Device for inline monitoring of free nucleosomes in the blood.

Abstract

The present invention relates to an extracorporeal device or ex vivo organ perfusion device that includes an in-line monitoring method or device for measuring cell-free nucleosomes, and in particular to an apheresis device that includes a monitoring method or device for measuring cell-free nucleosomes in the blood of a subject.

Description

Technical Field

[0001] (Field of the Invention) The present invention particularly relates to an in-line monitoring device for measuring cell-free nucleosomes for use in an apheresis device.

Background Art

[0002] (Background of the Invention) Many diseases are associated with a pathological condition in which the subject's blood contains abnormal or pathogenic blood components that threaten the subject's health or even life. Toxic components may not be found in the circulation of healthy individuals but may be present in affected subjects (e.g., monoclonal IgG or IgM paraprotein in patients with multiple myeloma), or may be present in both healthy and affected subjects but at elevated levels in affected subjects (e.g., platelets, cytokine molecules, and nucleosomes). Toxic components can be any of various types, including but not limited to cells, platelets, small molecules, or macromolecules (e.g., proteins, lipoproteins, or nuclear proteins). Apheresis is an extracorporeal method for treating such diseases, in which case the blood is removed from the subject and the toxic component is removed from the blood before it is returned to the subject.

[0003] One form of apheresis is whole blood apheresis. Another form of apheresis is plasma pheresis, which involves removing whole blood from the subject and placing it into an extracorporeal device, separating abnormal plasma from the whole blood, discarding the abnormal plasma, and returning the remaining whole blood to the subject, or alternatively, combining the plasma with the whole blood again and removing abnormal substances from the plasma before returning it to the subject. Solid-phase adsorbents that bind to abnormal substances in the plasma can be used to remove such plasma components by extracorporeal plasma pheresis before combining the plasma with the whole blood again and returning it to the subject. Renal dialysis for the removal of physiological waste products from the circulation is a common form of apheresis.

[0004] Sepsis is a fatal inflammatory disease triggered by bacterial, fungal, viral, or protozoan infections, characterized by a cytokine storm with elevated levels of circulating cytokines, as well as increased production of neutrophil extracellular traps (NETs) and elevated levels of circulating nucleosomes. Elevated cytokine and NET production due to NETosis is a pathological condition, leading to thrombosis, hypotension, hyperlactate, and hypourinary excretion, ultimately resulting in dyspnea, fainting, and multiple organ failure. Sepsis often requires emergency treatment in an intensive care setting with intravenous fluids and antimicrobial agents. Mechanical ventilation and dialysis may be necessary to support lung and kidney function and to prevent thrombosis. The risk of death from severe sepsis can be 50%, making it a leading cause of death worldwide.

[0005] Emergency treatment of sepsis to reduce circulating cytokine levels may be influenced by the removal of cytokines, including interleukin-6 (IL-6) and tumor necrosis factor (TNF-α), from circulation via plasmapheresis by using a solid-phase adsorbent contained in a cartridge through which the isolated plasma of the subject is passed before being returned to the body along with whole blood (Honore et al. (2019) Ann. Intensive Care 9: 56). Another adsorbent has been described for the removal of elevated levels of cell-free DNA (cfDNA) or NETs and circulating NETs degradation products from the plasma of subjects with sepsis. In this technique, histone H1.3 is used as a binder for cfDNA or NETs in plasmapheresis (WO2019053243). Because H1 has adverse effects on the cell membrane, H1.3 cannot be used as an adsorbent for NETs or cfDNA in whole blood apheresis.

[0006] One limitation of these devices is the inadequate monitoring of current treatments. The progression or absence of treatment over time is not monitored. In particular, there is currently no method for monitoring cfDNA or NETs levels for use in in vitro hematopoietic therapy, including when the treatment is designed to remove cfDNA or NETs. This results in disadvantages such as (i) the efficacy of the treatment being unknown and potentially continuing if other treatments are preferable; (ii) the time required to reach threshold depletion of the pathogen being unknown and therefore potentially continuing the treatment when it is no longer necessary; (iii) the treatment being stopped too early for the same reasons; or (iv) the real-time saturation level of the pathogen adsorbent during the treatment period being unknown and therefore potentially continuing the treatment with reduced efficacy if the column is saturated. Therefore, there remains a need in the art to provide a method for monitoring the progression of apheresis to help improve patient safety and treatment efficacy.

[0007] Extracorporeal hematopoiesis includes a wide variety of apheresis types, including, but are not limited to, the removal of physiological waste products, the collection of stem cells for autologous transplantation, the collection of leukocytes (e.g., in CAR T-cell therapy), the exchange of red blood cells (e.g., in the treatment of sickle cell disease), the exchange of plasma, and the supply of oxygen to the blood (as seen in extracorporeal membrane oxygenation or ECMO).

[0008] Extracorporeal hematopoietic therapy is known to cause inflammation, which can itself lead to elevated levels of NETs. Therefore, monitoring levels of circulating NETs or cfDNA is useful to improve patient safety and treatment efficacy in all such therapies. [Overview of the project]

[0009] (Summary of the invention) According to a first aspect of the present invention, an extracorporeal device is provided which includes a monitoring method or apparatus for measuring the level of cell-free nucleosomes present in the blood of a subject.

[0010] In a further embodiment, an in vitro method for treating a blood sample is provided, comprising a monitoring method or in-line device for measuring the level of cell-free nucleosomes or NETs present in the blood sample.

[0011] In a further embodiment, a device is provided for in vitro treatment of a target blood, comprising a monitoring method or in-line device for measuring the level of cell-free nucleosomes or NETs present in the target blood.

[0012] In a further embodiment, an apheresis device is provided that includes one or more affinity matrices for removing one or more pathogenic substances from the blood of a target, and includes an inline monitoring method or device for measuring the level of cell-free nucleosomes present in the blood of a target.

[0013] A further embodiment provides a method for monitoring a subject during an in vitro procedure, comprising: passing the subject's blood through an in vitro device described herein; and monitoring the level of cell-free nucleosomes in the subject's blood using a monitoring method or apparatus of the in vitro device.

[0014] In a further embodiment, a method is provided for treating a disease of a subject requiring such treatment, comprising: passing the blood of the subject through an external device described herein; and monitoring the level of cell-free nucleosomes in the blood of the subject using a monitoring method or apparatus for the external device.

[0015] According to another aspect of the present invention, an in vitro organ perfusion method or apparatus is provided, which includes a monitoring method or apparatus for measuring the level of cell-free nucleosomes present in a liquid perfusion solution. [Brief explanation of the drawing]

[0016] (Brief explanation of the drawing) [Figure 1] A simplified diagram of a plasma ferresis setup for removing NETs from the target blood, including in-line nucleosome measurement. An anticoagulant is added to the plasma separator, and blood is pumped from the body. The separated plasma is pumped into a solid-phase nucleosome / NETs adsorption matrix. The NETs / nucleosome-removed plasma is returned to the body along with the rest of the target blood. In the diagram, in-line nucleosome measurement is performed in the plasma at position 1 before nucleosome adsorption. Plasma nucleosome measurement may also be performed at position 2. Plasma nucleosome measurement may also be performed at both positions 1 and 2 to provide information on both current circulating nucleosome levels in the target and to monitor the effect of nucleosome / NETs removal by the NETs adsorbent. Whole blood nucleosome measurement may also be performed at positions 3, 4, or 5. [Figure 2] Results from the experiment described in Example 2, showing the mean levels of nucleosomes containing histone isoform H3.1 measured in 16 pigs induced by plasmapheresis-induced sepsis. 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 3] The experimental results described in Example 2 and shown in Figure 2, except for the levels in each individual test object. [Figure 4] H3.1-nucleosome levels measured in human subjects diagnosed with sepsis and in healthy human subjects. [Modes for carrying out the invention]

[0017] (Detailed explanation) In vitro therapies may involve various treatments that require monitoring of NETs levels or cfDNA levels to ensure patient safety and optimal treatment outcomes. Therefore, according to a first aspect of the present invention, an in vitro device is provided that includes a monitoring method or apparatus for measuring the level of cell-free nucleosomes or NETs present in the blood or blood plasma of a subject circulating in and / or outside the body.

[0018] Apheresis is an extracorporeal treatment that removes pathogenic substances or components from the blood of a patient in order to treat a disease (e.g., Ward MD (2011), Conventional Apheresis Therapies: A Review Journal of Clinical Apheresis 26:230-238). Therefore, references to “apheresis device” in this specification refer to a device for modifying the composition of blood outside the body before returning it to the subject.

[0019] Current technologies focus on apheresis treatment, but it is important to consider monitoring the progress of treatment to ensure that patients receive adequate treatment and improve the overall efficiency of the procedure. This is especially important as the demand for apheresis increases; the increased burden on apheresis resources can be minimized by ensuring that apheresis is performed only on patients as needed.

[0020] According to one embodiment, an apheresis device is provided that includes one or more affinity matrices for removing one or more pathogenic substances from a target blood and an in-line monitoring device for measuring the level of cell-free nucleosomes present in the target blood. Therefore, in one embodiment, an extracorporeal device is provided that further includes one or more affinity matrices for removing one or more pathogenic substances from a target blood.

[0021] In particular, the pathogenic substances are cell-free DNA (cfDNA) and / or neutrophil extracellular traps (NETs). Measuring the level of cell-free nucleosomes can be used to monitor the progress of treatment and to determine when the level of pathogenic substances, particularly NETs, reaches an acceptable level at which apheresis can be terminated.

[0022] (Monitoring method or device) In one embodiment, the monitoring method or device measures in real time the level of cell-free nucleosomes present in the blood of a subject. In some embodiments, the monitoring device is an in-line monitoring device. In one aspect of the invention, careful measurements of plasma or whole blood nucleosome levels are made at intervals, for example, at 15-minute intervals or at 30- or 60-minute intervals. In one embodiment, the monitoring method or device is a disposable or reusable measuring device. It can be plugged into an extracorporeal device for measurement, such as an apheresis device. Therefore, the measurement can be carried out in-line using any device attached to receive blood or plasma from the subject.

[0023] In one embodiment, the measurement is carried out using a lateral flow immunoassay cartridge or device that rapidly (e.g., in 5 minutes, or in 10, 15, or 20 minutes) measures the nucleosome level. In this embodiment, the lateral flow device may be designed similarly to a common lateral flow test used, for example, in urine pregnancy tests or other home tests involving finger prick blood tests. Such tests are well described in the art (e.g., by Koczula and Gallotta, Essays in Biochemistry (2016) 60: 111-120). [[ID=十二]]

[0024] In another embodiment, the measurement is performed using a microfluidic immunoassay device. A microfluidic immunoassay device is a small-scale biomarker immunoassay and is well-known in the art. Microfluidic devices have several advantages over lateral flow assays, including greater analytical quantification accuracy, simplified fluidics, reduced reagent volumes, and often shorter assay times of less than 5 minutes (see Barbosa and Reis, Analyst (2017) 142: 858). Microfluidic devices targeting many protein biomarkers, including cardiac biomarkers (e.g., troponin I, troponin T, creatine kinase, and myoglobin), cancer biomarkers (e.g., prostate-specific antigen, carcinoembryonic antigen, α-fetoprotein, and cancer antigen 125), and inflammatory biomarkers (e.g., C-reactive protein, TNF-α, IL-1, IL-4, and IL-6) have been produced. to A reusable precision microfluidic device for rapid measurement of insulin within 30 seconds from sampling has been reported (Cohen et al., Microchim. Acta (2017) 184: 835-841). This device utilizes polystyrene microspheres coated with anti-insulin antibodies within the microfluidic device to provide insulin measurements in about 30 seconds, which can be repeated as often as desired using a reagent pool to provide near real-time measurements of insulin. Therefore, in one embodiment of the present invention, rapid real-time measurements of nucleosomes (and optionally other inflammatory markers) are provided by a microfluidic device, e.g., an in-line microfluidic device.

[0025]

[0026] In a further embodiment of the present invention, a biosensor device (e.g., an in-line biosensor device) is used to detect the level (or concentration) of nucleosomes. A biosensor is an analytical device that converts a biological response into an electrical signal. Typically, a biosensor intended for use as a monitor of nucleosome levels in apheresis or plasmapheresis equipment consists of a sensing component that recognizes an analyte nucleosome (e.g., a nucleosome-specific binder such as an antibody or a chromatin protein such as histone H1 immobilized on a solid phase) that generates a signal, and a signal converter that provides an electrical output. This can be used in a repeating discontinuous measurement mode or a continuous mode (in which case, minute nucleosome bindings are measured in a moving timeframe to provide real-time electrical signal monitoring). In one embodiment, DNA is detected using a biosensor because nucleic acid-based sensing systems are more sensitive than antibody-based detection methods. In one embodiment, the present invention utilizes a DNA sensing component that detects DNA by binding to DNA insertion sites. Biosensors have been well described in this art, for example, by Mehrotra and J. Oral Biol. Craniofac. Res. (2016) 6(2): 153-9.

[0027] In one embodiment, the inline monitoring device includes a solid phase having an immobilized binder for binding to cell-free nucleosomes. In a further embodiment, the monitoring method or device includes a solid phase having an immobilized binder for binding to cell-free nucleosomes.

[0028] 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 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 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 narrowed airways, epithelial cell damage, inflammatory response, and thrombus formation (Marcos et al., Nat. Med. (2010) 16: 1018-23; Hoeksema et al., Future Microbiol. (2016) 11: 441-53).

[0029] 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).

[0030] 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.

[0031] It will be understood that cell-free nucleosomes can be detected by binding to their components. As used herein, the term “components” may refer 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.

[0032] The monitoring method or apparatus of the present invention, for example, an in-line monitoring apparatus, can measure the level of (cell-free) nucleosomes themselves. The reference to “nucleosomes themselves” refers to the total nucleosome level or concentration present in the sample, regardless of whether the nucleosomes contain any epigenetic features or not. Therefore, in one embodiment, the binder binds to core nucleosome features common to all or most nucleosomes. Detection of total nucleosome levels typically involves the detection of a histone protein common to all nucleosomes, such as histone H4. Thus, 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.

[0033] Cell-free nucleosomes may be mononucleosomes, oligonucleosomes, components of larger chromatin fragments, or components of NETs, ​​or mixtures thereof.

[0034] Mononucleosomes and oligonucleosomes have been detected by enzyme-linked immunosorbent assay (ELISA), and several methods have been reported (e.g., Salgame et al., Nucleic Acids Research, 25(3), 680-681 (1997); Holdenrieder et al., Int. J. Cancer 95, 114-120 (2001); van Nieuwenhuijze et al., Ann Rheum Dis; 62: 10-14 (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.

[0035] 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, and contain a vast array of diverse epigenetic structures, including specific histone isoforms (or variants), post-translational histone modifications, nucleotides or modified nucleotides, and protein adducts. In one embodiment, the binder binds to the epigenetic features of cell-free nucleosomes. It will be apparent to those skilled in the art that an increase in nucleosome levels is associated with an increase in certain subsets of circulating nucleosomes 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, WO2005 / 019826, WO2013 / 030579, WO2013 / 030578, and WO2013 / 084002, which are incorporated herein by reference).

[0036] A monitoring method or apparatus, such as an in-line monitoring apparatus, can measure the level of cell-free nucleosomes themselves and / or the epigenetic features of cell-free nucleosomes. The terms “epigenetic signal structure” and “epigenetic features” will be understood to be used interchangeably herein. These refer to specific features of nucleosomes 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 nucleosomes in nucleosome-protein adducts.

[0037] 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, and H3t. In one embodiment, the histone isoform is H3.1.

[0038] The structure of a nucleosome can vary due to post-translational modifications (PTMs) of histone proteins. Histone protein PTMs typically occur at the tail of the core histone, and common modifications include acetylation, methylation, or ubiquitination of lysine residues, as well as methylation 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 a cell-free nucleosome may be histone post-translational modifications (PTMs). The histone PTM may be a core nucleosome, e.g., H3, H2A, H2B, or H4, and in particular, a histone PTM of H3, H2A, or H2B. In particular, the histone PTM is a histone H3 PTM. Examples of such PTMs are described in WO 2005 / 019826.

[0039] 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, for example, H3R8cit.

[0040] 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 can be stimulated by exposure to lipopolysaccharides, resulting in increased transcription, and may possess antiviral properties.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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 attached to nucleosomes in NETs, ​​and therefore, nucleosome-CRP adducts are useful adducts for detecting elevated NET levels in the method of the present invention.

[0046] In a preferred embodiment, the protein adduct associated with the detected cell-free nucleosome is an MPO-nucleosome adduct or an NE-nucleosome adduct.

[0047] The term "biomarker" means a differential biological or biologically 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.

[0048] The methods and uses described herein are tested with bodily fluid samples taken from blood passing through an apheresis device, particularly blood, serum, or plasma samples. Preferably, plasma samples are used.

[0049] The subject may be, for example, a human subject or a (non-human) animal subject. The terms "subject" and "patient" are used interchangeably herein. In one embodiment, the subject is human. In one embodiment, the subject is a (non-human) animal.

[0050] (Further biomarkers) In one embodiment, a monitoring method or apparatus, such as an inline monitoring apparatus, includes a panel of markers to be measured in the blood of a subject.

[0051] Therefore, cell-free nucleosome levels can be detected or measured as one of the parameters in a panel of measurements. The panel may include various epigenetic features of nucleosomes as described above (e.g., histone isoforms and PTMs). Useful biomarkers in panel tests for the detection of infections requiring medical intervention include, but are not limited to, cytokine moieties (especially interleukins), C-reactive proteins, myeloperoxidases, D-dimers, factor VII-activated proteases (FSAPs), fibrinogens, and fibrin / fibrinogen disruption products. In one embodiment, the panel includes one or more cytokines, for example, one or more interleukins.

[0052] Interleukins (ILs) are a group of cytokines that act as signaling molecules and are usually secreted by white blood cells. They play an important role in stimulating immune responses and inflammation. They were first identified in the 1970s and have been designated with numbers as more interleukin types have been discovered. 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.

[0053] In one embodiment, one or more interleukins are selected from the group consisting of interleukin-6 (IL-6) and interleukin-12 (IL-12).

[0054] Interleukin can 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 under UniProt accession number P05231. In a particular embodiment, interleukin can be IL-6.

[0055] Alternatively, or even more precisely, 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 a particular embodiment, the interleukin may be IL-12.

[0056] 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 multiple interleukin measurements, e.g., IL-6 and IL-12. In a further embodiment, the epigenetic features of cell-free nucleosomes are selected from histone isoforms, e.g., H3.1 and post-translational modified histones, e.g., H3cit. In yet another embodiment, the measurement panel is H3.1, H3cit, H4cit, and IL-6.

[0057] In one embodiment, the panel includes C-reactive protein (CRP). CRP is a pentameric protein found in plasma, and levels of CRP (not attached to nucleosomes) 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 for determining disease progression and the effectiveness of treatment, and elevated CRP levels have been shown in patients with diabetes, hypertension, and increased risk of cardiovascular disease. Elevated CRP levels have also been observed in patients with renal failure and inflammatory bowel disease (IBD, including Crohn's disease and ulcerative colitis), and roughly correlate with coronary heart disease. However, elevated CRP is not directly associated with heart disease, and therefore 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.

[0058] In one embodiment, the panel contains myeloperoxidase (MPO). MPO is expressed in neutrophils and granulocytes and performs its antimicrobial activity by producing hypohalite. It is stored in azurophilic granules and released into the extracellular space upon degranulation. Levels of MPO (not measured when attached to nucleosomes) have been shown to be a useful predictor of myocardial infarction and have been combined with the measurement of CRP (not measured when attached to nucleosomes) to improve accuracy in predicting myocardial infarction risk in patients.

[0059] It will be apparent to those skilled in the art that any combination of the biomarkers disclosed herein can be used in monitoring methods or apparatus, such as in-line monitoring devices, and that additional markers can be added to panels containing these markers.

[0060] (Apheresis device) The present invention provides a device configured to perform apheresis or in vitro organ perfusion comprising one or more affinity matrices for binding to and removing pathogenic substances. The term "affinity matrix" may refer to a solid support (e.g., Sepharose or polystyrene support) on which a ligand (e.g., a cfDNA-binding molecule) is immobilized, or a solid support (e.g., a water-insoluble DNA-binding polymer) formed by the ligand itself.

[0061] Affinity matrices can be placed in various affinity columns or cartridges. For example, an apheresis in vitro organ perfusion device may include one or more affinity columns having a filtration cartridge and inlet and outlet ports, where the device can capture target pathogenic substances and remove them from the patient's blood or plasma or from the organ's fluid perfusion. The inlet and outlet ports can be positioned relative to the affinity matrix such that the blood / fluid perfusion entering the inlet port must come into contact with the affinity matrix before exiting the outlet port. Preferably, the shape of the device is designed to maximize contact between the blood (or plasma) or fluid perfusion and the affinity matrix as it passes through the device. Various such designs are known in the art.

[0062] In some embodiments, the apheresis apparatus includes two or more affinity matrices. For example, a first affinity matrix can capture a first pathogen, such as nucleosome-bound cfDNA and / or exosome-bound cfDNA, and a second affinity matrix can capture unbound cfDNA. The first and second affinity matrices are arranged in any order within the apparatus.

[0063] Affinity matrices include, for example, DNA-binding proteins (e.g., H1 histone), anti-histone antibodies (e.g., anti-histone H2A antibodies), anti-nucleosome antibodies (e.g., AN-1, AN-44), DNA intercalators (e.g., Hoechst dyes), DNA-binding polymers (e.g., cationic / basic polymers, e.g., polyethyleneimine, poly-L-lysine, poly-L-arginine, hexadimethrin bromide, amino-terminated (-NH2) polyamidoamine (PAMAM) dendrimers, polypropyleneimine (PPI) dendrimers; nonionic / neutral polymers, e.g., poly Vinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP), poly(4-vinylpyridine-N-oxide); anionic / acidic polymers; linear polymers, e.g., polyethyleneimine, poly-L-lysine, poly-L-arginine; branched polymers, e.g., polybranched poly-L-lysine, polybranched polyethyleneimine; or dendrimer polymers, e.g., polyamidoamine (PAMAM) dendrimers, polypropyleneimine (PPI) dendrimers); anti-DNA antibodies (e.g., mouse monoclonal IgM anti-ds+ssDNA antibodies); lectins (e.g., snowdrop (Galanthus nivalis) lectin (GNA), daffodil (Narcissus Pseudonarcissus) lectin (NPA), etc.) mosquito Navarin A, Phytohemagglu blood Examples include nin (or cyanobilin) ​​and any combination thereof. Apheresis devices, for example, the device described in WO2019 / 053243 incorporated herein by reference, can be used together with the monitoring method or apparatus of the present invention, for example, an inline monitoring device.

[0064] (pathogen) The pathogenic material that will be removed from the target blood or organ perfusion fluid during apheresis may be cell-free DNA (cfDNA), such as nucleosome-bound cfDNA, exosome-bound cfDNA, and / or unbound cfDNA.

[0065] As described herein, nucleosome-bound cfDNA may circulate in the blood as mononucleosomes or higher-order structures, such as oligonucleosomes or chromatin fragments. Exosome-bound DNA refers to cfDNA present in exosomes, which are small membrane vesicles (30–100 nm) of exocytosis origin secreted by most cell types. Exosomes may contain single-stranded DNA (ssDNA), mitochondrial DNA (mtDNA), and / or double-stranded DNA (dsDNA) in their internal or external space. Unbound cfDNA refers to cfDNA that is not associated with another entity (i.e., circulating cfDNA that does not contain particles) and may include dsDNA, ssDNA, and oligonucleotides.

[0066] The pathogenic substances removed from the target blood or fluid perfusion during apheresis may be neutrophil extracellular traps (NETs). While NETs protect against infection by trapping invading pathogens, excessive or inadequate NETosis can be a major cause of disease, and is involved in a long and ever-growing list of disease processes, including, but not limited to, all autoimmune diseases, all inflammatory conditions, Alzheimer's disease, atherosclerosis, bacterial infections, cystic fibrosis, pancreatitis, viral infections, diabetes, cancer, thrombosis, pneumonia, respiratory infections, gout, and sepsis (see, e.g., Sollberger et al. (2018) Developmental Cell 44(5):542-553; Thalin et al. (2019) Arterioscler. Thromb. Vasc. Biol. 39:1724-1738; and Neubert et al. (2019) Frontiers in Immunology 10:12).

[0067] Inadequate production of NETs is not only associated with these diseases but is also a causative factor. The long-term presence of NETs can lead to tissue damage and the development of autoimmune reactions against NET components, resulting in inflammation, autoimmunity, and vascular disease. Cytotoxic proteases in NETs can cause endothelial damage in sepsis and small vessel vasculitis. In severe influenza, the surface of alveolar capillaries in the lungs becomes trapped in NETs and can be damaged by cytotoxic NETs-related proteins, including histones and MPO. NETs measurements in bronchoalveolar lavage fluid samples taken from patients with pneumonia and serum samples taken from patients with COVID-19 infection have shown that NETs levels are higher in hospitalized patients receiving mechanical ventilation compared to hospitalized patients breathing room air. Therefore, NETs levels can predict which patients will require high levels of respiratory support.

[0068] Most individuals infected with influenza or coronavirus experience only mild illness. However, certain 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 serious consequences, including ARS, SARS, pneumonia, and death. While the exact mechanisms by which influenza or coronavirus infection leads to complications, including pneumonia, are not clear, it is thought that an excess of NETs (Neuronal Epileptic Spectrum Disorders) triggers an excessive immune response to the viral infection, contributing to pneumonia and, in the worst cases, acute lung damage that can lead to death.

[0069] In cancer, NETs are involved as a cause of cancer-associated thrombosis and as facilitators of metastatic stage IV cancer progression through various mechanisms, including the uptake of tumor cells into NETs, ​​the promotion of NET-bound tumor cell dispersal within the body, and the establishment of metastatic cell proliferation in new sites. Therefore, it is clear that NETs are associated with and / or causative factors in a wide variety of disease processes.

[0070] In one embodiment, the subjects have a disease characterized by elevated levels of NETs and / or cfDNA in the blood. Elevated circulating cfDNA levels are recognized markers for several diseases and conditions, including, but not limited to, sepsis, cancer (including metastatic cancer), acute organ failure, organ infarction (including myocardial infarction and ischemic stroke), hemorrhagic stroke, autoimmune disorders, graft-versus-host disease (GVHD), transplant rejection, systemic inflammatory response syndrome (SIRS), multiple organ failure syndrome (MODS), traumatic injury, pro-inflammatory conditions in the elderly, diabetes mellitus, atherosclerosis, neurodegenerative diseases, eclampsia, infertility, coagulation disorders, pregnancy-related complications, and infections. Therefore, cfDNA can be removed from subjects suffering from one of these diseases using apheresis devices, such as those incorporated herein by reference WO2019 / 053243, US 9,364,601, US2007 / 0092509, or those described in the literature by Kusaoi et al., Ther. Apher. Dial. (2016) 20: 348-353.

[0071] In one embodiment, the subject is suffering from an infectious disease. In a further embodiment, the subject is suffering from sepsis or septic shock.

[0072] Infections can be, for example, respiratory tract infections. In one embodiment, respiratory tract infections are selected from influenza, pneumonia, and severe acute respiratory syndrome (SARS). SARS is a respiratory infection caused by SARS coronavirus (SARS-CoV), among others, and is associated with known coronaviruses (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 may progress to pneumonia (e.g., direct viral pneumonia or secondary bacterial pneumonia).

[0073] (Monitoring method) A further embodiment provides a method for monitoring a subject during an in vitro procedure, comprising: passing the subject's blood through an in vitro device described herein; and monitoring the level of cell-free nucleosomes in the subject's blood using a monitoring method or device of the in vitro device. In one embodiment, the in vitro procedure is an apheresis procedure, and the in vitro device is an apheresis device. In a further embodiment, the level of cell-free nucleosomes in the subject's blood is monitored using an in-line monitoring device of the apheresis device.

[0074] In one embodiment, the duration of the apheresis procedure, e.g., length, is determined based on the level of cell-free nucleosomes in the subject's blood measured using a monitoring method or device, e.g., an in-line monitoring device. The monitoring device is particularly used to monitor when the apheresis procedure should be terminated, for example, when the level of cell-free nucleosomes has reached an acceptable level. Since the subject is only attached to the apheresis device for as long as necessary, this makes the treatment more efficient and effective.

[0075] In one embodiment, multiple measurements, for example, two in-line measurements, are performed in the blood, plasma, or fluid perfusion fluid before and after the point where the blood, plasma, or fluid perfusion fluid comes into contact with the solid-phase binder of nucleosomes (for example, at positions 1 and 2 in Figure 1) to monitor the levels of both nucleosomes in the blood or fluid perfusion fluid of the organ in question, and also to monitor the effectiveness of the solid-phase binder in removing nucleosomes.

[0076] Detecting and / or quantifying the level of cell-free nucleosomes present may include determining the concentration of cell-free nucleosomes.

[0077] In one embodiment, the detection or measurement method includes (i) contacting a sample with a first binder that binds to cell-free nucleosomes or their components; and (ii) detecting or quantifying the binding of a second binder in the sample. In one embodiment, the monitoring method or apparatus, for example, an in-line monitoring apparatus, is configured to measure the level of cell-free nucleosomes by an immunochemical method or a biosensor method.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] As used herein, the term “biosensor” means any device capable of detecting the presence of a biomarker. Examples of biosensors are described herein. A biosensor may include a ligand binder or ligand described herein, capable of specific binding to a biomarker. Such biosensors are useful in the detection and / or quantification of biomarkers of the present invention.

[0084] 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.

[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] The method of the present invention, involving the detection and / or quantification of one or more biomarkers, can be carried out using a tabletop device or incorporated into a disposable diagnostic or monitoring platform that can be used in an out-of-laboratory environment, such as a physician's office or at the patient's bedside. 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 the electronic transmission of collected data to a physician for interpretation, and thus can form the basis of e-medicine.

[0089] 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.

[0090] 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 infectious diseases.

[0091] Therefore, in one embodiment, the method further includes comparing the level of cell-free nucleosomes or their components with one or more controls. For example, the method may include comparing the level of cell-free nucleosomes obtained from a subject with the level of cell-free nucleosomes obtained from a normal subject. The controls may be healthy subjects.

[0092] 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.

[0093] 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).

[0094] (Treatment method) A further embodiment provides a method for treating a disease of interest requiring the treatment thereof, comprising: passing the blood of the subject through an external device described herein; and monitoring the level of cell-free nucleosomes in the blood of the subject using a monitoring method or apparatus for the external device. In one embodiment, the external device is an apheresis device, and by passing the blood of the subject through the apheresis device, one or more pathogenic substances are removed from the blood of the subject. In a further embodiment, the level of cell-free nucleosomes in the blood of the subject is monitored using an in-line monitoring device of the apheresis device.

[0095] Apheresis is performed by passing the target blood through an apheresis device to remove one or more pathogenic substances from the target blood. Therefore, in one embodiment, the apheresis procedure is stopped (i.e., terminated) when the level of cell-free nucleosomes in the target blood is determined to be at an acceptable level.

[0096] As described herein, subjects may have a disease characterized by elevated levels of neutrophil extracellular traps and / or cfDNA in the blood. Therefore, methods of treatment involve removing these substances from the blood in order to treat the disease. Accordingly, in another embodiment, a method is provided for reducing the level of cfDNA in a patient's blood, comprising: (a) performing an apheresis procedure which involves diverting blood or plasma from the patient to an apheresis device described herein to produce purified blood or plasma with reduced levels of cfDNA; (b) returning the purified blood or plasma to the patient; and (c) monitoring the level of cell-free nucleosomes in the subject's blood using a monitoring method or device for the apheresis device, e.g., an in-line monitoring device, to determine when the level of cfDNA in the patient's blood has been sufficiently reduced. The apheresis procedure can reduce the levels of substantially all types of cfDNA in the patient's blood, including nucleosome-bound cfDNA, exosome-bound cfDNA, and unbound cfDNA (including dsDNA, ssDNA, and oligonucleotides).

[0097] The duration (i.e., length) of the apheresis procedure can be determined using a monitoring method or device, for example, based on the level of cell-free nucleosomes in the subject's blood measured using an in-line monitoring device. Furthermore, it will be understood that this method may include repeating the apheresis procedure one or more times.

[0098] Diseases that can be treated with apheresis include neurological disorders (including acute inflammatory demyelinating polyneuropathy or Guillain-Barré syndrome, chronic inflammatory demyelinating polyradiculoneuritis, demyelinating polyneuropathy with paraproteinemia, PANDAS, Sydenham chorea, chronic focal encephalitis, Lambert-Eaton myasthenia pallidum syndrome, multiple sclerosis or MS, and neuromyelitis optica), renal disorders (anti-neutrophil cytoplasmic antibody (ANCA)-associated rapidly progressive glomerulonephritis or Wegener's granulomatosis, anti-glomerular basement membrane antibody or Goodpasture syndrome, antibody-mediated kidney graft rejection, and recurrent focal neuropathy). These include atypical segmental glomerulosclerosis, hematological disorders (including atypical hemolytic uremic syndrome or aHUS, autoantibodies against factor H, hyperviscosity, severe / symptomatic cryoglobulinemia in monoclonal gammaglobulinemia or paraproteinemia, thrombotic thrombocytopenic purpura, ABO-incompatible hematopoietic stem cell transplantation, myeloma with cylindrical nephropathy, and erythroimmunoimmunity during pregnancy), immunological disorders (including fulminant antiphospholipid syndrome and systemic lupus erythematosus or SLE), and metabolic disorders (including familial hypercholesterolemia and Wilson's disease) (de Back et al. (2019) Transfusion and Apheresis Science 58: 254-257).

[0099] In some embodiments, the method is effective in treating a patient's disorder, where the disorder is selected from sepsis, cancer (including metastatic cancer), acute organ failure, organ infarction (including myocardial infarction, ischemic stroke, and hemorrhagic stroke), transplant rejection, systemic inflammatory response syndrome (SIRS); multiple organ failure syndrome (MODS); graft-versus-host disease (GVHD), traumatic injury, pro-inflammatory conditions in the elderly, diabetes mellitus, atherosclerosis, neurodegenerative diseases, autoimmune diseases, eclampsia, infertility, coagulation disorders, pregnancy-related complications, and infections. In further embodiments, the disorder is selected from sepsis, cancer, or acute organ failure. In yet another embodiment, the disorder is sepsis. In an alternative embodiment, the disorder is cancer.

[0100] In a further aspect of the present invention, an in vitro organ perfusion method or apparatus is provided that incorporates the measurement of NETs, ​​nucleosomes, or cfDNA. Organs obtained from organ donors for transplantation into recipient subjects may deteriorate during storage or transport. Mild deterioration can lead to worse outcomes for recipient patients. Severely deteriorated organs cannot be used for transplantation and must be discarded. In the United States in 2018, 13.2% of organs recovered for transplantation, including 3,755 kidneys, 278 pancreases, 707 livers, 3 intestines, 23 hearts, and 317 lungs, were discarded (Israni et al.; Am J Transplant 2019; 20(Suppl 1): 509-541. doi: 10.1111 / ajt.15678). This organ deterioration is associated with an inflammatory response in the donor organ, and the formation of NETs is a major cause of donor organ failure (Caldarone et al., Eur Respir J 2019; 53: 1801736 [https: / / doi.org / 10.1183 / 13993003.01736-2018]). To help maintain donor organ survival, organs can be perfused with plasma in vitro, and further protection can be provided to the donor organ by monitoring and removing NETs in the device and within the organ. In this embodiment of the present invention, the health and survival of donor organ grafts perfused with an extracorporeal perfusion device can be monitored by measuring NETs, ​​nucleosomes, or cfDNA. This monitoring also provides an early warning of the risk of donor organ deterioration and an indicator of the need for organ treatment (e.g., by removal of NETs).

[0101] 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.

[0102] The present invention will now be illustrated with reference to the following non-limiting embodiments. [Examples]

[0103] (Example 1) Figure 1 shows an example of inline nucleosome measurement in plasma before nucleosome and NETs adsorption by the matrix in a NETs adsorbent (e.g., containing immobilized histone H1) at position 1. Measurements may be performed at other locations (e.g., positions 2-5 shown in Figure 1, but not limited to these). Measurements may also be performed at multiple locations. Measurements are performed in whole blood or plasma before and after the NETs adsorbent to provide real-time information on both the current circulating nucleosome levels in the subject and the effect of NUCRESOME / NETs removal by the NETs adsorbent.

[0104] (Example 2) Sepsis was induced in 16 pigs by infection with a high dose of Escherichia coli (E. coli) administered intravenously over a period of 3 hours (Figures 2 and 3, 0-3 hours). The septic pigs were treated by plasmapheresis as described in WO2019053243 to remove NETs from the bloodstream. Briefly, whole blood was taken 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 a period of 5 hours (Figures 2 and 3, 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).

[0105] To determine whether the method of the present invention is effective (i) as a monitor during infection and (ii) as a monitor of therapeutic efficacy, eight plasma samples were collected hourly from each pig for measurement of circulating nucleosomes at 0–7 hours after the onset of infection.

[0106] 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 plasmapheresis 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 2 and 3).

[0107] Plasma samples were assayed for the level of nucleosomes containing histone isoform H3.1 (H3.1-nucleosome). The assay was 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.

[0108] The mean results for circulating H3.1-nucleosome levels in control and treated pigs are shown in Figure 2a. 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.

[0109] The mean results for 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 2b. These results are similar to those observed for the mean circulating H3.1-nucleosome levels measured, shown in Figure 2a.

[0110] 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 2c. For control pigs, the results in Figure 2c are similar to those in Figures 2b (and 2a), 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 2c 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.

[0111] Therefore, the combined results of Figures 2b and 2c indicate that measuring H3.1-nucleosome levels is useful as a monitor and guide for the treatment of NETosis and sepsis.

[0112] The results for circulating H3.1 nucleosome levels measured in samples taken from all 16 pigs are individually shown in Figure 3a. 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 27–526 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 3a 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.

[0113] 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 3b for all 16 pigs. As described above for Figure 2, the results shown in Figure 3b are similar to those in Figure 3a. 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 a mean result of 143 ng / ml (range 34–497 ng / ml).

[0114] During the procedure, the results for plasma H3.1-nucleosome levels measured in samples taken from within the plasmaferesis device downstream of the cartridge are individually shown in Figure 3c for all 16 pigs. The mean H3.1-nucleosome level measured 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.

[0115] The combined results of Figures 3b and 3c indicate that measuring H3.1-nucleosome levels is useful as a monitor and guide for the treatment of NETosis and sepsis.

[0116] (Example 3) 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 2. Elevated levels were observed in sepsis samples compared to healthy subjects. This is likely due to the effects of NETosis at various stages of this disease (Figure 4).

[0117] (Example 4) Plasma samples were obtained from 52 patients hospitalized at German Heart Center, Clinic at the Technical University Munich. Fourteen patients, including five non-hospitalized patients with mild illnesses who were receiving outpatient treatment or in the emergency room (ER), three patients with more serious illnesses (but not requiring intensive care) who were hospitalized in a standard, routine ward, and six patients with severe illnesses who were hospitalized and in the intensive care unit, tested positive for COVID-19 by qRTPCR. Of the six patients requiring intensive care, four did not survive.

[0118] Plasma samples were also obtained from 38 patients who tested negative for COVID-19 by qRTPCR, including 11 recruited from outpatient clinics or emergency rooms, 22 in standard wards, and 5 in intensive care units.

[0119] The plasma samples were assayed for H3.1-nucleosome levels using various formats of immunoassays described in Example 2, which utilize the same antibody. H3.1-nucleosome levels were lower in COVID-negative patients and higher in COVID-positive patients. H3.1-nucleosome levels measured in COVID-positive patients with more severe illness (in standard wards) were higher than those measured in non-hospitalized patients with mild illness. H3.1-nucleosome levels measured in COVID-positive patients with severe illness (in the intensive care unit) were higher than those measured in patients hospitalized in standard wards. Furthermore, four patients who did not survive showed four peak levels among all subjects. These data indicate that H3.1-nucleosome levels (and H3R8Cit-nucleosome levels) track the disease trajectory in human subjects and can be used to monitor nucleosome and NETs levels in response to treatment. The data for this Example 4 is published in Cavalier et al.'s publication (2021) Front. Mol. Biosci. 8:600881. doi: 10.3389 / fmolb.2021.600881.

[0120] (Example 5) Plasma samples were obtained from 20 hospitalized patients with severe COVID-19 requiring organ support in the intensive care unit (ITU), and 28 hospitalized patients with non-severe COVID-19 who did not require organ support.

[0121] The plasma samples were assayed for H3.1-nucleosome levels using various immunoassay formats described in Example 2, which utilizes the same antibody. H3.1-nucleosome levels measured in non-severe COVID-19 patients were highly elevated. However, levels measured in severe COVID-19 patients were extremely elevated and higher than those in non-severe COVID-19 patients. These data indicate that H3.1-nucleosome levels track the disease trajectory in human subjects and can be used to monitor nucleosome and NETs levels in response to treatment. The data from this Example 5 are published in Rea et al. (2021) ISTH Academy. Rea C. 07 / 17 / 21; 326469; PB0268.

[0122] (Example 6) Serial plasma samples were obtained on days 3, 7, and 10 from 20 hospitalized patients with severe COVID-19 requiring organ support. Six of these patients died within 28 days.

[0123] The plasma samples were assayed for H3.1-nucleosome levels using various immunoassay formats described in Example 2, which utilizes the same antibody. H3.1-nucleosome levels measured at admission in severely ill COVID-19 patients were extremely elevated. Furthermore, the highest levels were observed in patients who later died, and this difference was maintained for 1–7 days from admission to the ITU. These data indicate that H3.1-nucleosome levels track the disease trajectory in human subjects and can be used to monitor nucleosome and NETs levels in response to treatment and for prognosis assessment. The data from this Example 6 are published in Rea et al. (2021) ISTH Academy. Rea C. 07 / 17 / 21; 326469; PB0268.

[0124] (Example 7) Serial plasma samples were obtained from three patients with COVID-19 upon admission, daily until day 10, and weekly thereafter until discharge. One of the patients was admitted directly to the ITU. The second patient was admitted to a regular internal medicine ward and remained there for the duration of their hospital stay. The third patient was admitted to a regular ward but was transferred to the ITU on day 5 of their hospital stay.

[0125] The plasma sample was assayed for H3.1-nucleosome levels using various immunoassay formats described in Example 2, which utilize the same antibody.

[0126] In patients admitted directly to the ITU, H3.1-nucleosome levels were high upon admission (approximately 1000 ng / ml) and remained high for 10-12 days. Following successful treatment, the levels dramatically decreased thereafter (to approximately 500 ng / ml) and remained at this level. The patients were discharged from the ITU after approximately three weeks.

[0127] In the second patient admitted to a regular internal medicine ward and remaining in the hospital for the duration of their stay, the H3.1-nucleosome level measured was approximately 500 ng / ml upon admission and remained at this level for about a week. After successful treatment, the level decreased to approximately 300 ng / ml by the time of discharge on the 10th day.

[0128] In the third patient, the measured H3.1-nucleosome level was approximately 1200 ng / ml upon admission to a regular internal medicine ward. However, treatment in the internal medicine ward was unsuccessful, and the patient was transferred to the ITU on day 5. Simultaneously, the H3.1-nucleosome level increased, peaking at approximately 1400 ng / ml on day 5, reflecting the patient's deteriorating condition. Subsequent treatment in the ITU was successful, and the measured H3.1-nucleosome level decreased to approximately 800 ng / ml by day 8 and to approximately 300 ng / ml by day 24. Lower levels were maintained, and the patient was discharged from the ITU on approximately day 33.

[0129] These data demonstrate that H3.1-nucleosome levels track the disease trajectory in individual human subjects, both in terms of disease improvement and exacerbation, and can be used to monitor nucleosome and NETs levels in response to treatment and to assess prognosis. The data from this Example 7 are published in Stanford et al. (2021) ISTH Academy. Rea C. 07 / 17 / 21; 326469; PB0268.

[0130] (Clause) 1. An apheresis apparatus comprising one or more affinity matrices for removing one or more pathogenic substances from the target blood, and an in-line monitoring device for measuring the level of cell-free nucleosomes present in the target blood. 2. The apheresis apparatus according to Clause 1, wherein the inline monitoring device measures the level of cell-free nucleosomes present in the target blood in real time. 3. The apheresis apparatus according to Clause 1 or Clause 2, wherein the inline monitoring device comprises a solid phase having an immobilized binder for binding to cell-free nucleosomes. 4. The apheresis apparatus according to Clause 3, wherein the binder binds to core nucleosome features common to all or most of the nucleosomes. 5. The apheresis apparatus according to Clause 3, wherein the binder binds to the epigenetic features of cell-free nucleosomes. 6. The apheresis apparatus according to Clause 5, wherein the epigenetic features are histone isoforms (e.g., histone isoforms of coanucleosomes, in particular histone H3 isoforms) or histone post-translational modifications (e.g., histone PTMs of coanucleosomes, in particular histone H3 or H4 PTMs), specific nucleotides associated with cell-free nucleosomes, and protein adducts associated with cell-free nucleosomes. 7. The apheresis apparatus according to Clause 5 or Clause 6, wherein the epigenetic feature is a post-translational modification of histone selected from citrullination. 8. The apheresis apparatus according to any one of Clauses 1 to 7, wherein the in-line monitoring device is configured to measure the level of cell-free nucleosomes by immunochemical or biosensor method. 9. An apheresis apparatus according to any one of Clauses 1 to 8, wherein the inline monitoring device includes a panel of markers to be measured in the blood of the subject. 10. The apheresis apparatus according to any one of the clauses 1 to 9, wherein the inline monitoring device further comprises a binder for detecting one or more interleukins present in the blood of the subject, such as IL-6 and / or IL-12. 11. The apheresis apparatus according to any one of the claims 1 to 10, wherein the in-line monitoring apparatus further comprises a binder for detecting one or more protein markers, such as C-reactive protein (CRP), myeloperoxidase (MPO), D-dimer, and / or factor VII-activated protease (FSAP). 12. An apheresis apparatus according to any one of the clauses 1 to 11, wherein the pathogenic substance is cell-free DNA (cfDNA), for example, nucleosome-bound cfDNA, exosome-bound cfDNA, and / or unbound cfDNA. 13. An apheresis apparatus according to any one of the clauses 1 to 12, wherein one or more affinity matrices are arranged in one or more affinity columns. 14. An apheresis apparatus as described in any one of the clauses 1 to 13, wherein the subject is a human or an animal. 15. A method for monitoring a subject during an apheresis procedure, comprising: passing the subject's blood through an apheresis apparatus described in any one of Clauses 1 to 14; and monitoring the level of cell-free nucleosomes in the subject's blood using an in-line monitoring device of the apheresis apparatus. 16. The method according to Clause 15, wherein the length of the apheresis procedure is determined based on the level of cell-free nucleosomes in the subject's blood measured using the in-line monitoring device. 17. A method for treating a disease of a subject that requires the treatment thereof, comprising: passing the blood of the subject through an apheresis apparatus described in any one of the clauses 1 to 14; and monitoring the level of cell-free nucleosomes in the blood of the subject using an in-line monitoring device of the apheresis apparatus. 18. The method according to Clause 17, wherein the apheresis procedure is terminated when the level of cell-free nucleosomes in the blood of the subject is determined to be at an acceptable level. 19. The method according to Clause 17 or Clause 18, wherein the subject has a disease characterized by elevated levels of extracellular neutrophil traps and / or cfDNA in the blood. 20. The method described in any one of paragraphs 15 to 19, wherein the subject is a human or an animal. This application provides the invention in the following embodiments. (Aspect 1) An extracorporeal device comprising a monitoring method or apparatus for measuring the level of cell-free nucleosomes present in the blood of a target. (Aspect 2) An in vivo organ perfusion device comprising a monitoring method or apparatus for measuring the level of cell-free nucleosomes present in a liquid perfusion fluid. (Aspect 3) The apparatus according to embodiment 1, wherein the external device is an apheresis device. (Aspect 4) The apparatus according to any one of embodiments 1 to 3, further comprising one or more affinity matrices for removing one or more pathogenic substances from the target blood or the liquid perfusion fluid. (Appendix 5) The apparatus according to any one of embodiments 1 to 4, wherein the monitoring method or apparatus measures the level of cell-free nucleosomes present in the blood of the subject in real time. (Aspect 6) The apparatus according to any one of embodiments 1 to 5, wherein the monitoring method or apparatus comprises a solid phase having an immobilized binder for binding to cell-free nucleosomes. (Aspect 7) The apparatus according to embodiment 6, wherein the binder binds to core nucleosome features common to all or most nucleosomes. (Pattern 8) The apparatus according to embodiment 6, wherein the binder binds to the epigenetic features of cell-free nucleosomes. (Aspect 9) The apparatus according to embodiment 8, wherein the epigenetic features are histone isoforms (e.g., histone isoforms of coanucleosomes, particularly histone H3 isoforms) or histone post-translational modifications (e.g., histone PTMs of coanucleosomes, particularly histone H3 or H4 PTMs), specific nucleotides associated with cell-free nucleosomes, and protein adducts associated with cell-free nucleosomes. (Aspect 10) The apparatus according to embodiment 8 or embodiment 9, wherein the epigenetic feature is a post-translational modification of histone selected from citrullination. (Aspect 11) The apparatus according to any one of embodiments 1 to 10, wherein the monitoring method or apparatus is configured to measure the level of cell-free nucleosomes by an immunochemical method or a biosensor method. (Aspect 12) The apparatus according to any one of embodiments 1 to 11, wherein the monitoring method or apparatus includes a panel of markers to be measured in the blood of the subject. (Aspect 13) The apparatus according to any one of embodiments 1 to 12, wherein the monitoring method or apparatus further comprises a binder for detecting one or more interleukins, such as IL-6 and / or IL-12, present in the blood of the subject. (Aspect 14) The apparatus according to any one of embodiments 1 to 13, wherein the monitoring method or apparatus further comprises a binder for detecting one or more protein markers, such as C-reactive protein (CRP), myeloperoxidase (MPO), D-dimer, and / or factor VII-activated protease (FSAP). (Aspect 15) The apparatus according to any one of embodiments 4 to 14, wherein the pathogenic substance is cell-free DNA (cfDNA), for example, nucleosome-bound cfDNA, exosome-bound cfDNA, and / or unbound cfDNA. (Aspect 16) The apparatus according to any one of embodiments 4 to 15, wherein one or more affinity matrices are arranged in one or more affinity columns. (Aspect 17) The apparatus according to any one of embodiments 1 to 16, wherein the subject is a human or an animal. (Aspect 18) A method for monitoring a subject during an in vitro procedure, comprising: passing the subject's blood through an in vitro device described in any one of embodiments 1 to 17; and monitoring the level of cell-free nucleosomes in the subject's blood using a monitoring method or device for the in vitro device. (Aspect 19) The method according to embodiment 18, wherein the in vitro procedure is an apheresis procedure and the in vitro device is an apheresis device. (Aspect 20) The method according to embodiment 19, wherein the duration of the apheresis treatment is determined based on the level of cell-free nucleosomes in the blood of the subject measured using the monitoring method or apparatus. (Aspect 21) A method for treating a disease of which the treatment is required, comprising: passing the blood of the subject through an extracorporeal device described in any one of embodiments 1 to 17; and monitoring the level of cell-free nucleosomes in the blood of the subject using a monitoring method or apparatus for the extracorporeal device. (Aspect 22) The method according to embodiment 21, wherein the external device is an apheresis device, and by passing the target blood through the apheresis device, one or more pathogenic substances are removed from the target blood. (Aspect 23) The method according to any one of embodiments 18 to 22, wherein the in vitro treatment is stopped when it is determined that the level of cell-free nucleosomes in the blood of the subject is at an acceptable level. (Aspect 24) The method according to any one of embodiments 21 to 23, wherein the subject has a disease characterized by elevated levels of neutrophil extracellular traps and / or cfDNA in the blood. (Aspect 25) The method according to any one of embodiments 18 to 24, wherein the subject is a human or an animal.

Claims

1. An in vitro method for monitoring a subject during an apheresis procedure, the in vitro method comprising passing a sample of blood obtained from the subject during the apheresis procedure through the subject and measuring the level of cell-free nucleosomes using a monitoring device, the monitoring device comprising a solid phase having an immobilized binder that binds to the epigenetic features of cell-free nucleosomes.

2. The in vitro method according to claim 1, wherein the duration of the apheresis treatment is determined based on the level of cell-free nucleosomes in the blood of the subject measured using the monitoring device.

3. The in vitro method according to claim 1 or 2, wherein the monitoring device measures the level of cell-free nucleosomes present in the blood of the subject in real time.

4. The in vitro method according to claim 1 or 2, wherein the monitoring device is an inline monitoring device.

5. The in vitro method according to claim 1 or 2, wherein the epigenetic feature is selected from histone isoforms or histone post-translational modifications.

6. The in vitro method according to claim 5, wherein the histone isoform is H3.

1.

7. The in vitro method according to claim 5, wherein the post-translational modification of the histone is citrullination.

8. The in vitro method according to claim 1 or 2, wherein the monitoring device is configured to measure the level of cell-free nucleosomes by an immunochemical method or a biosensor method.

9. The in vitro method according to claim 1 or 2, wherein the monitoring device includes a panel of markers to be measured in the blood of the subject.

10. The in vitro method according to claim 1 or 2, wherein the monitoring device further comprises a binder for detecting one or more protein markers.

11. The in vitro method according to claim 10, wherein the protein marker is selected from C-reactive protein (CRP), myeloperoxidase (MPO), D-dimer, and / or factor VII-activated protease (FSAP).

12. The in vitro method according to claim 1 or 2, wherein the apheresis procedure comprises an apheresis device comprising one or more affinity matrices for removing one or more pathogenic substances from the blood of the subject.

13. The in vitro method according to claim 12, wherein the pathogenic substance is cell-free DNA (cfDNA).

14. The in vitro method according to claim 12 or 13, wherein the one or more affinity matrices are arranged in one or more affinity columns.

15. The in vitro method according to claim 1 or 2, wherein the subject is a human or an animal.

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