Cell-free nucleosome-level triage method

Abstract

The present invention relates to the use of cell-free nucleosome levels to identify patients at risk for developing NETosis-associated adverse responses to infectious diseases. The method is used to monitor disease progression and assign risk of adverse outcomes in patients suffering from infectious diseases.

Description

[Technical Field]

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

[0002] (Background of the invention) Influenza spreads globally in annual epidemics, resulting in approximately 3 to 5 million severe illnesses and 290,000 to 650,000 deaths. More recently, the emergence and rapid progression of a new infectious disease, COVID-19, has developed into a pandemic. Some infections can lead to acute respiratory syndrome (ARS), acute respiratory distress syndrome (ARDS), or severe acute respiratory syndrome (SARS), which can be life-threatening disease progressions requiring medical intervention. Because infectious disease outbreaks and pandemics place a significant burden on international healthcare services, methods for triaging patients to identify those most likely to require hospital intervention are crucial in helping healthcare providers prioritize patients, save lives, and more effectively manage the increased demand for medical services.

[0003] COVID-19, influenza, and other infections can progress to NETosis-related complications, which can be severe and potentially fatal. Such complications include sepsis, a life-threatening organ failure that can occur as a complication of infection. There is currently a lack of methods for treating inadequate NETosis, identifying individuals at high risk of NETosis-related complications, monitoring the progression of such complications requiring such treatment, monitoring the efficacy of treatment, and monitoring the progression of such diseases.

[0004] Previously, Holdenrieder et al., in Int. J. Cancer (2001) 95: 114-120, described the detection of nucleosome levels in serum samples from patients with benign and malignant diseases. The epigenetic composition of circulating cell-free nucleosomes, including their histone modifications, histone variants, DNA modifications, and adduct content, has also been studied as a blood-based biomarker for cancer. See WO 2005 / 019826, WO 2013 / 030577, WO 2013 / 030579, and WO 2013 / 084002.

[0005] There remains a need in the art to provide effective treatments for NETosis-related conditions, simple and cost-effective methods for identifying and prioritizing individuals at high risk of developing NETosis-related complications with poor prognosis during infection, and methods for monitoring disease treatment and 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 PCR-confirmed COVID-19 infections, from 15 patients: 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 6] Results from the experiment described in Example 12, showing the mean levels of nucleosomes containing histone isoform H3.1 measured in 16 pigs induced by sepsis treated with plasmapheresis. In 9 pigs, plasma was passed through a cartridge containing NETs binder (treated, black bar), and in 7 pigs, plasma was passed through a control cartridge without NETs binder (control, white bar). [Figure 7] The experimental results described in Example 12 and shown in Figure 6, except for the levels in each individual test object. [Figure 8]H3.1-nucleosome levels measured in human subjects diagnosed with sepsis and in healthy human subjects. [Overview of the project]

[0007] (Summary of the invention) According to the first aspect, 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) Monitoring the progression of infection in the subject using any changes at the level of the cell-free nucleosome or its components. A method is provided that includes:

[0008] In a further embodiment, a method for assigning the risk of developing or progressing medical complications 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; and (ii) Using the detected cell-free nucleosome levels, assign the likelihood of medical complications developing or progressing in the subject. A method is provided that includes:

[0009] In a further embodiment, a method for assigning the risk of adverse outcomes to subjects 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; and (ii) Using the detected cell-free nucleosome levels, assign the potential adverse outcomes to the subject. : includes, Here, a method is provided in which subjects identified as having a high potential for adverse outcomes are assigned to medical intervention.

[0010] According to a further aspect, a method of selecting a subject suffering from an infectious disease that requires medical treatment of a medical complication of the 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 indicate the presence, progression, or onset of a medical complication that requires treatment in the subject : A method is provided.

[0011] In a preferred embodiment, the infectious disease is respiratory influenza or coronavirus infection, and the medical complication is ARS, ARDS, or SARS, or pneumonia. Therefore, in one embodiment, a method of detecting a subject who requires medical treatment for pneumonia, ARS, ARDS, or SARS, 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 level of the cell-free nucleosomes as an indicator that the subject requires medical treatment for pneumonia, ARS, ARDS, or SARS : A method is provided.

[0012] In a preferred embodiment, the infectious disease is respiratory influenza or coronavirus infection, and the medical complication is ARS, ARDS, SARS, or pneumonia.

[0013] In another preferred embodiment, the infectious disease is sepsis. Therefore, in one embodiment, 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 binding agent to detect or measure the level of cell-free nucleosomes or components thereof; and (ii) using the level of the cell-free nucleosome as an indicator that the subject requires medical treatment for sepsis or septic shock A method is provided that includes:

[0014] According to a further aspect of the invention, a method of monitoring an infectious disease in a subject, 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 a component thereof; (ii) repeating the detection or measurement of the level of cell-free nucleosomes or a component thereof in the body fluid obtained from the subject on one or more occasions; (iii) using any change in the level of the cell-free nucleosome or a component thereof to monitor the progression of the infectious disease in the subject A method is provided that includes:

BEST MODE FOR CARRYING OUT THE INVENTION

[0015] (Detailed Description) Nucleosomes are released into circulation when chromatin fragments during cell death. Many infections, such as viral infections, initiate cell death through various mechanisms (cell binding and entry, endosomal TLR3 activation, and gene expression), thereby increasing the number of circulating nucleosomes in the blood (Danthi et al., Annu. Rev. Virol. (2016) 3: 533-53). Furthermore, infections can induce NETosis, which promotes post-translational histone modifications such as acetylation or hypercitrullination of histones H3 and H4 (Wang Y et al., J. Cell Biol. (2009) 184(2): 205-213), leading to the decondensation of chromatin, which is released en masse into circulation as the initial response to infection. However, extracellular nucleosomes and neutrophil extracellular traps (NETs) can cause severe complications if not promptly removed. 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), while NETs have been shown to enhance lung injury during viral pneumonia (Ashar et al., Am. J. Pathol. (2018) 188(1): 135-148). In fact, host-targeted NET toxicity is associated with dyspnea, obstruction of narrow airways, endothelial and epithelial cell damage, inflammatory responses, thrombosis, and other pathological conditions (Marcos et al., Nat. Med. (2010) 16: 1018-23; Hoeksema et al., Future Microbiol. (2016) 11: 441-53).

[0016] Most individuals infected with influenza or coronavirus experience only mild illness. However, several population subgroups, including those over 60 years of age, and those with underlying conditions such as diabetes, chronic lung disease, and especially chronic heart disease, are at risk of severe effects, including ARS, SARS, pneumonia, and death. While the exact mechanism by which influenza or coronavirus infection leads to complications, including pneumonia, is unknown, it is thought to be caused by an excessive immune response to the viral infection, where excess NETs contribute to pneumonia and, in the worst cases, acute lung damage leading to death.

[0017] This invention predicts disease severity and outcomes in infectious diseases by using elevated levels of cell-free nucleosomes, including NETs.

[0018] Therefore, according to one aspect, a method of assigning the risk of adverse outcomes to subjects 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; and (ii) Using the detected cell-free nucleosome levels, assign the potential adverse outcomes to the subject. A method is provided which includes: The method can be used to assign subjects identified as having a high potential for adverse outcomes to medical interventions.

[0019] In one embodiment, a method for assigning the risk of adverse outcomes to subjects suffering from infectious diseases, (i) Contacting a body fluid sample obtained from the subject with a binder to detect or measure the level of neutrophil extracellular trapping material or its components; and (ii) Using the detected levels of extracellular neutrophil trapping material, assign the potential for adverse outcomes to the subject. A method is provided that includes:

[0020] Nucleosomes are the basic units of chromatin structure and consist of protein complexes of eight highly conserved core histones (each composed of a pair of histones H2A, H2B, H3, and H4). Approximately 146 base pairs of DNA are wrapped around this complex. Another histone, H1 or H5, acts as a linker and participates in chromatin condensation. The DNA wraps around a sequence of nucleosomes in a structure often described as "beads on a string," which forms the basic structure of open chromatin, or euchromatin. In compressed chromatin, or heterochromatin, these strings form coils and supercoils, resulting in a closed, complex structure (Herranz and Esteller, Methods Mol. Biol. (2007) 361: 25-62).

[0021] When "nucleosome" is detected in a bodily fluid sample, it may refer to a "cell-free nucleosome." Throughout this document, the term "cell-free nucleosome" is intended to include any cell-free chromatin fragment containing one or more nucleosomes.

[0022] It will be understood that cell-free nucleosomes can be detected by binding to their components. As used herein, the term “components” refers to a portion of a nucleosome; i.e., the entire nucleosome does not need to be detected. Components of cell-free nucleosomes may be selected from the group consisting of histone proteins (i.e., histone H1, H2A, H2B, H3, or H4), histone post-translational modifications, histone variants or isoforms, proteins bound to nucleosomes (i.e., nucleosome-protein adducts), DNA fragments associated with nucleosomes, and / or modified nucleotides associated with nucleosomes. For example, the components may be histone (isoform) H3.1 or histone H1 or DNA.

[0023] The methods and uses of the present invention can measure the level of (cell-free) nucleosomes themselves. The reference to “nucleosomes themselves” refers to the total nucleosome level or concentration present in a sample, regardless of whether the nucleosomes contain any epigenetic features or not. Detection of total nucleosome levels typically involves the detection of a histone protein common to all nucleosomes, such as histone H4. Therefore, nucleosomes themselves can be measured by detecting a core histone protein, such as histone H4. As described herein, histone proteins form structural units known as nucleosomes, which are used to package DNA in eukaryotic cells.

[0024] Normal cell turnover in adults involves the daily generation of a vast number of cells through cell division and the death of a similar number, primarily through apoptosis. During apoptosis, chromatin is broken down into mononucleosomes and oligonucleosomes released from the cell. Under normal conditions, levels of circulating nucleosomes in healthy subjects have been reported to be low. Elevated levels are seen in subjects with various conditions, including many cancers, autoimmune diseases, inflammatory conditions, strokes, and myocardial infarctions (Holdenreider and Stieber, Crit. Rev. Clin. Lab. Sci. (2009) 46(1): 1-24).

[0025] Historically, nucleosome ELISA has primarily been used in cell culture as a method for detecting apoptosis (Salgame et al., Nucleic Acids Res. (1997) 25(3): 680-681; Holdenrieder et al. (2001), see above; van Nieuwenhuijze et al., Ann. Rheum. Dis. (2003) 62: 10-14), but it is also used to measure circulating cell-free nucleosomes in serum and plasma (Holdenrieder et al. (2001)). Levels of cell-free nucleosomes released into circulation by dying cells in serum and plasma are measured by ELISA in many different cancer studies to evaluate their use as potential biomarkers.

[0026] Cell-free nucleosomes may be mononucleosomes, oligonucleosomes, components of larger chromatin fragments, or components of NETs, ​​or mixtures thereof.

[0027] Mononucleosomes and oligonucleosomes can be detected by enzyme-linked immunosorbent assay (ELISA), and several methods have been reported (e.g., Salgame et al. (1997); Holdenrieder et al. (2001); van Nieuwenhuijze et al. (2003)). These assays typically utilize anti-histone antibodies (e.g., anti-H2B, anti-H3, or anti-H1, H2A, H2B, H3, and H4) as capture antibodies and anti-DNA or anti-H2A-H2B-DNA complex antibodies as detection antibodies.

[0028] Circulating nucleosomes are not a homogeneous group of protein-nucleic acid complexes. Rather, they are a heterogeneous group of chromatin fragments resulting from the digestion of chromatin during cell death, containing a vast array of epigenetic structures, including specific histone isoforms (or variants), post-translational histone modifications, nucleotides or modified nucleotides, and protein adducts. It will be apparent to those skilled in the art that an increase in nucleosome levels is associated with an increase in certain circulating nucleosome subsets containing specific epigenetic signals, including nucleosomes containing specific histone isoforms (or variants), nucleosomes containing specific post-translational histone modifications, nucleosomes containing specific nucleotides or modified nucleotides, and nucleosomes containing specific protein adducts. Assays of these types of chromatin fragments are known in the art (see, for example, WO 2005 / 019826, WO 2013 / 030579, WO 2013 / 030578, and WO 2013 / 084002, which are incorporated herein by reference).

[0029] Some proteins are generated in NETs that are directly or indirectly attached to nucleosomes. These proteins include, but are not limited to, myeloperoxidase (MPO), neutrophil elastase (NE), lactotransferrin, azulocidine, cathepsin G, leukocyte proteinase 3, lysozyme C, neutrophil defensin 1, neutrophil defensin 3, myeloid cell nuclear differentiation antigen, S100 calcium-binding protein A8, S100 calcium-binding protein A9, S100 calcium-binding protein A12, actin β, actin γ, α-actin, plastin-2, cytokeratin-10, catalase, α-enolase, and transketolase (Urban et al., PLOS Pathogens. (2009) 10: e1000639). Any nucleosome-protein adducts generated in NETs are useful adducts for detecting elevated NET levels in the method of the present invention. C-reactive protein (CRP) can also be added to nucleosomes in NETs, ​​and therefore, nucleosome-CRP adducts are useful adducts for detecting elevated NET levels in the method of the present invention.

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

[0031] In one embodiment, the components of the cell-free nucleosome include the epigenetic characteristics of the cell-free nucleosome.

[0032] The biomarkers used in the method of the present invention may be at the level of the cell-free nucleosome itself and / or epigenetic features of the cell-free nucleosome. The terms “epigenetic signal structure” and “epigenetic features” will be understood to be used interchangeably herein. These refer to specific features of a nucleosome that can be detected. In one embodiment, the epigenetic features of a nucleosome are selected from the group consisting of post-translational histone modifications, histone isoforms, modified nucleotides, and / or proteins bound to the nucleosome in a nucleosome-protein adduct.

[0033] In one embodiment, the epigenetic features of a nucleosome include one or more histone variants or isoforms. The epigenetic features of a cell-free nucleosome may be histone isoforms, for example, histone isoforms of core nucleosomes, in particular histone H3 isoforms. The terms “histone variant” and “histone isoform” may be used interchangeably herein. The structure of a nucleosome may also differ by the inclusion of another histone isoform or variant, which is a different gene or splicing product and has a different amino acid sequence. Many histone isoforms are known in the art. Histone variants can be classified into several families, which are subdivided into individual types. Nucleotide sequences of numerous histone variants are publicly known and are publicly available, for example, in the National Human Genome Research Institute (NHGRI) histone database (Marino-Ramirez et al., "The Histone Database: an integrated resource for histones and histone fold-containing proteins," Database Vol. 2011, and http: / / genome.nhgri.nih.gov / histones / complete.shtml), the GenBank (NIH gene sequence) database, the EMBL nucleotide sequence database, and the DNA Databank of Japan (DDBJ). For example, histone H2 variants include H2A1, H2A2, mH2A1, mH2A2, H2AX, and H2AZ. In another example, histone isoforms of H3 include H3.1, H3.2, H3.3, and H3t.

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

[0035] The structure of nucleosomes can vary due to post-translational modifications (PTMs) of histone proteins. Histone protein PTMs typically occur at the tail of core histones, and common modifications include acetylation, methylation, or ubiquitination of lysine residues, as well as methylation or citrullination of arginine residues, and phosphorylation of serine residues, among many others. Many histone modifications are known in the art, and their number is increasing as new modifications are identified (Zhao and Garcia (2015) Cold Spring Harb Perspect Biol, 7: a025064). Therefore, in one embodiment, the epigenetic features of cell-free nucleosomes may be histone post-translational modifications (PTMs). Histone PTMs may be histone PTMs of core nucleosomes, e.g., H3, H2A, H2B, or H4, and in particular, H3, H2A, or H2B. In particular, histone PTM is histone H3 PTM. Examples of such PTM are described in WO 2005 / 019826.

[0036] For example, post-translational modifications may include methylation (which may be acetylation, monomethylation, dimethylation, or trimethylation), phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamation, and / or isomerization (see Ausio's reference (2001) Biochem Cell Bio 79: 693). In one embodiment, the histone PTM is selected from citrullination or ribosylation. In a further embodiment, the histone PTM is H3 citrulline (H3cit) or H4 citrulline (H4cit). In yet another embodiment, the histone PTM is H3cit.

[0037] In one embodiment, histone PTM is ribosylation, also known as ADP-ribosylation. Posttranslational histone ADP-ribosylation of nucleosomes occupying promoters of macrophage inflammatory response markers is stimulated by exposure to lipopolysaccharides, resulting in increased transcription, and may possess antiviral properties. Furthermore, all members of the coronavirus family contain a highly conserved macrodomain within non-structural protein 3 (nsp3) that regulates posttranslational ADP-ribosylation by enzymatic removal of covalently attached ADP-ribose from protein targets. Recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) strains containing mutated macrodomains with reduced nsp3 de-ADP-ribosylation activity are less infectious and induce early enhanced interferon (IFN), interferon-stimulated gene (ISG), and pro-inflammatory cytokine responses. Therefore, modifying the level of circulating ADP-ribosylated nucleosomes released from macrophages is considered useful in the methods of the present invention.

[0038] It is also possible to detect a group or class of related histone post-translational modifications (rather than a single modification). Typical examples, though not limited to them, include two-site immunoassays that utilize one antibody or other selective binder directed to bind to a nucleosome and another antibody or other selective binder directed to bind to a group of histone modifications of interest. Examples of such antibodies directed to bind to a group of histone modifications, though not limited to them, include, for illustrative purposes, anti-panacetylated antibodies (e.g., panacetyl H4 antibodies [H4panAc]), anti-citrullinated antibodies, or anti-ubiquitin antibodies.

[0039] In one embodiment, the epigenetic features of a nucleosome include one or more DNA modifications. In addition to epigenetic signaling mediated by nucleosome histone isoforms and PTM composition, nucleosomes also differ in their nucleotide and modified nucleotide composition. Some nucleosomes may contain more 5-methylcytosine residues (or 5-hydroxymethylcytosine residues or other nucleotides or modified nucleotides) than other nucleosomes. In one embodiment, the DNA modification is selected from 5-methylcytosine or 5-hydroxymethylcytosine.

[0040] In one embodiment, the epigenetic features of a nucleosome include one or more protein-nucleosome adducts or complexes. A further subset of circulating nucleosomes is nucleosome protein adducts. It has long been known that chromatin contains numerous non-histone proteins bound to its constituent DNA and / or histones. These chromatin-related proteins are of a wide variety of types, possessing diverse functions and including transcription factors, transcription enhancers, transcription repressors, histone regulatory enzymes, DNA damage repair proteins, and many others. These chromatin fragments, including nucleosomes and other non-histone chromatin proteins or DNA and other non-histone chromatin proteins, have been described in the Art.

[0041] In one embodiment, the protein attached to the nucleosome (and therefore potentially used as a biomarker) is selected from transcription factors, high-mobility group proteins, or chromatin regulatory enzymes. The term "transcription factor" refers to a protein that binds to DNA and regulates gene expression by promoting transcription (i.e., activating) or repressing transcription (i.e., repressing). A transcription factor contains one or more DNA-binding domains (DBDs) that bind to specific sequences of DNA adjacent to the gene it regulates. All circulating nucleosomes and nucleosome parts, types, or subgroups described herein may be useful in the present invention.

[0042] It will be understood that multiple epigenetic features of cell-free nucleosomes can be detected in the methods and uses of the present invention. Multiple biomarkers can be used as a combination biomarker. Therefore, in one embodiment, the use includes multiple epigenetic features of cell-free nucleosomes as a combination biomarker. The epigenetic features may be of the same type (e.g., PTMs, histone isoforms, nucleotides, or protein adducts) or of different types (e.g., PTMs combined with histone isoforms). For example, post-translational histone modifications and histone variants can be detected (i.e., multiple types of epigenetic features can be detected). Or, further, multiple types of post-translational histone modifications or multiple types of histone isoforms can be detected. In one embodiment, the use includes post-translational histone modifications and histone isoforms as a combination biomarker in a sample for the diagnosis, detection, treatment, selection, prediction, or monitoring of infectious diseases. In one embodiment, the combination biomarkers are H3.1 and H3cit. As an alternative example, the combined biomarkers are H3.1 and H4cit.

[0043] The term "biomarker" refers to a differential biological or bio-derived indicator of a process, event, or state. Biomarkers can be used in diagnostic methods, such as clinical screening and prognosis assessment, as well as in monitoring treatment outcomes, identifying patients most likely to respond to specific therapeutic interventions, and screening and developing drugs. Biomarkers and their use are beneficial in identifying new drug therapies and discovering new targets for drug treatment.

[0044] Biomarkers are also useful as companion diagnostic products for selecting patients suitable for treatment with specific therapies. The inventors hereby demonstrate that testing circulating nucleosome levels, or levels of nucleosomes containing specific epigenetic signals or structures, is a useful companion product for the therapy of NETs or NETosis-related diseases.

[0045] The present invention relates to a method for assigning patients at risk of adverse outcomes. Adverse outcomes include death and / or acute events requiring emergency medical attention, such as hospitalization (i.e., inpatient treatment) and / or surgery. In many patients, infections are overcome by the person's own immune system without requiring medical intervention. However, in some patients, infections may progress or increase in severity without being overcome by the immune system, or the patient's own immune response to the infection may lead to adverse outcomes. For example, adverse outcomes may include acute coronary or cardiac events (e.g., myocardial infarction and / or stroke), acute multi-organ or single-organ failure (e.g., renal failure, hepatic failure, and / or heart failure), onset of debilitating acute illness, and / or acute respiratory illness (e.g., pneumonia, hypoventilation / bradypnea, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), bronchiolitis, and / or bronchitis). Therefore, in one embodiment, the method described herein assigns patients or subjects at risk of developing an acute respiratory disease. In a further embodiment, the acute respiratory disease is pneumonia. In a further embodiment, the acute respiratory disease is hypoventilation / bradypnea. In yet another embodiment, the acute respiratory disease is acute respiratory distress syndrome (ARDS) and / or severe acute respiratory syndrome (SARS).

[0046] Assigning patients at risk of adverse outcomes may involve assigning immediate or short-term risks, or medium-term risks. Immediate or short-term risks include cases where a patient may develop an adverse outcome within 30 days of symptom onset or confirmed diagnosis, for example, within 2 weeks or 14 days, 1 week or 7 days, or 5 days. Such immediate or short-term risks may also include cases where a patient may develop an adverse outcome within 30 days of implementing the method described herein, for example, within 2 weeks or 14 days, 1 week or 7 days, or 5 days. An example of a short-term risk is the development of NETs-related complications for COVID infection requiring hospitalization. Medium-term risks include cases where a patient may develop an adverse outcome more than 30 days after symptom onset, confirmed diagnosis, and / or implementation of the method described herein. An example of a medium-term risk is the development of so-called long-term COVID, where the effects of COVID infection can last for several months. Therefore, in one embodiment, the method described herein assigns patients or subjects at risk of developing an adverse outcome within 2 weeks or 14 days of symptom onset or confirmed diagnosis. In a further embodiment, the method described herein assigns the risk of developing an adverse outcome within one week or seven days from the onset of symptoms or a definitive diagnosis. In yet another embodiment, the method described herein assigns the risk of developing an adverse outcome within five days from the onset of symptoms or a definitive diagnosis.

[0047] Therefore, in another aspect of the present invention, a method for identifying a subject having an infectious disease requiring hospitalization, (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; and (ii) Using the detected cell-free nucleosome levels, determine whether the subject should be hospitalized for treatment. A method is provided that includes:

[0048] It will be understood that the method of the present invention can also be used to identify patients who do not require hospitalization, that is, to determine whether a subject should be hospitalized for treatment using the detected cell-free nucleosome levels. This aspect of the present invention would be useful in identifying patients who, even if already hospitalized, can be discharged early.

[0049] The methods and uses described herein can be used to test body fluid samples, particularly blood, serum, or plasma samples. Preferably, plasma samples are used. Plasma samples can be collected in a collection tube containing one or more anticoagulants, such as ethylenediaminetetraacetic acid (EDTA), heparin, or sodium citrate, particularly EDTA.

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

[0051] An infectious disease can be defined by the tissue affected by the disease. For example, a disease may affect the heart, brain, kidneys, liver, pancreas, lungs, and / or blood, and an infectious disease may be a bacterial, viral, fungal, or microbial infection that is generally known to affect such tissues or organs. In one embodiment, the infectious disease is a respiratory tract infection. According to this embodiment, the infectious disease affects the lungs, upper and / or lower respiratory tract.

[0052] Other tissues that may be affected by the disease include peripheral tissues such as the limbs, hands, and feet, and the infection may be a bacterial infection (e.g., gangrene). In one embodiment, the infection and / or disease may affect multiple tissues or organs simultaneously. For example, the infection may be a bacterial infection of the limbs, hands, or feet, and the disease may also affect the blood (e.g., sepsis); in one embodiment, the infection is sepsis. In another example, the disease may be heart failure or coronary artery failure, and other tissues or organs affected by the disease may include the kidneys and renal system, and / or the brain (e.g., stroke). In yet another example, the disease may affect the lungs, or the infection may be a respiratory tract infection, and other tissues or organs affected may include the heart, coronary artery system, and / or the brain (e.g., heart failure, myocardial infarction, and / or stroke).

[0053] In one embodiment, circulating nucleosome levels are measured in samples taken from an infected subject to determine the prognosis of the disease. In another embodiment, circulating nucleosome levels are measured in multiple samples taken at intervals from an infected subject to monitor disease progression and / or evaluate the efficacy of treatment.

[0054] In a further embodiment, circulating nucleosome levels are measured in samples taken from subjects suffering from sepsis or septic shock, particularly to assess the prognosis of the disease. Further measurements in a number of samples taken at intervals from subjects suffering from sepsis or septic shock can be performed to monitor disease progression and / or to assess the efficacy of treatment.

[0055] In one embodiment, the respiratory tract infection is selected from influenza, pneumonia, and severe acute respiratory syndrome (SARS). SARS is a respiratory infection caused by SARS coronavirus (SARS-CoV), and related coronaviruses are known (e.g., COVID-19 (also known as SARS-CoV-2, and formerly known as 2019-nCoV)). It is known to cause fever, flu-like symptoms, cough, and malaise, and can lead to pneumonia (e.g., direct viral pneumonia or secondary bacterial pneumonia).

[0056] The emergence of COVID-19 and its rapid progression into a pandemic situation are placing a significant burden on international healthcare services. Infectivity predictions range from 70-80% of a country's population. While most people experience mild symptoms, it appears that a double-digit percentage of those infected may be severely affected.

[0057] Identifying COVID-19-positive individuals at high risk of severe reactions or complications, including pneumonia, enables triage, facilitating the allocation of urgent medical resources, including emergency beds and ventilators, until herd immunity is established, thus protecting communities from future large-scale pandemics. Therefore, in a preferred embodiment, a method for identifying individuals infected with influenza or coronavirus infection requiring medical treatment, (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; and (ii) Using the detected cell-free nucleosome levels, determine whether the subject requires medical treatment. A method is provided that includes:

[0058] (Diagnosis and monitoring methods) In a further embodiment, a method for monitoring the severity of an infectious disease in a subject, (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) Repeated detection or measurement of the level of cell-free nucleosomes or their components in the body fluids obtained from the subject on one or more occasions; (iii) Monitoring the progression of infection in the subject using any changes at the level of the cell-free nucleosome or its components. A method is provided that includes:

[0059] In a further embodiment, a method for monitoring the progression of an infectious disease in a subject who has an infectious disease, is suspected of having an infectious disease, or has a tendency to have a poor prognosis for an infectious disease, (i) A step of contacting a sample obtained from the subject with a binder to detect or measure the level of cell-free nucleosomes; and (ii) A step of monitoring the progression of the infection by comparing the detected cell-free nucleosome levels with earlier samples taken from the subject. A method is provided that includes:

[0060] In a further embodiment, 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) Monitoring the progression of infection in the subject using any changes at the level of the cell-free nucleosome or its components. A method is provided that includes:

[0061] Even if a subject is determined to be free from or have a mild infection, the present invention can still be used to monitor disease progression for the future development of medical complications. For example, if the method includes a sample from a subject determined to have a mild infection, the biomarker level can be repeated at a later time to determine whether the biomarker level has changed.

[0062] Detection and / or quantification can be performed directly on purified or concentrated nucleosome samples, or indirectly on extracts or dilutions thereof from such samples. Quantifying the amount of biomarkers present in a sample may include determining the concentration of biomarkers present in the sample. The detection, monitoring, and diagnostic uses and methods described herein are useful for confirming the presence of a disease, monitoring disease development by assessing onset and progression, or assessing improvement or regression of a disease. The detection, monitoring, and diagnostic uses and methods are also useful in methods for evaluating clinical screening, prognosis, therapy selection, and therapeutic benefit, i.e., for drug screening and development.

[0063] In one embodiment, the disease is a condition characterized by high levels of NETs or NETosis with pathological and clinical complications.

[0064] Detection or measurement may include immunoassays, immunochemistry, mass spectrometry, chromatography, chromatin immunoprecipitation, or biosensor methods. In particular, detection and / or measurement may include two-site immunoassays of nucleosome moieties. Such methods are preferred for measuring nucleosomes or epigenetic features incorporated into nucleosomes in situ, utilizing two anti-nucleosome conjugates or one anti-nucleosome conjugate in combination with an anti-histone modification or anti-histone variant or anti-DNA modification or anti-addition protein detection conjugate. Detection and / or measurement may also include two-site immunoassays utilizing, for example, a combination of labeled or immobilized: anti-nucleosome, anti-histone modification, anti-histone variant / isoform, anti-DNA modification, or anti-addition protein conjugate.

[0065] The inventors herein captured cleaved and uncleaved nucleosomes using a two-site immunoassay for H3.1-nucleosomes, utilizing immobilized anti-histone H3.1 antibodies directed to bind to epitopes around amino acids 30-33 of the histone H3.1 protein, along with labeled anti-nucleosome antibodies directed to bind to epitopes present in intact nucleosomes but not on isolated (free) histone or DNA nucleosome components. This type of epitope requires that the native three-dimensional configuration of the target nucleosome be intact, and therefore may be referred to herein as a “stereostructural nucleosome epitope.”

[0066] The H3R8Cit nucleosome measurements described herein were performed using a two-site immunoassay utilizing immobilized antibodies directed to bind to citrullinated nucleosomes with arginine 8 of histone H3, along with the same labeled anti-nucleosome antibody directed to bind to structurally nucleosomal epitopes.

[0067] In one embodiment, the detection or measurement method includes contacting a body fluid sample with a solid phase containing a binder for detecting cell-free nucleosomes or their components, and detecting binding to the binder.

[0068] In one embodiment, the detection or measurement method includes: (i) contacting a sample with a first binder that binds to the epigenetic features of cell-free nucleosomes; (ii) contacting the sample bound by the first binder in step (i) with a second binder that binds to cell-free nucleosomes; and (iii) detecting or quantifying the binding of the second binder in the sample.

[0069] In another embodiment, the detection or measurement method includes: (i) contacting a sample with a first binder that binds to cell-free nucleosomes; (ii) contacting the sample bound by the first binder in step (i) with a second binder that binds to the epigenetic features of cell-free nucleosomes; and (iii) detecting or quantifying the binding of the second binder in the sample.

[0070] The detection or measurement of biomarker levels can be carried out using one or more reagents, for example, suitable binders. For example, one or more binders may include, optionally combined with one or more interleukins, a desired biomarker, such as a nucleosome or its constituent parts, epigenetic features of a nucleosome, or a ligand or binder specific to a structural / shape mimic of a nucleosome or its constituent parts.

[0071] As used herein, the terms “antibody,” “binder,” or “ligand” are intended to be non-limiting and include any binder capable of binding to a particular molecule or entity, and it will be apparent to those skilled in the art that any suitable binder may be used in the methods of the present invention. It will also be apparent that the term “nucleosome” is intended to include mononucleosomes, oligonucleosomes, NETs, ​​and any protein-DNA chromatin fragments that can be analyzed in a fluid medium.

[0072] Methods for detecting biomarkers are known in the art. The reagent may comprise one or more ligands or binders, e.g., natural compounds or chemically synthesized compounds, capable of specific binding to a desired target. The ligand or binder may comprise peptides, antibodies, or fragments thereof, or synthetic ligands such as plastic antibodies, or aptamers or oligonucleotides, capable of specific binding to a desired target. The antibody may be a monoclonal antibody or a fragment thereof. When using an antibody fragment, it will be understood that it retains the ability to bind to a biomarker so that the biomarker can be detected (according to the present invention). The ligand / binder may be labeled with a detectable marker, e.g., a luminescent, fluorescent, enzyme, or radioactive marker; or, further, the ligand according to the present invention may be labeled with an affinity tag, e.g., biotin, avidin, streptavidin, or His (e.g., hexa-His) tag. Alternatively, ligand binding may be determined using label-free techniques, e.g., ForteBio's label-free techniques.

[0073] As used herein, the terms “detect” or “diagnose” encompass the identification, confirmation, and / or characterization of a disease state. The detection, monitoring, and diagnostic methods according to the present invention are useful for confirming the presence of a disease, for monitoring the development of a disease by assessing its onset and progression, or for assessing the improvement or regression of a disease. The detection, monitoring, and diagnostic methods are also useful for evaluating clinical screening, prognosis, therapy selection, and assessment of therapeutic benefits, i.e., for drug screening and development.

[0074] The method of the present invention may involve standardization of marker levels. For example, the level of cell-free nucleosomes containing a specific epigenetic feature can be standardized against the level of the nucleosome itself (or some other type of nucleosome or parameter), and the level can be expressed as a ratio of nucleosomes containing the feature. For example, to express the level of citrullinated nucleosomes as a ratio of citrullinated nucleosomes.

[0075] In one embodiment, the method described herein is repeated on multiple occasions. This embodiment offers the advantage of enabling monitoring of detection results over a period of time. Such preparations offer the benefit of monitoring or evaluating the efficacy of treatment for a disease state. Using such a monitoring method of the present invention, onset, progression, stabilization, improvement, relapse, and / or remission can be monitored.

[0076] In the monitoring method, the test sample may be collected on two or more occasions. The method may further include comparing the level of biomarkers present in the test sample with one or more controls and / or one or more past test samples collected early, for example, from the same subject before the start of treatment and / or from the same subject at an earlier stage of treatment. The method may include detecting changes in the properties or amounts of biomarkers in test samples collected on different occasions.

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

[0078] Methods for monitoring the efficacy of therapies can be used to monitor the therapeutic effectiveness of existing and new therapies in human subjects and non-human animals (e.g., animal models). These monitoring methods can be incorporated into the screening of new drug substances and substance combinations.

[0079] In a further embodiment, monitoring of more rapid changes due to immediate-acting therapy can be performed at shorter 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 a kit preferably includes one or more ligands for the detection and / or quantification of biomarkers according to the present invention, and / or biosensors and / or arrays described herein, optionally together with instructions for use of the kit.

[0081] A further aspect of the present invention is a kit for detecting the presence of an infectious disease, comprising a biosensor capable of detecting and / or quantifying one or more of the biomarkers defined herein. As used herein, the term “biosensor” means anything capable of detecting the presence of a biomarker. Examples of biosensors are described herein. A biosensor may comprise a ligand binder or ligand described herein, capable of specific binding to a biomarker. Such a biosensor is useful in the detection and / or quantification of the biomarkers of the present invention.

[0082] Preferably, a biosensor for detecting one or more biomarkers combines the recognition of biomolecules with appropriate means for converting the detection of the presence of a biomarker in a sample or the quantification of a biomarker in a sample into a signal. The biosensor can be adapted for "alternative site" diagnostic testing in, for example, hospital wards, outpatient departments, operating rooms, homes, the field, and workplaces. Biosensors for detecting one or more biomarkers according to the present invention include acoustic sensors, plasmon resonance sensors, holographic sensors, biolayer interferometry (BLI) sensors, and microengineering sensors. Imprint recognition elements, thin-film transistor technology, magnetoacoustic resonator devices, and other novel acoustic-electric systems can be utilized in biosensors for detecting one or more biomarkers.

[0083] Biomarkers for detecting the presence of disease are essential targets for the discovery of novel targets and drug molecules that can delay or halt the progression of the disorder. Since the levels of biomarkers indicate disorder and drug response, biomarkers are useful for identifying novel therapeutic compounds in in vitro and / or in vivo assays. The biomarkers described herein can be used in methods for screening compounds that modulate the activity of the biomarkers.

[0084] Accordingly, in a further embodiment of the present invention, the use of the described binder or ligand, which may be a peptide, antibody or fragment thereof, or aptamer or oligonucleotide directed to a biomarker according to the present invention; or the use of a biosensor, array or kit according to the present invention for identifying a substance that can promote and / or inhibit the generation of a biomarker.

[0085] Immunoassays described herein include any method utilizing one or more antibodies or other specific binders directed to bind to biomarkers as defined herein. Immunoassays include two-site immunoassays or immunoassays utilizing enzyme detection methods (e.g., ELISA), fluorescently labeled immunoassays, time-resolved fluorescently labeled immunoassays, chemiluminescent immunoassays, immunoturbidimetric assays, particulate-labeled immunoassays, and immunoradioanalysis assays, as well as single-site immunoassays, reagent-limited immunoassays, competitive immunoassays including labeled antigens and labeled antibodies, and single-antibody immunoassays using various labeling types, including radioactive, enzyme, fluorescent, time-resolved fluorescence, and particulate labeling. All of these immunoassays are well known in the art; see, for example, Salgame et al. (1997) and van Nieuwenhuijze et al. (2003).

[0086] Identification, detection, and / or quantification can be carried out by any method suitable for identifying the presence and / or amount of a specific protein in a biological sample derived from the subject, or in a purified or extracted product or dilution thereof of the biological sample. In particular, quantification can be carried out by measuring the concentration of the target in one or more samples. Biological samples that can be examined in the method of the present invention include the biological samples defined above herein. Samples can be prepared by conventional methods, and for example, diluted or concentrated as appropriate, and stored. The present invention is particularly used in plasma samples that can be obtained from the subject.

[0087] Identification, detection, and / or quantification of biomarkers can be carried out by detecting the biomarker or a fragment thereof, for example, a fragment having a C-terminal or N-terminal cleavage. The fragments are preferably longer than 4 amino acids, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. It should be noted that peptides with sequences identical to or related to the histone tail sequence are particularly useful histone protein fragments.

[0088] For example, detection and / or quantification can be performed by one or more methods selected from the group consisting of immunoassays, immunochromatography, SELDI(-TOF), MALDI(-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 or LC-MS-based techniques. Suitable LC-MS techniques include ICAT® (Applied Biosystems, CA, USA) or iTRAQ® (Applied Biosystems, CA, USA). Liquid chromatography (e.g., 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] A method for detecting and / or quantifying one or more biomarkers according to the present invention can be carried out on a benchtop device or incorporated into a disposable diagnostic or monitoring platform that can be used in an out-of-laboratory environment, e.g., a physician's office or at the bedside of a subject. A suitable biosensor for carrying out the method of the present invention is a “credit” card equipped with an optical or acoustic reader. The biosensor is configured to enable electronic transmission of the collected data to a physician for interpretation, and thus can form the basis of e-medicine. Therefore, in a further embodiment of the present invention, the use of a bedside or point-of-care immunoassay method for measuring biomarkers according to the present invention is provided. In one embodiment, the bedside immunoassay method includes a point-of-care immunoassay device (e.g., Abbott i-STAT or LightDeck Diagnostics point-of-care immunoassay device). In one embodiment, the bedside immunoassay method includes a lateral flow test. In a preferred embodiment, the biomarker is a nucleosome or a nucleosome containing epigenetic features.

[0090] Identifying biomarkers of disease status enables the integration of diagnostic procedures and treatment regimens. Biomarkers provide a means of indicating treatment response, response failure, undesirable side effect profiles, degree of medication compliance, and achievement of appropriate serum drug levels. Biomarkers can be used to provide warnings of adverse drug responses. Biomarkers are useful in the development of personalized therapies because evaluation of response allows for fine-tuning of dosage, minimizing the number of prescribed medications, reducing delays in achieving effective therapy, and avoiding adverse drug reactions. Therefore, by monitoring the biomarkers of this invention, care for a subject can be precisely tailored to the needs determined by the subject's disability and pharmacological profile, and thus, biomarkers can be used to adjust optimal doses, predict positive treatment responses, and identify subjects at high risk of severe side effects.

[0091] Biomarker-based testing provides first-line assessment for "new" subjects and offers an objective means for accurate and rapid diagnosis that is not achievable with current methods.

[0092] Biomarker monitoring methods, biosensors, point-of-care tests, lateral flow tests, and kits are also important as targeted monitoring tools to enable physicians to determine whether a relapse is due to an exacerbation of the disability. If pharmacological treatment is deemed inadequate, therapy can be reinstated or increased; if appropriate, a change in therapy can be made. Because biomarkers are sensitive to the state of the disability, they provide an indicator of the impact of drug therapy.

[0093] References to "subject" or "patient" are used interchangeably herein. A subject may be a human or an animal subject. In one embodiment, the subject is human. In one embodiment, the subject is a (non-human) animal. The panels and methods described herein may be carried out in vitro or ex vivo.

[0094] Detection and / or quantification can be compared to a cutoff level. The cutoff value can be predetermined by analyzing results from multiple patients and controls to determine a suitable value for classifying subjects as having or not having the disease. For example, for diseases where biomarker levels are higher in patients with the disease, a detected level higher than the cutoff indicates that the patient has the disease. Alternatively, for diseases where biomarker levels are lower in patients with the disease, a detected level lower than the cutoff indicates that the patient has the disease. Advantages of using a simple cutoff value include the ease with which clinicians can understand the test and the elimination of the need for software or other aids in interpreting the test results. The cutoff level can be determined using methods available in the art.

[0095] Detection and / or quantification may also be compared with controls. For example, it will be apparent to those skilled in the art that control subjects may be selected according to various criteria, which may include subjects known to be disease-free or subjects with different diseases (e.g., for differential diagnosis studies). "Controls" may include healthy subjects, unaffected subjects, and / or subjects without infection. Controls may also include subjects with infections exhibiting asymptomatic or mild symptoms, for example, subjects infected with respiratory viruses exhibiting asymptomatic or mild symptoms. Mild symptoms may include manageable symptoms that do not require hospital intervention and / or intensive medical treatment.

[0096] In one embodiment, a subject who tests positive by the method of the present invention may be infected with a viral disease and may develop further medical complications or subsequently develop further medical complications. In contrast, a control subject may also be infected with a viral disease but will not develop medical complications or subsequently develop medical complications. Comparison with controls is well known in the field of diagnosis. The range of values ​​observed in the control group can be used as a normal, healthy, or reference range for comparison with the values ​​observed in the test subject. For example, if the reference range is less than 10 units, a test value of 5 units is considered normal or does not require treatment, while a value of 11 units is considered abnormal and indicates the need for treatment.

[0097] Therefore, in one embodiment, the method further includes comparing the level of cell-free nucleosomes or their components in a body fluid sample of a subject with one or more controls. For example, the method may include comparing the level of cell-free nucleosomes present in a sample obtained from a subject with the level of cell-free nucleosomes present in a sample obtained from a normal subject. The controls may be healthy subjects.

[0098] In one embodiment, cell-free nucleosome or its component levels are elevated compared to the control.

[0099] It will be understood that in all cases it is not necessary to measure control levels for comparative purposes. For example, once a “normal range” is established for healthy / uninfected controls, it can be used as the standard for all subsequent tests. The normal range can be established by obtaining samples from multiple control subjects without infection and testing for biomarker levels. The results (i.e., biomarker levels) of subjects suspected of having infection can then be examined to determine whether they fall within or outside their respective normal ranges. The use of “normal ranges” is standard practice for disease detection.

[0100] In one embodiment, the method further includes determining at least one clinical parameter of the patient. This parameter can be used in interpreting the results. Clinical parameters may include any relevant clinical information, such as, but are not limited to, body temperature, sex, weight, body mass index (BMI), smoking status, and dietary habits. Therefore, in one embodiment, the clinical parameter is selected from the group consisting of body temperature, age, sex, and body mass index (BMI).

[0101] In one embodiment, the method of the present invention is performed to identify subjects at high risk of developing a severe reaction to an infection, and therefore, subjects requiring medical intervention. Such medical intervention may include one or more of the therapies described herein.

[0102] According to another aspect of the present invention, a method for assigning the risk of adverse outcomes to subjects 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; and (ii) Using the detected cell-free nucleosome levels, assign the potential adverse outcome to the subject. The use of a binder in the manufacture of a kit for use in a method including the following is provided.

[0103] A further aspect of the present invention relates to a method for detecting a person who requires medical treatment for pneumonia, acute respiratory syndrome (ARS), or severe acute respiratory syndrome (SARS), (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; and (ii) Using the cell-free nucleosome level as an indicator that the subject requires medical treatment for pneumonia, ARS, or SARS. The use of a binder in the manufacture of a kit for use in a method including the following is provided.

[0104] A further aspect of the present invention provides a method for detecting a subject who requires medical treatment for sepsis or septic shock, (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; and (ii) Using the cell-free nucleosome level 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 including the following is provided.

[0105] A further aspect of the present invention relates to a method for detecting a person who requires medical treatment for pneumonia, acute respiratory syndrome (ARS), or severe acute respiratory syndrome (SARS), (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; and (ii) Using the cell-free nucleosome level as an indicator that the subject requires medical treatment for pneumonia, ARS, or SARS. A method is provided that includes:

[0106] A further aspect of the present invention provides a method for detecting a subject who requires medical treatment for sepsis or septic shock, (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; and (ii) Using the cell-free nucleosome level as an indicator that the subject requires medical treatment for sepsis or septic shock. A method is provided that includes:

[0107] (Further biomarkers) Cell-free nucleosome levels can be detected or measured as one of the measurement panels. The panel may include different epigenetic features of nucleosomes described above (e.g., histone isoforms and PTMs). Useful biomarkers in panel testing for the detection of severe respiratory infections requiring medical intervention include, but are not limited to, cytokine moieties (particularly interleukins), C-reactive proteins, myeloperoxidases, D-dimers, factor VII-activated proteases (FSAPs), fibrinogens, and fibrin / fibrinogen degradation products. In one embodiment, the panel includes C-reactive proteins. In one embodiment, the panel includes one or more cytokines, for example, one or more interleukins.

[0108] Interleukins (ILs) are a group of cytokines, usually secreted by white blood cells, that act as signaling molecules. Interleukins play an important role in stimulating immune responses and inflammation. Interleukins were first identified in the 1970s, and as more interleukin species have been discovered, they have been given numerical names. Examples of interleukins include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15.

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

[0110] The interleukin may be IL-6. Interleukin-6 (IL-6) is a cytokine with a wide variety of biological functions. 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 described in UniProt accession number P05231. In one particular embodiment, the interleukin may be IL-6.

[0111] Alternatively, or further, the interleukin may be IL-12. Interleukin-12 (IL-12) is a T cell stimulator because it stimulates the proliferation and function of T cells. It is a heterodimeric cytokine composed of IL-12A and IL-12B. The sequence of human IL-12A is publicly known in the art and is described in UniProt accession number P29459, and the sequence of human IL-12B is also publicly known and is described in UniProt accession number P29460. In one particular embodiment, the interleukin may be IL-12.

[0112] In one embodiment, the panel includes cell-free nucleosomes or their epigenetic features and interleukins. In another embodiment, the panel includes cell-free nucleosome epigenetic features and two interleukins. For example, cell-free nucleosome measurement can be combined with the measurement of multiple interleukins such as IL-6 and IL-12. In a further embodiment, the cell-free nucleosome epigenetic features are selected from histone isoforms such as H3.1 and post-translational modified histones such as H3cit. In yet another embodiment, the measurement panel is H3.1, H3cit, H4cit, and IL-6.

[0113] In one embodiment, the panel includes C-reactive protein (CRP). CRP is a pentameric protein found in plasma, and levels of CRP (regardless of whether it is attached to a nucleosome) increase in the plasma in response to inflammation, for example, in bacterial, viral, fungal, and microbial infections. CRP levels increase after IL-6 secretion by macrophages and T cells, and its physiological role is to bind to lysophosphatidylcholine expressed on the surface of dead or dying cells in order to activate the complement system via C1q. It also binds to phosphocholine on the surface of some bacteria, enhancing phagocytosis. Measuring CRP levels is useful in determining disease progression and the effectiveness of treatment, and elevated CRP levels have been shown in patients with diabetes, hypertension, and an increased risk of cardiovascular disease. Elevated CRP levels are also seen in patients with renal failure and inflammatory bowel disease (IBD, including Crohn's disease and ulcerative colitis), and generally correlate with coronary heart disease. However, when elevated CRP is not directly associated with heart disease, it is not a specific prognostic marker. Since CRP increases during inflammation, viral infections such as SARS or coronaviruses (e.g., COVID-19) can also lead to elevated plasma CRP levels.

[0114] In one embodiment, the panel includes myeloperoxidase (MPO). MPO is expressed in neutrophil granulocytes and performs its antimicrobial activity by producing hypohalite. This is stored in azurophilic granules and released into the extracellular space upon degranulation. MPO levels are known to be a useful predictor of myocardial infarction and are combined with CRP measurement to increase the accuracy of predicting a patient's risk of myocardial infarction. In one embodiment, the panel includes neutrophil elastase (NE).

[0115] Models can be derived using the biomarkers of the present invention. Methods for deriving models or algorithms are well known in the art, and suitable software packages are available. Typical software tools for this purpose include SPSS (Statistical Package for Social Sciences) and "R". These software packages provide linear and nonlinear data modeling for clinical data.

[0116] It will be apparent to those skilled in the art that any combination of the biomarkers disclosed herein can be used in panels and algorithms for the detection or prediction of complications of infectious diseases, and that additional markers can be added to panels containing these markers.

[0117] According to aspects of the present invention, a use is provided for a panel test for detecting or predicting complications of an infectious disease in a patient, wherein the panel test comprises reagents for detecting nucleosomes or their components and one or more interleukins in a sample obtained from the patient. In one embodiment, the complications are NETs-related complications. In one embodiment, the complications are 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; (ii) Using the levels measured in step (i) to indicate the presence and / or severity and / or medical complications of the infection in the subject; and (iii) If the subject is determined to have a severe infection or medical complication in process (ii), administer therapy. A method is provided that includes this.

[0119] In a further embodiment, a method is provided for treating an infection in a subject in need thereof, comprising the step of administering a therapy (e.g., a therapeutic agent) to a subject that has been identified to have varying levels of cell-free nucleosomes in a sample obtained from the subject, compared to the level of cell-free nucleosomes in a sample obtained from a control subject. The therapy may include, but is not limited to, one or more suitable treatments for the disease, including drugs (e.g., anti-inflammatory drugs, anticoagulants, therapeutic anti-NETs antibody drugs, DNase drugs, NETosis inhibitors, antibacterial drugs, or antiviral drugs), apheresis, mechanical ventilation, fluid support, or others.

[0120] In one embodiment, the treatment includes antibiotic therapy (e.g., penicillin, cephalosporin, tetracycline, aminoglycoside, macrolide, clindamycin, sulfonamide, trimethoprim, metronidazole, tinidazole, quinolone, and / or nitrofurantoin), antimicrobial therapy (e.g., ethambutol, isoniazid, pyrazinamide, rifampicin, aminoglycoside (amikacin, kanamycin), polypeptide (capreomycin, biomycin, enviomycin), fluoroquinolone (ciprofloxacin, levofloxacin, moxifloxacin), thioamide (ethionamide, prothionamide), cycloserine (croserine), terizidone, rifabutin, macrolide (clarithromycin), linezolid) One or more of the following are selected: thioacetazone, thioridazine, arginine, vitamin D and / or R207910), antiviral treatment for COVID-19 (e.g., remdesivir), antiviral treatment for influenza (e.g., amantadine, umifenovir, moloxidine, rimantadine, umifenovir, zanamivir and neuraminidase inhibitors, cap-dependent endonuclease inhibitors, adamantane, peramivir, zanamivir, oseltamivir phosphate, and baloxavir marboxil), and antiviral treatment for other viral diseases that may result in high levels of NETosis, as well as antifungal treatment (e.g., clotrimazole, econazole, miconazole, terbinafine, fluconazole, ketoconazole, and amphotericin).

[0121] In one embodiment, the treatment is an anti-inflammatory drug. Many steroidal and non-steroidal anti-inflammatory drugs are known in the art. Some examples of steroidal anti-inflammatory drugs include, but are not limited to, dexamethasone, hydrocortisone, cortisone, betamethasone, prednisone, prednisolone, triamcinolone, and methylprednisolone. Some examples of non-steroidal anti-inflammatory drugs include, but are not limited to, aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, CD24Fc (CD24 protein bound to the Fc region of immunoglobulin G), and EXO-CD24 (CD24 exosome).

[0122] In one embodiment, the treatment is DNase therapy to digest excess NETs, ​​or an inhibitor of NETosis, such as an anthracycline drug. In a further embodiment, the anthracycline drug is selected from epirubicin, daunorubicin, doxorubicin, and idarubicin.

[0123] In one embodiment, the treatment is a therapeutic antibody directed to bind to NETs or constituent parts of NETs, ​​or, but not limited to, a therapeutic antibody directed to bind to nucleosomes or any constituent part of nucleosomes. Examples include therapeutic antibodies directed to bind to nucleosomes containing histone isoform H3.1, citrullinated histones, myeloperoxidase, neutrophil elastase, or C-reactive proteins.

[0124] In one embodiment, the treatment is a nucleic acid scavenger, such as a DNA scavenger polyamidoamine, that adsorbs and / or removes nucleic acids from circulation or from the body.

[0125] No treatments inhibiting NETosis have been used, or to the best of our knowledge, have been tested for use in humans to date. There are several reasons for this. Firstly, as mentioned above, NETosis is a vital component of the immune system. This is especially important in patients who may not yet have developed an antibody response to infectious pathogens and in patients where the NETosis process, along with phagocytosis, may be the primary mode of combating infection and preventing its spread. Furthermore, this is even more important in the case of NETosis inhibitor therapy, because it does not remove the products of NETosis (which can be replaced), but rather disables the immune process itself, preventing further production of NETs. Secondly, while the half-life of intravenously administered DNases has been reported to be 3-4 hours, the half-life of anthracyclines exceeds 40 hours, and anthracyclines remain in circulation for at least several days. Disabling the immune system for several days in patients with severe infections would be enough time for a significant worsening of the disease. Thirdly, anthracyclines are cytotoxic drugs commonly used to treat cancer by killing cancer cells. It is clear that drugs that disable one of the key mechanisms of action of the immune system, and that are long-lasting and cytotoxic, should not be administered to subjects with diseases that are not NETosis-related or who do not require such treatment. Therefore, the treatment of patients with normal or low NETs with drugs that inhibit NETosis is inappropriate and potentially dangerous. Thus, the administration of such therapies requires careful selection of patients with high NETs levels who require treatment using the methods described herein.

[0126] Therefore, in one embodiment of the present invention, a combination product is provided comprising a drug for the treatment of NETosis-related diseases and a companion diagnostic test for the detection or measurement of cell-free nucleosomes. In some embodiments, the combination product may comprise several drugs (e.g., NETosis inhibitors and cardioprotective agents and / or antibiotics) and / or several companion tests (e.g., cell-free nucleosome testing and cytokine fraction testing). In one embodiment, the combination product comprises a DNase drug and a companion diagnostic test for the detection or measurement of cell-free nucleosomes.

[0127] In one embodiment, the combination product includes a drug that digests NETs, ​​for example, a DNase drug that digests NETs composed of longer nucleosome chains into smaller fragments containing shorter oligonucleosome chains or mononucleosomes, and a companion diagnostic test for the detection or measurement of cell-free nucleosomes.

[0128] In one embodiment, the combined product includes a nucleic acid scavenger and a companion diagnostic test for the detection or measurement of cell-free nucleosomes.

[0129] In one embodiment, the combination product includes a therapeutic antibody agent directed to bind to NETs, ​​constituent parts of NETs, ​​MPO, NE, or CRP, and a companion diagnostic test for the detection or measurement of cell-free nucleosomes.

[0130] In one embodiment, the combination product includes a drug therapy that inhibits or interferes with NETs formation by neutrophil cells and a companion diagnostic test for the detection or measurement of cell-free nucleosomes. Drugs that inhibit or interfere with NETs formation are not currently used to treat NETosis-related diseases. Anthracycline drugs were reported to be effective in treating sepsis induced in mice by Figueiredo et al., Immunity (2013) 39: 874-884. Lethal sepsis was induced in mice by cecal ligation and puncture, resulting in death in 100% of mice within 72 hours. Treatment with the anthracycline drug epirubicin resulted in a 75% survival rate and reduced inflammation, as measured by various circulating inflammation biomarkers. However, the mechanism of action of anthracycline in achieving this effect could not be elucidated, and Figueiredo said nothing about NETs or NETosis. Later, Khan et al., Cancers (2019) 11: 1328, investigated the effects of anthracycline drugs on neutrophil cells in cell culture in vitro and reported that they inhibited NETosis. Khan concluded that anthracyclines and other DNA chelating agents could be considered potential therapeutic agents for suppressing unwanted NETosis in NETs-related diseases. However, since the main toxic side effect of anthracyclines is cardiotoxicity, Khan proposed that anthracyclines could be administered in combination with dexrazoxane, a cardioprotective agent used to limit the side effects of anthracyclines, which does not affect NETosis or the ability of anthracyclines to suppress NETosis.

[0131] Anthracycline drugs are extremely effective chemotoxic agents for cancer treatment. However, it has several side effects, including alopecia, skin rash, nausea and vomiting, discomfort, fever, peripheral neurotoxicity, secondary leukemia, and cardiotoxicity. Cardiomyotoxicity is dose-limiting because it can potentially lead to life-threatening congestive heart failure (CHF) during or several months to years after treatment. For example, the probability of developing cardiomyopathy based on a composite index of signs, symptoms, and reduction in left ventricular ejection fraction is 1-2% at a total cumulative dose of doxorubicin of 300 mg / m 2 ², 3-5% at a dose of 400 mg / m 2 ², 5-8% at 450 mg / m 2 ², and 6-20% at 500 mg / m 2 ². The risk of developing CHF increases rapidly when the total cumulative dose of doxorubicin exceeds 400 mg / m 2 ². Typical doses used in chemotherapy are 60-75 mg / m 2 per treatment cycle.

[0132] The dose of anthracycline observed to be useful in the treatment of a mouse model of sepsis is 0.6 μg / g (in the literature of Figueiredo et al.), which the inventors have clarified corresponds to approximately 25 mg / m 2 ² in an 80 kg human male. Therefore, a single intravenous dose is less than 1 / 10 of the dose that causes 1-2% cardiotoxicity. The concentration of anthracycline drug required for almost complete inhibition of neutrophil cell NETosis in vitro is 5 μM (in the literature of Khan et al.), which corresponds to approximately 3 μg / ml. Studies on the pharmacokinetic properties of doxorubicin have shown that a single 25 mg / m 2 bolus intravenous dose produces a serum concentration of 10 μg / ml serum. Furthermore, the half-life of doxorubicin in circulation is approximately 41 hours, and the serum level remains above 3 μg / ml for approximately 4 days. Therefore, a single 25 mg / m 2The bolus dose inhibits NETosis for several days and can be used as a treatment for sepsis or other NETosis-related disorders. 25 mg / m² 2 Single bolus dose levels or repeated cumulative dose levels below a certain level may also be effective.

[0133] Nanoparticle anthracycline drug formulations have been reported to offer a targeted NETosis inhibitory approach, in which the active drug is released only in activated neutrophils, thereby avoiding toxic side effects while preserving the NETosis ability of remaining neutrophils to respond to further infection (Zhang et al., Science Advances 2019;5: eaax7964). Therefore, in one embodiment, a combination product is provided comprising 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] A therapy involving the removal of NETs and NET degradation products from circulation by plasmapheresis has been reported to provide a treatment for NETosis that avoids pharmacotoxic side effects (WO2019053243). Therefore, in one embodiment, a combination product is provided that includes plasmapheresis therapy for the treatment of NETosis-related diseases and a companion diagnostic test for the detection or measurement of cell-free nucleosomes.

[0135] Anti-inflammatory therapies based on the CD24 protein have been reported for subjects suffering from COVID-19. One example is CD24-exosomes, which have been reported to cure 29 out of 30 moderate / severe COVID cases within a few days (Times of Israel 5 Feb 2021 and ClinicalTrials.gov Identifier: NCT04747574), and involve the delivery of CD24 in exosomes. Another such therapy, CD24Fc, contains a non-polymorphic region of CD24 bound to the fragment crystallizable region (Fc) of human IgG1 (ClinicalTrials.gov Identifier: NCT04317040). Thus, in one embodiment, a combination product is provided comprising plasmapheresis therapy for the treatment of NETosis-related disorders and a companion diagnostic test for the detection or measurement of cell-free nucleosomes.

[0136] NETs or NETosis-related diseases include infectious diseases such as sepsis, pneumonia, COVID, and influenza, as well as, but are not limited to, NETosis-related complications of other diseases including pneumonia, SARS or ARDS of any cause, thrombotic or microthrombotic conditions, many inflammatory disease conditions, and amputation, as well as thrombotic complications of diabetes and cancer, and many other diseases that involve pathological increases in NETs production.

[0137] This method, (i) Measure the level of cell-free nucleosomes in the sample obtained from the subject (optionally in combination with the level of one or more interleukins); (ii) Identifying the subject as having a NETosis-related disease (e.g., an infection) requiring treatment based on a higher level of cell-free nucleosomes compared to the control; and (iii) Administering the treatment to the subject It may include a colon (:).

[0138] In preferred embodiments, the treatment is DNase therapy for digesting excess NETs, ​​anti-nucleosome or anti-NETs therapeutic antibody therapy, apheresis or plasmapheresis therapy for removing excess NETs, ​​or an inhibitor of NETosis as described herein.

[0139] In one embodiment, a method for identifying a subject suffering from an infectious disease who has a medical complication requiring treatment or is at risk of developing such a medical complication, (i) A step of measuring the level of cell-free nucleosomes in the sample obtained from the subject (optionally in combination with the level of one or more interleukins); (ii) The step of identifying the subject as having an infection requiring treatment based on a higher level of cell-free nucleosomes compared to a control; 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, a method for identifying a subject infected with a respiratory virus who has a medical complication requiring treatment or is at risk of developing such a medical complication, (i) A step of measuring the level of cell-free nucleosomes in the sample obtained from the subject (optionally in combination with the level of one or more interleukins); (ii) The step of identifying the subject as being at risk of developing medical complications based on a higher level of cell-free nucleosomes compared to the control; 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, and the medical complication is pneumonia. Preferred treatments include, but are not limited to, respiratory support using extracorporeal oxygen supply, respiratory support using a medical ventilator designed to provide artificial respiration of air into and out of the lungs of patients who are physically unable to breathe adequately without assistance, and / or the provision of oxygen and / or antiviral, antifungal, or anti-inflammatory drugs.

[0143] According to another aspect of the present invention, a method for treating an infectious disease is provided, comprising using a panel test to identify a patient in need of treatment for the infectious disease and providing the treatment, wherein the panel test comprises reagents for detecting the measurement of nucleosomes or their components. Patients with the infectious disease are thought to have higher levels of cell-free nucleosomes compared to a control.

[0144] (therapeutic antibody) Therapeutic antibodies can be administered intravenously to neutralize the portion of a target that causes damage or disease. Therapeutic antibodies, as well as other similar or derived therapeutic binders such as Fab and Fv fragments, are typically human or humanized in nature of the amino acid sequences of their heavy and light chains. Therapeutic antibodies and methods for their development and production are well known in the art. Therefore, in a further embodiment of the present invention, an anti-nucleosomal antibody for the treatment of severe hyperimmune reactions, including pneumonia, is provided.

[0145] Therefore, in a further embodiment, a method for treating a subject infected with a respiratory virus having medical complications, (i) A step of measuring the level of cell-free nucleosomes in the sample obtained from the subject (optionally in combination with the level of one or more interleukins); (ii) The step of identifying the subject as having a medical complication based on a higher level of cell-free nucleosomes compared to a control; and (iii) The step of administering a therapeutic anti-nucleosome antibody to the subject. A method is provided that includes:

[0146] The prevention or inhibition of NETosis through treatment with NETosis inhibitors reduces the levels of NETs and nucleosomes in the circulation and / or tissues of subjects suffering from diseases with inadequately high levels of NETs. This has been shown to improve clinical outcomes in subjects suffering from NETosis-related disease conditions such as sepsis or stroke (Figueiredo et al., Immunity (2013) 39: 874-884 and Zhang et al., Science Advances (2019) 5: eaax7964). Similarly, the removal of NETs and nucleosomes from the circulation and / or tissues of subjects suffering from NETosis-related disease conditions leads to improved clinical outcomes.

[0147] The immunoassays described herein for the measurement of H3.1-nucleosomes, citrullinated nucleosomes, MPO, and NE utilize monoclonal antibodies with high binding affinity and specificity to nucleosomes and NETs. These antibodies strongly and specifically bind to NETs, ​​NET metabolites, and nucleosomes. Therefore, these antibodies can be used in vivo as therapeutic antibodies to bind to NETs, ​​neutralize them, and promote their clearance from the body, for example, by phagocytosis (Weiskopf and Weissman, Mabs (2015) 7:303-10).

[0148] CRP is an acute-phase protein known to be physically associated with NETs, ​​which can further induce NETosis. Therefore, antibodies against CRP can neutralize and remove NETs, ​​and neutralization and clearance of CRP can inhibit the induction of NETosis.

[0149] Therefore, in one aspect of the present invention, a method for treating NETosis-related diseases is provided, comprising the administration of a therapeutic antibody directed to bind to nucleosomes or their components, DNA, myeloperoxidase, neutrophil elastase, or C-reactive proteins. The therapeutic antibody can neutralize NETs derived from a diseased subject or promote the clearance of such NETs. Methods for administering therapeutic antibodies are well known in the art.

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

[0151] The anti-histone H3.1 antibody, anti-nucleosome antibody, and anti-citrullinated H3 antibody used by the inventors for the nucleosome assays described herein have been selected as highly avid and specific antibodies, and are therefore particularly useful as therapeutic antibodies.

[0152] In particular, anti-histone H3.1 antibodies can be highly specific. Nucleosomes undergo cleavage in which the histone tail is physically and irreversibly removed by regulatory proteolysis or cleavage. Furthermore, histone degradation has been shown to be involved in the formation of NETs (see Papayannopoulos et al. (2010) J. Cell Biol. 191(3): 677-691). Regarding histone H3, cleavage has been reported to occur around amino acid position 21 (Yi and Kim (2018) BMB 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 generally does not contain post-translationally modified amino acids (e.g., lysine, serine, or arginine). Therefore, an antibody directed to bind to this epitope (i.e., amino acid positions 29-35) is unaffected, or minimally affected, by the post-translational modification status of the nucleosome and binds to all or most nucleosomes containing histone H3.1, regardless of the PTM structure. Accordingly, the solid-phase capture antibody selected by the inventors for use in the immunoassays described herein was an anti-histone H3.1 antibody directed to bind to an epitope located inside the histone core near amino acid positions 30-33, so that both intact and cleaved nucleosomes are captured by the antibody, regardless of their PTM status. This maximizes the capture of H3.1-nucleosomes, and the effectiveness of this approach is evident in Figure 6. The amino acid sequence of histone H3.1 is publicly known in the art and is described in UniProt accession number P68431.

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

[0154] The labeled antibodies used herein for immunoassays were anti-nucleosome antibodies directed to bind to structural nucleosome epitopes present in intact nucleosomes containing a histone octamer core complexed with DNA. These antibodies do not bind (or bind weakly) to free histone octamer complexes, free histones (i.e., those without DNA), free DNA, or free histones. Even in this case, the antibodies may be relatively unaffected by the histone PTM composition of the nucleosome to which they bind.

[0155] Therefore, in one embodiment of the present invention, the therapeutic antibody is directed to bind to a three-dimensional nucleosome epitope present in an intact nucleosome containing a histone octameric core that has formed a complex with DNA.

[0156] In one embodiment of the present invention, a therapeutic antibody is directed to selectively bind to a cleaved nucleosome by binding to an epitope present in the cleaved nucleosome, for example, in which one or more histone tails have been removed. In this embodiment, the epitope may be pre-hidden in the intact nucleosome (and thus prevent antibody binding) by the presence of a complete histone tail. Therefore, an epitope bound by an antibody selective for cleaved nucleosomes may be an inaccessible epitope in the intact (i.e., complete or uncleaved) nucleosome. In one embodiment, the cleaved nucleosome comprises histone H3, H2A, and / or H4 proteins from which histone tails have been removed.

[0157] In one embodiment, a mixture of two or more therapeutic antibodies can be administered to a target. For example, but not limited to, a mixture of one antibody directed to bind to a nucleosomal epitope present in an intact nucleosome and another antibody directed to bind to a core histone epitope of histone H3.1.

[0158] The antibodies used by the inventors for immunoassays are mouse monoclonal antibodies. These mouse monoclonal antibodies would be useful therapeutic antibodies in mice. (Since monoclonal antibodies are exogenous proteins) their use in humans or other animals results in an antigenic immune response and the production of anti-mouse immunoglobulin antibodies. To avoid an antigenic response, non-human monoclonal antibodies may be humanized. Humanized antibodies are non-human antibodies whose protein composition is modified to resemble that of naturally occurring human antibodies, and are well known in the art. Antibodies consist of a variable domain that is unique to the antibody and contains a complementarity-determining region (CDR) that determines the epitope binding specificity and binding affinity of the antibody, and a species-specific constant domain. One method of humanization involves the fusion of DNA encoding the variable CDR of a non-human antibody with DNA encoding the human constant domain. Using this method, it is possible to produce a DNA vector encoding a predominantly human antibody that is not antigenic to human targets and has the binding specificity and binding affinity of the original non-human monoclonal antibody. Humanized antibodies can be manufactured on a large scale using the resulting DNA vector.

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

[0160] It will be understood that the embodiments described herein may apply to all aspects of the present invention, that is, embodiments described for use may apply equally to the claimed methods, etc.

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

[0162] (Example 1) The inventors demonstrated that heparin addition induces NET formation in leukocytes in fresh healthy whole blood samples, followed by detection of NETs produced in plasma (Lelliott et al., International Immunology (2019) pii: dxz084). The inventors collected whole blood samples from two healthy volunteers, expected to contain low levels of circulating NETs, ​​in EDTA plasma blood collection tubes and heparin plasma blood collection tubes. The two EDTA plasma blood collection tubes were immediately centrifuged to separate the cell fraction from the plasma fraction, minimizing contamination with large chromatin (containing NETs), and the plasma was transferred to freezing tubes and frozen. The two heparin plasma blood collection tubes were gently rotated and incubated at room temperature for 1 hour. The tubes were then centrifuged, and the plasma was transferred to freezing tubes and frozen.

[0163] The samples were assayed for nucleosomes containing histone isoform H3.1 (H3.1-nucleosomes) using a two-run ELISA procedure. Briefly, 20 μl of the sample was added to a microtiter well containing magnetic particles pre-coated with anti-histone H3.1 antibody. The sample was incubated, the magnetic particles were isolated, and washed. Anti-nucleosome antibodies, directed to bind to three-dimensional nucleosome epitopes conjugated with horseradish peroxidase, were added to the magnetic particles. The particles were incubated, then isolated, and washed. The bound anti-nucleosome antibodies were measured using a colorimetric substrate reaction. The results are shown in Figure 1, which indicate that NET material levels were high in heparin tubes but low in EDTA tubes. This clearly demonstrates that elevated levels of circulating NET material can be detected by a simple, low-cost immunoassay test.

[0164] (Example 2) DNA was extracted from the two heparin and plasma samples described in Example 1 and analyzed by fragment size using a tip-based capillary electrophoresis system (Agilent Bioanalyzer). DNA fragments approximately 150 bp in size, corresponding to mononucleosomes, had a retention time of approximately 60 seconds. As shown in Figure 2, the observed levels of mononucleosome-associated DNA were low (as expected for healthy volunteers). DNA fragments corresponding to NET material had a longer retention time of approximately 110 seconds. As shown in Figure 2, the levels of NET material were low in EDTA plasma (as expected for healthy volunteers), but high in heparin plasma tubes where NET formation was stimulated by heparin exposure. This result supports the idea that the increase in nucleosome levels observed in heparin plasma in Example 1 was due to NETs, ​​not mononucleosomes.

[0165] (Example 3) EDTA plasma samples were collected from 100 subjects who had all tested positive for coronavirus infection, including 50 control subjects with mild symptoms and 50 test subjects with respiratory complications requiring mechanical ventilation. Circulating NET material was measured using the nucleosome immunoassay method described in Example 1. The 50 control subjects were found to have low NET nucleosome levels, and these levels were used to establish the control range. The 50 test subjects were found to have higher NET nucleosome levels, and these higher levels were used as an indicator that, in addition to viral infection, the subjects had respiratory complications requiring treatment.

[0166] (Example 4) The experiment performed in Example 3 was repeated, but the immunoassay performed measured citrullinated nucleosomes. Fifty control subjects were found to have low citrullinated nucleosome levels, and these levels were used to establish the control range. Fifty test subjects were found to have higher citrullinated nucleosome levels, and these higher levels were used as an indicator that, in addition to viral infection, the subjects had respiratory complications requiring treatment.

[0167] (Example 5) The experiment performed in Example 3 is repeated, but the immunoassay performed measures myeloperoxidase-nucleosome adduct levels. Fifty control subjects were found to have low myeloperoxidase-nucleosome levels, and these levels are used to establish the control range. Fifty test subjects were found to have higher myeloperoxidase-nucleosome levels, and these higher levels are used as an indicator that, in addition to viral infection, the subject has respiratory complications requiring treatment.

[0168] (Example 6) The experiment performed in Example 3 is repeated, but the immunoassay performed measures neutrophil elastase-nucleosome adducts. Fifty control subjects were found to have low neutrophil elastase-nucleosome levels, and these levels are used to establish the control range. Fifty test subjects were found to have higher neutrophil elastase-nucleosome levels, and these higher levels are used as an indicator that, in addition to viral infection, the subject has respiratory complications requiring treatment.

[0169] (Example 7) The experiment conducted in Example 5 is repeated, but with the additional measurement of CRP and IL6 levels in EDTA plasma samples or serum samples from the same subject. An algorithm is developed using logistic regression analysis of the results to maximize the clinical sensitivity and specificity for identifying the target of the test.

[0170] (Example 8) EDTA plasma samples were collected in 2019, prior to the COVID-19 pandemic, from 50 healthy individuals and 50 individuals hospitalized with symptoms suspected to be COVID-19. Of the 50 individuals hospitalized with symptoms of COVID-19, 34 tested positive for COVID-19 using polymerase chain reaction (PCR) testing, and 16 tested negative for COVID-19 using PCR testing.

[0171] Patients with symptoms were selected to include both those who had experienced severe illness and those who had experienced milder illness. 40 patients were hospitalized, including 5 who were admitted to the ICU, two of whom received mechanical ventilation for 9 and 10 days, respectively. Hospitalized patients who tested negative for COVID-19 via PCR still received treatment for symptoms of respiratory infection.

[0172] H3.1-nucleosome levels were measured in plasma samples. The results are shown in Figure 3. All PCR-positive COVID-19 patients had elevated plasma H3.1-nucleosome levels, which allowed 100% of PCR-positive COVID-19 patients to be distinguished from normal controls with a specificity of 94% (3 false positives out of 50 control subjects) and an AUC of 98.7%.

[0173] PCR-positive COVID-19 patients could be divided into two groups based on their H3.1-nucleosome levels. The first group, consisting of 15 out of 34 subjects, had H3.1-nucleosome levels below 600 ng / ml. The second group, consisting of 19 out of 34 subjects, had H3.1-nucleosome levels higher than the upper limit of the assay (>700 ng / ml).

[0174] A similar pattern was observed in 16 patients who had COVID-19 symptoms but tested negative by PCR, with 6 having H3.1 nucleosome levels below 600 ng / ml and 10 having H3.1 nucleosome levels >700 ng / ml.

[0175] (Example 9) To determine whether circulating H3.1-nucleosome levels predict the severity of COVID-19 disease, EDTA plasma samples were collected from 14 subjects diagnosed with COVID-19 infection by positive PCR COVID-19 virus test results. Of these, five samples were collected from subjects who were presenting by outpatient hospital appointments or being examined in the hospital emergency room (ER), three samples were collected from patients admitted to regular psychiatric wards (i.e., not in the intensive care unit), and six samples were collected from patients with extremely severe illness who were transferred to the intensive care unit (ICU) of a tertiary hospital center for advanced respiratory support, including mechanical ventilation and extracorporeal membrane oxygenation (where the patient is cannulated and the function of the patient's lungs is replaced by blood supply through an artificial lung), and other clinical support. The mortality rate for 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. There was a clear increase in levels between subjects examined in outpatient / ER settings and those admitted to general wards for COVID-19 infection. The area under the curve (AUC) for this distinction was 100%. There was also a clear increase in levels between subjects admitted to general wards for COVID-19 infection compared to those admitted to the ICU. The area under the curve for this distinction was also 100%. Furthermore, the four patients who died were found to have the four highest circulating H3.1-nucleosome levels. The AUC for predicting mortality was also 100%.

[0177] These results indicate that circulating H3.1-nucleosome levels predict disease severity and mortality, and can be used for prognosis assessment. Therefore, the method of the present invention can be used to predict the level of care required for patients or subjects at various stages of the clinical process, including the later stages of examination for respiratory infections or determining the level of clinical support and / or the nature of the treatment required.

[0178] Similarly, these results indicate that circulating H3.1-nucleosome levels can be used to monitor patients through serial sampling to determine whether levels are elevated or decreasing, informing clinical decisions regarding future treatment and clinical support regimens. For example, decreasing levels may indicate a decision that intensive respiratory support is no longer essential and / or that the treatment used is effective.

[0179] Conversely, rising levels may indicate a decision that intensive respiratory support is required and / or that the current treatment used is ineffective and additional or alternative treatments should be considered. Therefore, the method of the present invention can be used to select patients who require treatment for NETosis-related conditions, including, but not limited to, NETosis inhibitors, treatments to eliminate NETs by apheresis or plasmapheresis, or anti-inflammatory treatments, such as CD24 therapy (e.g., EXO-CD24 or CD24Fc). Accordingly, the method of the present invention can be used as a companion diagnostic product for drugs and treatments for diseases associated with NETosis.

[0180] Similarly, this also indicates that the method of the present invention may be suitable for use in evaluating the efficacy of investigational drugs for treating respiratory disease conditions. In one embodiment, circulating H3.1-nucleosome levels can be used as a surrogate endpoint in a clinical trial, either alone or in combination with other parameters.

[0181] (Example 10) To determine whether the method of the present invention is effective for other nucleosome regions, the same subjects described in Example 9 were also examined for the level of circulating nucleosomes (H3R8Cit-nucleosomes) containing histone modifications of citrullination of an arginine residue at position 8 of histone H3. This nucleosome region was selected because NETs chromatin is known to be citrullinated. The results are shown in Figure 5, supporting the effectiveness of the method of the present invention using citrullinated nucleosome measurements, and more generally, using measurements of any circulating nucleosome region or NETs region containing MPO, NE, or other NETs component regions.

[0182] These results indicate that circulating H3R8Cit-nucleosome levels predict disease severity and mortality and can be used for prognosis. Similarly, H3R8Cit-nucleosome levels can be used to predict the level of care required by patients or subjects at various stages of the clinical process, and patients can be monitored for H3.1-nucleosome levels by sequential sampling as described in Example 9 above.

[0183] (Example 11) Blood samples are collected from subjects with NETosis-related disorders and assayed for nucleosomes as described herein as indicators of overproduction or excess production of NETs by the subject. If the measured NET levels are below the threshold cutoff, the patient will not receive NETosis inhibitory therapy, such as anthracycline therapy. If the measured NET levels are above the threshold cutoff, the patient will receive NETosis inhibitory therapy, such as anthracycline therapy. Further blood samples are collected at appropriate intervals (e.g., every 4 hours or daily) to monitor the decline in the subject's circulating NET levels and determine the effectiveness of treatment. When used in this manner, the present invention provides a combination product comprising NETosis inhibitor therapy and a companion diagnostic for patient selection and monitoring of patients with pathological NETs levels requiring NETosis therapy (e.g., antiviral or antibacterial agents, anti-inflammatory agents, anticoagulants or anti-hemitogamous agents, NETosis inhibitors, DNase agents, anti-nucleosome therapeutic antibodies, anti-MPO therapeutic antibodies, and anti-NE therapeutic antibodies, or apheresis or plasmapheresis therapy to remove NETs from circulation).

[0184] (Example 12) Sepsis was induced in 16 pigs by infection with Escherichia coli (E. coli) administered by intravenous infusion over 3 hours (Figures 6 and 7, 0-3 hours). The septic pigs were treated by plasmapheresis as described in WO2019053243 to remove NETs from the bloodstream. Briefly, whole blood was removed from the pig's body through a tube into a plasmapheresis device, separated into cell and plasma fractions, and the plasma was passed through a plasmapheresis cartridge containing a NETs binder to remove NETs. The plasma was then recombined with blood cells and returned to the pig's body. The plasmapheresis treatment was performed over 5 hours (Figures 6 and 7, 2-7 hours). The plasmapheresis cartridges used on 9 pigs contained the NETs binder (treatment pigs), while the cartridges used on the other 7 pigs did not contain the binder (control pigs).

[0185] Eight plasma samples were collected hourly from each pig at 0–7 hours after the onset of infection. Circulating nucleosomes were measured to determine whether the method of the present invention is effective (i) as a monitor during the infection period and (ii) as a monitor for the efficacy of treatment.

[0186] Furthermore, to determine whether the degree of plasma removal by the NETs binder in the cartridge can be monitored by the method of the present invention, plasma samples were collected from a plasmaferesis device both upstream of the cartridge (to sample plasma entering the NETs binder cartridge) and downstream of the cartridge (to sample plasma leaving the NETs binder cartridge). Five upstream samples and five downstream samples were collected hourly at 3 to 7 hours after the onset of infection (3 to 7 hours in Figures 6 and 7).

[0187] Plasma samples were assayed for nucleosomes containing histone isoform H3.1 (H3.1-nucleosome). Assay measurements were performed using an automated immunoassay instrument. Briefly, a calibration sample (50 μl) was incubated with acridinium ester-labeled anti-nucleosome antibody (50 μl) and assay buffer (100 μl) at 37°C for 1800 seconds. Magnetic beads coated with anti-histone H3.1 antibody (20 μl) were added, and the mixture was incubated for a further 900 seconds. The magnetic beads were then isolated, washed three times, and the magnetically coupled acridinium ester was determined by luminescence output over 7000 milliseconds.

[0188] The mean results for circulating H3.1-nucleosome levels in control and treated pigs are shown in Figure 6a. Control pigs (infected and sepsis induced but not treated) subsequently developed sepsis over several hours, which was reflected in the observed increase in circulating H3.1-nucleosome levels. The increase in mean H3.1-nucleosome levels was evident at 1 hour (after the onset of infection) and accelerated at 3 hours, which is consistent with the time course of the NETosis process. H3.1-nucleosome levels continued to rise, reaching 361 ng / ml at 7 hours. A similar initial increase in mean circulating H3.1-nucleosome levels was observed in treated pigs from 0–2 hours. Initiation of plasmapheresis treatment at 2 hours resulted in a slowdown in the increase in nucleosome levels, with the mean level observed at 7 hours being 150 ng / ml. This is considerably lower than the average level observed in control pigs, demonstrating the effectiveness of plasmapheresis and showing that H3.1 nucleosome levels are an effective monitor and treatment guide for the course and severity of septic disease, as well as an effective monitor for the in vivo NETosis process.

[0189] The mean results of plasma H3.1-nucleosome levels measured in samples taken from within the plasmaferesis device upstream of the cartridge during the procedure are shown in Figure 6b. These results are similar to those observed for the mean circulating H3.1-nucleosome levels measured, shown in Figure 6a.

[0190] During the procedure, the mean results for plasma H3.1-nucleosome levels measured in samples taken from within the plasmaferesis device downstream of the cartridge are shown in Figure 6c. For control pigs, the results in Figure 6c are similar to those in Figure 6b (and 6a), indicating that passing plasma through a cartridge without a NETs binder did not significantly affect the observed H3.1-nucleosome levels. This is consistent with the expected result that the level of NETs in plasma was not significantly affected by passing through a cartridge without a NETs binder. For treated pigs, the results in Figure 6c are all low. This is consistent with the expected result that passing plasma through a cartridge containing a NETs binder removed most or all of the NETs from the plasma. Furthermore, these results indicate that the NETs binder in the cartridge was not saturated with NETs after 7 hours and continued to bind to all or most of the NETs present in the plasma entering the device. Therefore, measuring the level of H3.1 nucleosomes is useful in determining when the binding material in the cartridge becomes saturated and, consequently, is no longer useful as a tool for removing NETs and should be replaced with a fresh cartridge.

[0191] Therefore, the combined results of Figures 6b and 6c indicate that measuring H3.1-nucleosome levels is useful as a monitor and guide for the treatment of NETosis and sepsis.

[0192] The results for circulating H3.1 nucleosome levels measured in samples taken from all 16 pigs are individually shown in Figure 7a. The mean H3.1 nucleosome level observed in control pigs at 7 hours was 361 ng / ml, which was above 120 ng / ml in all control pigs (range 123–743 ng / ml). In contrast, the mean H3.1 nucleosome level observed in treated pigs at 7 hours was 150 ng / ml, which was 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 also indicate that H3.1 nucleosome levels are an effective monitoring and treatment guide for the course and severity of septic disease, and an effective monitoring and treatment guide for excessive NETosis in vivo. Furthermore, the results in Figure 7a demonstrate that, using the measurement of circulating H3.1-nucleosome levels, individuals with elevated NETs levels can be identified as suitable candidates for therapies aimed at reducing NETs or NETosis levels.

[0193] Seven control pigs exhibited elevated nucleosome levels and increased clinical stress indicators, requiring more intensive medical support than the seven treated pigs with lower nucleosome levels. Furthermore, two treated pigs observed to have H3.1-nucleosome levels above 120 ng / ml also exhibited elevated clinical stress indicators and required more intensive medical support. Therefore, the method of the present invention is a good method for monitoring the efficacy of NETosis treatment.

[0194] During the procedure, the results for plasma H3.1-nucleosome levels measured in samples taken from within the plasmaferesis device upstream of the cartridge are individually shown in Figure 7b for all 16 pigs. As described above for Figure 6, the results shown in Figure 7b are similar to those in Figure 7a. The mean H3.1-nucleosome level observed in control pigs at 7 hours was 368 ng / ml (range 121–629 ng / ml). In contrast, the H3.1-nucleosome level observed in treated pigs at 7 hours was lower, with an average result of 143 ng / ml (for all 9 treated pigs). For the 7 responder pigs, the range of results at 7 hours was 34–127 ng / ml.

[0195] During the procedure, the results for plasma H3.1-nucleosome levels measured in samples taken from the plasmaferesis device downstream of the cartridge are individually shown in Figure 7c for all 16 pigs. The mean H3.1-nucleosome level observed in control pigs at 7 hours was 378 ng / ml (range 147–617 ng / ml). In contrast, the mean H3.1-nucleosome level observed in plasma downstream of the cartridge in treated pigs at 7 hours was 2.4 ng / ml (range 0.7–6.5 ng / ml), and was less than 7 ng / ml at all time points for all 9 treated pigs.

[0196] The combined results of Figures 7b and 7c indicate that measuring H3.1-nucleosome levels is useful as a monitor and guide for the treatment of NETosis and sepsis.

[0197] Combined, the results shown in Figure 7 indicate that the plasmapheresis treatment regime successfully removed NETs from the circulation of all nine treated pigs, and that seven of the nine pigs responded well to the treatment, while two were non-responders. These nucleosome results correlate very well with the clinical observations of the pigs.

[0198] Furthermore, the seven treated responder pigs could be clearly distinguished from treated non-responder pigs and untreated pigs based on the observed nucleosome results.

[0199] (Example 13) Plasma samples were obtained from 20 human subjects diagnosed with sepsis and 10 healthy human subjects. The plasma samples were assayed for the levels of nucleosomes containing histone isoform H3.1 (H3.1-nucleosomes) using the automated immunoassay apparatus described in Example 12. Elevated levels were observed in sepsis samples compared to healthy subjects. This is likely due to the effects of NETosis at various stages of the disease (Figure 8). This application provides the invention in the following embodiments. (Aspect 1) A method for monitoring the progression of disease in subjects 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) Monitoring the progression of infection in the subject using any changes at the level of the cell-free nucleosome or its components. The method including: (Aspect 2) A method of assigning the risk of adverse outcomes to individuals 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; and (ii) Using the detected cell-free nucleosome levels, assign the potential adverse outcomes to the subject. : includes, The method wherein subjects identified as having a high probability of adverse outcomes are assigned to medical intervention. (Aspect 3) The method according to embodiment 1 or embodiment 2, wherein the infectious disease is a viral, bacterial, fungal, or microbial infection. (Aspect 4) The method according to any one of embodiments 1 to 3, wherein the aforementioned infectious disease is a respiratory tract infection. (Aspect 5) The method according to embodiment 4, wherein the respiratory infection is selected from influenza, pneumonia, and severe acute respiratory syndrome (SARS). (Aspect 6) The method according to embodiment 1 or embodiment 2, wherein the subject is suffering from sepsis or septic shock. (Aspect 7) The method according to any one of embodiments 1 to 6, wherein the bodily fluid sample is a blood, serum, or plasma sample. (Pattern 8) The method according to any one of embodiments 1 to 7, wherein the cell-free nucleosome is part of or obtained from a neutrophil extracellular trap. (Aspect 9) The method according to any one of embodiments 1 to 8, wherein the components of the cell-free nucleosome include the epigenetic characteristics of the cell-free nucleosome. (Aspect 10) The method according to embodiment 9, wherein the epigenetic feature is a histone isoform, for example, a coanucleosome histone isoform, in particular a histone H3 isoform. (Aspect 11) The method according to embodiment 10, wherein the histone isoform is H3.1. (Aspect 12) The method according to embodiment 9, wherein the epigenetic feature is a post-translational modification (PTM) of histone, for example, a coanucleosome histone PTM, particularly a histone H3 or H4 PTM. (Aspect 13) The method according to embodiment 12, wherein the histone PTM is selected from citrullination or ribosylation. (Aspect 14) The method according to any one of embodiments 1 to 13, wherein the level of the cell-free nucleosome or its components is detected or measured using an immunoassay, immunochemistry, mass spectrometry, chromatography, chromatin immunoprecipitation, or biosensor method. (Aspect 15) The method according to any one of embodiments 1 to 14, wherein the detection or measurement method includes contacting the body fluid sample with a solid phase containing a binder for detecting cell-free nucleosomes or their components, and detecting binding to the binder. (Aspect 16) The method according to any one of embodiments 1 to 15, wherein the detection or measurement method comprises: (i) contacting the sample with a first binder that binds to the epigenetic features of cell-free nucleosomes; (ii) contacting the sample bound by the first binder in step (i) with a second binder that binds to cell-free nucleosomes; and (iii) detecting or quantifying the binding of the second binder in the sample. (Aspect 17) The method according to any one of embodiments 1 to 16, wherein the subject is a human or an animal. (Aspect 18) The method according to any one of embodiments 1 to 17, further comprising comparing the level of cell-free nucleosomes or their components in the body fluid sample of the subject with one or more controls. (Aspect 19) The method according to embodiment 18, wherein the control is a healthy subject. (Aspect 20) The method according to embodiment 18, wherein the control is a subject having an infectious disease that is asymptomatic or shows mild symptoms. (Aspect 21) The method according to any one of embodiments 1 to 20, wherein the level of the cell-free nucleosome or its components is elevated compared to a control. (Aspect 22) The method according to any one of embodiments 1 to 21, wherein the level of the cell-free nucleosome is detected or measured as one of the measurement panels. (Aspect 23) The method according to embodiment 22, wherein the panel contains one or more interleukins. (Aspect 24) The method according to embodiment 23, wherein the one or more interleukins are selected from the group consisting of IL-6 and IL-12. (Aspect 25) The method according to any one of embodiments 22 to 24, wherein the panel comprises C-reactive protein (CRP), myeloperoxidase (MPO), D-dimer, and / or factor VII-activated protease (FSAP). (Aspect 26) The method according to any one of embodiments 22 to 25, wherein the panel includes an MPO. (Aspect 27) A method for detecting individuals requiring medical treatment for pneumonia, acute respiratory syndrome (ARS), acute respiratory distress syndrome (ARDS), or severe acute respiratory syndrome (SARS), (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; and (ii) Using the cell-free nucleosome level as an indicator that the subject requires medical treatment for pneumonia, ARS, ARDS, or SARS. The method including: (Aspect 28) A method for detecting a subject who requires medical treatment for sepsis or septic shock, (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; and (ii) Using the cell-free nucleosome level as an indicator that the subject requires medical treatment for sepsis or septic shock. The method including: (Aspect 29) A method for treating NETosis-related diseases, comprising the administration of a therapeutic antibody directed to bind to nucleosomes or their components, myeloperoxidase, neutrophil elastase, or C-reactive proteins. (Aspect 30) The method according to embodiment 29, wherein the NETosis-related disease is accompanied by a high level of extracellular neutrophil trapping. (Aspect 31) The method according to embodiment 29 or embodiment 30, wherein the NETosis-related disease is a viral or bacterial infection. (Aspect 32) The method according to any one of embodiments 29 to 31, wherein the therapeutic antibody is directed to bind to an epitope present in an intact nucleosome. (Aspect 33) The method according to any one of embodiments 29 to 31, wherein the therapeutic antibody is directed to bind to an epitope present in the cleaved nucleosome. (Aspect 34) The method according to any one of embodiments 29 to 33, wherein the therapeutic antibody is directed to bind to a nucleosome component that is histone H3.1 or a citrullinated histone. (Aspect 35) The method according to any one of embodiments 29 to 34, wherein the therapeutic antibody is directed to bind to a histone H3.1 epitope located at amino acid positions 30 to 33 in the amino acid sequence of histone H3.1.

Claims

1. A method for obtaining data to monitor the progression of sepsis or septic shock in a subject, (i) Contacting a body fluid sample obtained from the subject with a binder that binds to histone H3.1 to detect or measure the level of cell-free nucleosomes containing histone H3.1; and (ii) Repeat step (i) at least once. : includes, The method wherein changes in the level of cell-free nucleosomes containing histone H3.1 serve as data for monitoring the progression of sepsis or septic shock in the subject.

2. A method for obtaining data to assign the risk of adverse outcomes to subjects suffering from sepsis or septic shock, The body fluid sample obtained from the subject is brought into contact with a binder that binds to histone H3.1, and the level of cell-free nucleosomes containing histone H3.1 is detected or measured. : includes, The method wherein the detected cell-free nucleosome levels serve as data for assigning potential adverse outcomes to the subject.

3. The method according to claim 1 or 2, wherein the bodily fluid sample is a blood, serum, or plasma sample.

4. The method according to any one of claims 1 to 3, wherein the level of cell-free nucleosomes containing histone H3.1 is detected or measured using an immunoassay, immunochemistry, mass spectrometry, chromatography, chromatin immunoprecipitation, or biosensor.

5. The method according to any one of claims 1 to 4, wherein the detection or measurement method includes contacting the body fluid sample with a solid phase containing a binder that binds to histone H3.1, and detecting the binding to the binder.

6. The method according to any one of claims 1 to 5, wherein the detection or measurement method comprises: (i) contacting the sample with a first binder that binds to histone H3.1; (ii) contacting the sample bound by the first binder in step (i) with a second binder that binds to cell-free nucleosomes; and (iii) detecting or quantifying the binding of the second binder in the sample.

7. The method according to claim 6, wherein the first binder is an anti-histone H3.1 antibody directed to bind to an epitope around amino acids 30-33 of the histone H3.1 protein.

8. The method according to claim 6 or 7, wherein the second binder is an anti-nucleosome antibody directed to bind to a three-dimensional nucleosome epitope.

9. The method according to any one of claims 1 to 8, wherein the subject is a human or an animal.

10. The method according to any one of claims 1 to 9, further comprising comparing the level of cell-free nucleosomes containing histone H3.1 in the body fluid sample of the subject with one or more controls.

11. The method according to claim 10, wherein the control is a healthy subject.

12. The method according to claim 10, wherein the control is a subject having sepsis or septic shock exhibiting no symptoms or mild symptoms.

13. The method according to any one of claims 1 to 12, wherein the level of cell-free nucleosomes containing the aforementioned histone H3.1 is elevated compared to a control.

14. The method according to any one of claims 1 to 13, wherein the level of cell-free nucleosomes containing histone H3.1 is detected or measured as one of the panels of measurement.

15. The method according to claim 14, wherein the panel contains one or more interleukins.

16. The method according to claim 15, wherein the one or more interleukins are selected from the group consisting of IL-6 and IL-12.

17. The method according to any one of claims 14 to 16, wherein the panel comprises C-reactive protein (CRP), myeloperoxidase (MPO), D-dimer, and / or factor VII-activated protease (FSAP).

18. The method according to any one of claims 14 to 17, wherein the panel includes MPO.

19. A method for obtaining data to detect subjects requiring medical treatment for sepsis or septic shock, The body fluid sample obtained from the subject is brought into contact with a binder that binds to histone H3.1, and the level of cell-free nucleosomes containing histone H3.1 is detected or measured. : includes, The method wherein the level of cell-free nucleosomes containing histone H3.1 serves as an indicator that the subject requires medical treatment for sepsis or septic shock.

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