

12. The aforementioned epigenetic features are post-translational modifications (PTMs) of histones, such as coanucles. The method according to claim 9, wherein the histone PTM is a some, particularly histone H3 or H4 PTM.

13. The method according to claim 12, wherein the histone PTM is selected from citrullination or ribosylation. 。

14. The level of the cell-free nucleosome or its components is used in immunoassays, immunochemistry, and quality Using quantitative spectroscopy, chromatography, chromatin immunoprecipitation, or biosensor methods A method according to any one of claims 1 to 13, wherein detection or measurement is performed.

15. The detection or measurement method involves detecting the body fluid sample as a cell-free nucleosome or its constituent elements. Contacting a solid phase containing a binder for detection, and detecting the binding to the binder. The method according to any one of claims 1 to 14, including.

16. The detection or measurement method is (i) the sample is an epigenetic test of cell-free nucleosomes (ii) bringing into contact with a first binder that binds to the characteristic; (ii) in step (i) the first binder The sample, bound by [method], is then brought into contact with a second binder that binds to cell-free nucleosomes. (iii) to detect or quantify the binding of the second binder in the sample; The method described in any one of the requests 1 to 15.

17. The method according to any one of claims 1 to 16, wherein the subject is a human or an animal.

18. The level of cell-free nucleosomes or their components in the aforementioned body fluid sample is set to 1 or more pairs The method according to any one of claims 1 to 17, further comprising comparing with light.

19. The method according to claim 18, wherein the control is a healthy subject.

20. The method according to claim 18, wherein the control is a subject having an infectious disease that is asymptomatic or shows mild symptoms. Law.

21. The levels of cell-free nucleosomes or their components are elevated compared to the control. The method according to any one of claims 1 to 20.

22. The level of the cell-free nucleosomes is detected or measured as one of the measurement panels. The method according to any one of claims 1 to 21.

23. The method according to claim 22, wherein the panel contains one or more interleukins.

24. Claim 23, wherein the one or more interleukins are selected from the group consisting of IL-6 and IL-12. Method of description.

25. The aforementioned panel includes C-reactive protein (CRP), myeloperoxidase (MPO), and D-dimer. , and / or comprising factor VII-activated protease (FSAP), as any one of claims 22 to 24 Method of loading.

26. The method according to any one of claims 22 to 25, wherein the panel includes MPO.

27. Pneumonia, acute respiratory syndrome (ARS), acute respiratory distress syndrome (ARDS), or severe acute respiratory syndrome A method for detecting individuals who require medical treatment for SARS, (i) The bodily fluid sample obtained from the subject is brought into contact with a binder to form cell-free nucleosomes or To detect or measure the level of the components; and (ii) The level of cell-free nucleosomes in the subject is a medical condition for pneumonia, ARS, ARDS, or SARS. To use as an indicator that medical treatment is needed. The method including:

28. This method was used to detect individuals who require medical treatment for sepsis or septic shock. hand, (i) The bodily fluid sample obtained from the subject is brought into contact with a binder to form cell-free nucleosomes or To detect or measure the level of the components; and (ii) The level of the cell-free nucleosomes in the medical field of sepsis or septic shock. To use as an indicator that appropriate treatment is needed. The method including:

29. A method for treating NETosis-related diseases, wherein nucleosomes or their components, To bind to eloperoxidase, neutrophil elastase, or C-reactive protein. The method comprising administering a directed therapeutic antibody.

30. The method according to claim 29, wherein the NETosis-related disease is accompanied by a high level of extracellular neutrophil trapping. 。

31. The claim 29 or 30, wherein the NETosis-related disease is a viral or bacterial infection. method.

32. The therapeutic antibody is directed to bind to an epitope present in an intact nucleosome. The method according to any one of claims 29 to 31.

33. The therapeutic antibody is designed to bind to epitopes present in the cleaved nucleosomes. The method according to any one of claims 29 to 31, which is directed.

34. The therapeutic antibody is a nucleosome component of histone H3.1 or citrullinated histone. The method according to any one of claims 29 to 33, wherein the components are oriented to bond to each other.

35. The aforementioned therapeutic antibody is located at amino acid positions 30-33 in the amino acid sequence of histone H3.1 The H3.1 epitope is oriented to bind to any one of claims 29 to 34. Method of loading.

Images