WO2022214873A1 - The method and device for purification of blood from circulating citrullinated histones and neutrophil extracellular traps (nets) - Google Patents
The method and device for purification of blood from circulating citrullinated histones and neutrophil extracellular traps (nets) Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
- A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
- A61M1/3486—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents
Definitions
- the invention provides devices and their use for removal of citrullinated histones and neutrophil extracellular traps (NETs) from patients’ blood, to limit the negative effects of circulating citrullinated histones and NETs and to treat various diseases.
- NETs neutrophil extracellular traps
- NETs Neutrophil extracellular traps
- PADs peptidyl arginine deiminases
- citrullinated histones are found in the extracellular space of neutrophils along with DNA as components of NETs (Obermayer A, et al. New aspects on the structure of neutrophil extracellular traps from chronic obstructive pulmonary disease and in vitro generation. PLoS ONE. (2014) 9:e97784. doi: 10.1371/joumal.pone.0097784).
- DNase I Endogenous deoxyribonuclease I (DNase I) enzyme activity is heavily suppressed in diseases accompanied by intensive NET formation. It was discovered that DNase I can effectively degrade established NETs, thereby abolishing their pathogenic effect.
- Fresenius (WO2017137495A1) has proposed to use a device for the extracorporeal treatment of blood comprising a solid phase on which a polypeptide is immobilized which is suitable for the inactivation of free nucleic acids. Suitable polypeptides are, for example, deoxyribonucleases.
- Santersus has provided apheresis devices and their use for substantial removal of all types of cell-free DNA (cfDNA) in patients’ blood, including nucleosome-bound cfDNA, exosome-bound cfDNA and unbound cfDNA (including double stranded DNA (dsDNA), single stranded DNA (ssDNA) and oligonucleotides), to limit the negative effects of the circulating cfDNA and to treat various diseases.
- cfDNA cell-free DNA
- dsDNA double stranded DNA
- ssDNA single stranded DNA
- oligonucleotides oligonucleotides
- DNase I-generated NET fragments can be highly cytotoxic and proinflammatory (Scozzi, D, Wang, X, Liao, F, et al. NET fragments stimulate innate immune responses that prevent lung transplant tolerance. Am J Transplant. 2019; 19: 1011- 1023; Podolska, MJ, Mahajan, A, Hahn, J, et al. Treatment with DNases rescues hidden neutrophil elastase from aggregated NETs. J Leukoc Biol. 2019; 106: 1359- 1366).
- Histones, DNA, and Citrullination Promote Neutrophil Extracellular Trap Inflammation by Regulating the Localization and Activation of TLR4, Cell Reports, Volume 31, Issue 5, 2020; Deng Q, et al., Citrullinated Histone H3 as a Therapeutic Target for Endotoxic Shock in Mice. Front. Immunol. 10:2957 (2020); Wolf Eilenberg et al., Histone citrullination as a novel biomarker and target to inhibit progression of abdominal aortic aneurysms, Translational Research, 2021). NETs are the major source of citrullinated histones in diseased patients.
- Citrullinated histones may exist in human blood being part of nucleosome particles, as part of nucleosome degradation products or being released fom nucleosome particles to the blood. Citrullinated histones may or may not be complexed with cfDNA.
- the invention provides a device configured to perform an extracorporeal blood purification comprising one or more affinity matrices, wherein said one or more affinity matrices are capable of selectively capturing citrullinated histones from blood of a subject.
- the first of said one or more affinity matrices comprises at least one antibody which specifically recognizes at least one citrullinated histone isoform.
- the first of said one or more affinity matrices comprises an antibody specific for histone HI, H1.0, HI.3, H3.1, H3R8cit, H3PanCit, H3.4 Pan Cit (R2, 8, 17, 26), H3cit (R2, R8, R17), H2Acit, or H4cit.
- the first of said one or more affinity matrices comprises an antibody specific for citrullinated residues present within the N-terminal domain and/or globular domain of Histone HI, Histone 2A, Histone 2B, Histone 3, or Histone 4.
- useful antibodies include, e.g., antibody specific for Histone 2A Cit R3, Rl l, R17, R20, Histone 3 Cit R2, 8, 17, 26, or Histone 4 Cit R3.
- said one or more affinity matrices are additionally capable of capturing endotoxin from blood of a subject.
- the invention provides a method of treating a disease in a subject in need thereof, the method comprising:
- step (b) returning the blood purified in step (a) to the subject.
- the invention provides a method of treating a disease in a subject in need thereof, the method comprising:
- step (b) returning the blood purified in step (a) to the subject, wherein the blood purification process is accompanied by an administration to the subject of a deoxyribonuclease (DNase) enzyme.
- DNase deoxyribonuclease
- Non-limiting examples of the diseases treatable by the methods of the invention include, e.g., a neurodegenerative disease, a cancer, a chemotherapy-related toxicity, an irradiation induced toxicity, an organ failure, an organ injury, an organ infarct, ischemia, an acute vascular event, a stroke, graft-versus-host-disease (GVHD), graft rejection, sepsis, systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), a traumatic injury, aging, diabetes, atherosclerosis, an autoimmune disorder, eclampsia, infertility, a pregnancy-associated complication, a coagulation disorder, and an infection.
- a neurodegenerative disease e.g., a cancer, a chemotherapy-related toxicity, an irradiation induced toxicity, an organ failure, an organ injury, an organ infarct, ischemia, an acute vascular event, a stroke, graft-versus-host-
- Figure 1 shows Table 2 as referenced in Example 2.
- the term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range.
- the allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.
- the term "device” as used herein refers to any assembly known in the art to enable the purification of liquid solutions, such as, without limitation, e.g., any hollow-ware, a column, a column matrix, a filter, a membrane, a semi-permeable material, a bead (e.g., a microbead or a nanobead), or a tubing.
- a column matrix e.g., a column matrix
- a filter e.g., a microbead or a nanobead
- a semi-permeable material e.g., a bead (e.g., a microbead or a nanobead)
- a bead e.g., a microbead or a nanobead
- affinity matrix refers to (i) a solid support into which a ligand is immobilized.
- citrullinated histones refers to any of histone molecules having arginine converted into citrulline.
- a prerequisite of NET formation is activation of the calcium-dependent peptidyl-arginine deiminase 4 (PAD4) enzyme, which mediates the conversion of positively charged arginine residues to citrulline on histone tails.
- PAD4 is known to hypercitrullinate multiple sites on histone tails including arginine residues on histones H3, H4, and H2A
- PAD4- mediated citrullination of histone H3 at arginine residues 2, 8, and 17 is predominantly associated with NET formation and associated pathologies.
- Nucleosome-bound cell-free DNA is cfDNA that is bound to a nucleosome.
- a nucleosome is a subunit of nuclear chromatin.
- Nucleosome-bound cfDNA might circulate in blood as mononucleosomes or higher order structures such as oligonucleososmes or even fragments of chromatin containing over 50-100 x 10 3 base pairs of DNA. Circulating nucleosome-bound cfDNA may originate from cells undergoing necrosis or apoptosis and from neutrophil NETosis.
- the terms “subject” and “patient” are used interchangeably and refer to animals, including mammals such as humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models.
- the subject refers to a human patient, including both genders in adult and child populations.
- the terms “treat”, “treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.
- the term “treat” also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease.
- a state, disorder or condition may also include (1) preventing or delaying the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms.
- John Wiley and Sons, Inc. Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc. : Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc. : Hoboken, NJ.
- apheresis device is configured to selectively capture citrullinated histones and nucleosomes containing citrullinated histones from blood of a subject.
- extracorporeal removal focused to maximise removal of citrullinated histones has a positive impact on the treatment of diseases characterized by elevated circulating levels of nucleosomes in the blood.
- the present disclosure provides a method for treating diseases characterized by elevated circulating citrullinated histones through the removal of substantially all types of citrullinated histones c, including those associated with nucleosome particles, nucleosome degradation products or circulating alone.
- affinity matrices or combinations thereof are able to effectively capture substantially all types of citrullinated histones
- Anti-histone antibody affinity matrix and filtration column were prepared as follows: 5 mL of spherical beads from highly cross-linked N-hydroxysuccinimide (NHS) activated 4% agarose, mean beads size of 90 micrometers (NHS-activated Sepharose 4 Fast Flow, GE Healthcare Life Sciences) were used. The activated matrix was washed twice with cold (2 - 4°C) coupling buffer (0.2 M NaHCCh, 0.5 M NaCl, pH 8.3).
- NHS N-hydroxysuccinimide
- affinity matrices wherein said affinity matrices are capable of selective capture of citrullinated histones, can efficiently remove citrullinated histones from blood of a subject presumabely through removal of free and nucleosome-bound citrullinated histones.
- Using a combination of matrixes capable of selective capture of citrullinated histones with matrixes capable of binding unmodified histones is able to almost completely remove citrullinated histones and nucleosomes from blood of a subject.
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Abstract
The invention provides devices and their use for removal of citrullinated histones and neutrophil extracellular traps (NETs) from patients' blood, to limit the negative effects of circulating citrullinated histones and NETs and to treat various diseases.
Description
THE METHOD AND DEVICE FOR PURIFICATION OF BLOOD FROM
CIRCULATING CITRULLINATED HISTONES AND M
EXTRACELLULAR TRAPS (NETs)
CROSS REFERENCE TO RELATED APPLICATIONS [001] This patent claims priority to U.S. Provisional Application No. 63/171,025, filed April 5, 2021, the disclosure of which is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[002] The invention provides devices and their use for removal of citrullinated histones and neutrophil extracellular traps (NETs) from patients’ blood, to limit the negative effects of circulating citrullinated histones and NETs and to treat various diseases.
BACKGROUND OF THE INVENTION
[003] Neutrophil extracellular traps (NETs) were discovered as extracellular strands of decondensed DNA, which were expelled from activated neutrophils. NETs have been implicated as key players into pathogenesis of an increasingly large number of human diseases including cancer, acute organ injury, kidney disease, GVH disease, stroke, thrombosis, autoimmunity, diabetes, atherosclerosis, sepsis, eclampsia, fertility, coagulopathies and neurodegeneration. Histone citrullination/deimination induced by peptidyl arginine deiminases (PADs) is an important posttranslational modification that facilitates chromatin decondensation during NET formation (6). Moreover, citrullinated histones are found in the extracellular space of neutrophils along with DNA as components of NETs (Obermayer A, et al. New aspects on the structure of neutrophil extracellular traps from chronic obstructive pulmonary disease and in vitro generation. PLoS ONE. (2014) 9:e97784. doi: 10.1371/joumal.pone.0097784).
[004] Endogenous deoxyribonuclease I (DNase I) enzyme activity is heavily suppressed in diseases accompanied by intensive NET formation. It was discovered that DNase I can effectively degrade established NETs, thereby abolishing their pathogenic effect. Fresenius (WO2017137495A1) has proposed to use a device for the extracorporeal treatment of blood comprising a solid phase on which a polypeptide is immobilized which is suitable for the inactivation of free nucleic acids. Suitable polypeptides are, for example, deoxyribonucleases.
Santersus (WO 2019/053243) has provided apheresis devices and their use for substantial removal of all types of cell-free DNA (cfDNA) in patients’ blood, including nucleosome-bound cfDNA, exosome-bound cfDNA and unbound cfDNA (including double stranded DNA (dsDNA), single stranded DNA (ssDNA) and oligonucleotides), to limit the negative effects of the circulating cfDNA and to treat various diseases.
[005] However it was recently discovered that upon digestion by DNase I, DNase I-generated NET fragments can be highly cytotoxic and proinflammatory (Scozzi, D, Wang, X, Liao, F, et al. NET fragments stimulate innate immune responses that prevent lung transplant tolerance. Am J Transplant. 2019; 19: 1011- 1023; Podolska, MJ, Mahajan, A, Hahn, J, et al. Treatment with DNases rescues hidden neutrophil elastase from aggregated NETs. J Leukoc Biol. 2019; 106: 1359- 1366). Also, DNases disintegrate the 3D structure of NETs and do not remove other NET components which remain in circulation causing substantial damage (Santocki, Michal; Kolaczkowska, Elzbieta. 2020. "On Neutrophil Extracellular Trap (NET) Removal: What We Know Thus Far and Why So Little" Cells 9, no. 9: 2079). Citrullinated histones have been recently recognized as important mediators of sepsis, septic shock and certain other debilitating conditions (Tsourouktsoglou et.al. Histones, DNA, and Citrullination Promote Neutrophil Extracellular Trap Inflammation by Regulating the Localization and Activation of TLR4, Cell Reports, Volume 31, Issue 5, 2020; Deng Q, et al., Citrullinated Histone H3 as a Therapeutic Target for Endotoxic Shock in Mice. Front. Immunol. 10:2957 (2020); Wolf Eilenberg et al., Histone citrullination as a novel biomarker and target to inhibit progression of abdominal aortic aneurysms, Translational Research, 2021). NETs are the major source of citrullinated histones in diseased patients. Citrullinated histones however may exist in human blood being part of nucleosome particles, as part of nucleosome degradation products or being released fom nucleosome particles to the blood. Citrullinated histones may or may not be complexed with cfDNA.
SUMMARY OF THU INVENTION
[006] As specified in the Background section, above, there is a need for new extracorporeal methods to maximise removal of citrullinated histones and for new more effective devices to realize such methods. The present invention addresses this and other needs by providing devices and associated processes.
[007] In one aspect, the invention provides a device configured to perform an extracorporeal
blood purification comprising one or more affinity matrices, wherein said one or more affinity matrices are capable of selectively capturing citrullinated histones from blood of a subject.
[008] In some embodiments, the first of said one or more affinity matrices comprises at least one antibody which specifically recognizes at least one citrullinated histone isoform.
[009] In some embodiments, the first of said one or more affinity matrices comprises an antibody specific for histone HI, H1.0, HI.3, H3.1, H3R8cit, H3PanCit, H3.4 Pan Cit (R2, 8, 17, 26), H3cit (R2, R8, R17), H2Acit, or H4cit.
[010] In some embodiments, the first of said one or more affinity matrices comprises an antibody specific for citrullinated residues present within the N-terminal domain and/or globular domain of Histone HI, Histone 2A, Histone 2B, Histone 3, or Histone 4. Non-limiting examples of useful antibodies include, e.g., antibody specific for Histone 2A Cit R3, Rl l, R17, R20, Histone 3 Cit R2, 8, 17, 26, or Histone 4 Cit R3.
[Oil] In some embodiments, said one or more affinity matrices are additionally capable of capturing endotoxin from blood of a subject.
[012] In a related aspect, the invention provides a method of treating a disease in a subject in need thereof, the method comprising:
(a) performing a blood purification procedure comprising diverting blood from the subject into the device of any of the above embodiments, and
(b) returning the blood purified in step (a) to the subject.
[013] In another related aspect, the invention provides a method of treating a disease in a subject in need thereof, the method comprising:
(a) performing a blood purification procedure comprising diverting blood from the subject into the device of of any of the above embodiments, and
(b) returning the blood purified in step (a) to the subject, wherein the blood purification process is accompanied by an administration to the subject of a deoxyribonuclease (DNase) enzyme.
[014] Non-limiting examples of the diseases treatable by the methods of the invention include, e.g., a neurodegenerative disease, a cancer, a chemotherapy-related toxicity, an irradiation induced toxicity, an organ failure, an organ injury, an organ infarct, ischemia, an acute vascular event, a stroke, graft-versus-host-disease (GVHD), graft rejection, sepsis, systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), a traumatic injury, aging,
diabetes, atherosclerosis, an autoimmune disorder, eclampsia, infertility, a pregnancy-associated complication, a coagulation disorder, and an infection.
[015] These and other aspects of the present invention will be apparent to those of ordinary skill in the art in the following description, claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS [016] Figure 1 shows Table 2 as referenced in Example 2.
PET ATT, ED DESCRIPTION OF THE INVENTION
Definitions
[017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [018] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure.
[019] The term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.
[020] The term "device" as used herein refers to any assembly known in the art to enable the purification of liquid solutions, such as, without limitation, e.g., any hollow-ware, a column, a column matrix, a filter, a membrane, a semi-permeable material, a bead (e.g., a microbead or a nanobead), or a tubing. The terms “column” and “cartridge” are used interchangeably herein in the context of an apheresis device.
[021] The term “affinity matrix” as used herein refers to (i) a solid support into which a ligand is immobilized.
[022] The term “citrullinated histones” refers to any of histone molecules having arginine converted into citrulline. A prerequisite of NET formation is activation of the calcium-dependent
peptidyl-arginine deiminase 4 (PAD4) enzyme, which mediates the conversion of positively charged arginine residues to citrulline on histone tails. While PAD4 is known to hypercitrullinate multiple sites on histone tails including arginine residues on histones H3, H4, and H2A, PAD4- mediated citrullination of histone H3 at arginine residues 2, 8, and 17 (H3R2,8,17Cit) is predominantly associated with NET formation and associated pathologies.
[023] “Nucleosome-bound cell-free DNA (cfDNA)” is cfDNA that is bound to a nucleosome. A nucleosome is a subunit of nuclear chromatin. Nucleosome-bound cfDNA might circulate in blood as mononucleosomes or higher order structures such as oligonucleososmes or even fragments of chromatin containing over 50-100 x 103 base pairs of DNA. Circulating nucleosome-bound cfDNA may originate from cells undergoing necrosis or apoptosis and from neutrophil NETosis.
[024] As used herein, the terms “subject” and “patient” are used interchangeably and refer to animals, including mammals such as humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models. In certain embodiments, the subject refers to a human patient, including both genders in adult and child populations.
[025] In the context of the present invention insofar as it relates to any of the disease conditions recited herein, the terms “treat”, “treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. Within the meaning of the present invention, the term “treat” also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease. The terms “treat”, “treatment”, and the like regarding a state, disorder or condition may also include (1) preventing or delaying the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms.
[026] The practice of the present invention employs, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, conjugation chemistry and biochemistry, which are within the skill of
the art. Such tools and techniques are described in detail in e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc. : Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc. : Hoboken, NJ. Hermanson (2013) Bioconjugate Techniques, 3rd ed., Academic Press; Niemeyer (2004) Bioconjugation Protocols: Strategies and Methods, Springer Science & Business Media and Hermanson et al. (1992) Immobilized Affinity Ligand Techniques, Academic Press. Additional techniques are explained, e.g., in U.S. Patent No. 7,912,698 and U.S. Patent Appl. Pub. Nos. 2011/0202322 and 2011/0307437.
Devices and Methods of the Invention
[027] As specified in the Background Section, there is a great need in the art to develop new methods and devices for maximise removal of citrullinated histones from the blood. The present disclosure addresses this and other needs by providing adevices and methods, wherein the apheresis device is configured to selectively capture citrullinated histones and nucleosomes containing citrullinated histones from blood of a subject.
[028] The use of extracorporeal removal technologies can provide an effective solution to eliminate citrullinated histones from circulation and, correspondingly, decrease the level and negative effects of circulating citrullinated histones. Haemofiltration is an extracorporeal treatment that removes blood components from patients; it is used for the treatment of conditions in which a pathogenic substance or component in the blood is causing development of diseases: see for example, Ward M.D., Conventional Apheresis Therapies: A Review Journal of Clinical Apheresis 26:230-238 (2011).
[029] Surprisingly, as demonstrated herein, extracorporeal removal focused to maximise removal of citrullinated histones has a positive impact on the treatment of diseases characterized by elevated circulating levels of nucleosomes in the blood.
[030] The present disclosure provides a method for treating diseases characterized by elevated
circulating citrullinated histones through the removal of substantially all types of citrullinated histones c, including those associated with nucleosome particles, nucleosome degradation products or circulating alone.
[031] Without wishing to be bound by theory, in certain diseases, wherein the level of circulating citrullinated histones is increased, different types of circulating citrullinated histones might act in concert by triggering different molecular pathways each leading to disease progression and patient mortality; different types and fractions of circulating citrullinated histones acting together might generate synergistic toxicity, i.e., toxic (negative) effect of two or more types of circulating citrullinated histones is greater than the sum of the negative effects of each fraction of citrullinated histones taken separately.
[032] It is further described herein that several affinity matrices or combinations thereof are able to effectively capture substantially all types of citrullinated histones,
EXAMPLES
[033] The present invention is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.
Example 1: Preparation of affinity matrixes and columns
[034] Anti-histone antibody affinity matrix and filtration column were prepared as follows: 5 mL of spherical beads from highly cross-linked N-hydroxysuccinimide (NHS) activated 4% agarose, mean beads size of 90 micrometers (NHS-activated Sepharose 4 Fast Flow, GE Healthcare Life Sciences) were used. The activated matrix was washed twice with cold (2 - 4°C) coupling buffer (0.2 M NaHCCh, 0.5 M NaCl, pH 8.3). 1000 pg of three difeerent antibodies anti-Histone H3 (Citrulline Argl7, Citrulline Arg2, Citrulline Arg8) antibody (Novus Biologicals, LLC; NB100- 57135), anti-Histone H4 (Citrulline Arg3) antibody (07-596 Sigma-Aldrich) and anti-Histone HI
antibody (sc-8030, Santa Cruz Biotechnology) were dialyzed against coupling buffer and then coupled according to the manufacturer's procedure to NHS activated Sepharose. Three cycles of washing with coupling buffer followed by 0.1 M acetate buffer (pH 4.0) were used to remove the excess of unbound antibodies.
[035] The resulting anti-Histone H3(Citl7.2.8.), anti-Histone H4(Cit3) and anti-Histone HI affinity matrixes were packed as follows:
Table 1
Example 2. Removal of citrullinated histones and nucleosomes from blood of septic patient
[036] Blood samples were collected from the patient admitted to North-Western Regional Scientific and Clinical Center (St.Petersburg, Russian Federation) with the diagnosis of COVID- 19 disease, severe course, complicated by community-acquired bilateral polysegmental viral pneumonia (80% lung damage), spontaneous tension left-sided pneumothorax, pyopneumothorax. Aliquotes of 5.0 mL of fresh heparinized blood was applied to the columns A, B, C, D, and E and allowed to flow through. Plasma was separated from collected blood and quantified for the presence of citrullinated histone H3 using an ELISA Kit (citrullinated histone H3 ELISA kit, Cayman Chemical) according to manufacturer’s instructions. The concentration of citrullinated H3 was measured by optical densitometry at 450 nm in a Multiskan FC microplate reader (Thermo Fisher). Nucleosomal DNA fraction were assested through 1% PAAG electrophoresis using E-Gel EX Invitrogen system. The results are summarized in the table below (see also Figure 1):
Table 2
[037] Thus, the use of blood-compatible affinity matrices, wherein said affinity matrices are capable of selective capture of citrullinated histones, can efficiently remove citrullinated histones from blood of a subject presumabely through removal of free and nucleosome-bound citrullinated histones. Using a combination of matrixes capable of selective capture of citrullinated histones with matrixes capable of binding unmodified histones is able to almost completely remove
citrullinated histones and nucleosomes from blood of a subject.
* * *
[038] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims. It is further to be understood that all values are approximate, and are provided for description.
[039] Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.
Claims
1. A device configured to perform an extracorporeal blood purification comprising one or more affinity matrices, wherein said one or more affinity matrices are capable of selectively capturing citrullinated histones from blood of a subject.
2. The device of claim 1, wherein the first of said one or more affinity matrices comprises at least one antibody which specifically recognizes at least one citrullinated histone isoform.
3. The device of claim 1 or claim 2, wherein the first of said one or more affinity matrices comprises an antibody specific for histone HI, H1.0, HI.3, H3.1, H3R8cit, H3PanCit, H3.4 Pan Cit (R2, 8, 17, 26), H3cit (R2, R8, R17), H2Acit, or H4cit.
4. The device of any one of claims 1-3, wherein the first of said one or more affinity matrices comprises an antibody specific for citrullinated residues present within the N-terminal domain and/or globular domain of Histone HI, Histone 2A, Histone 2B, Histone 3, or Histone 4.
5. The device of claim 4, wherein the antibody is specific for Histone 2A Cit R3, R11, R17, R20, Histone 3 Cit R2, 8, 17, 26, or Histone 4 Cit R3.
6. The device of any one of claims 1-5, wherein said one or more affinity matrices are additionally capable of capturing endotoxin from blood of a subject.
7. A method of treating a disease in a subject in need thereof, the method comprising:
(a) performing a blood purification procedure comprising diverting blood from the subject into the device of any one of claims 1-6, and
(b) returning the blood purified in step (a) to the subject.
8. A method of treating a disease in a subject in need thereof, the method comprising:
(a) performing a blood purification procedure comprising diverting blood from the subject into the device of any one of claims 1-6, and
(b) returning the blood purified in step (a) to the subject, wherein the blood purification process is accompanied by an administration to the subject of a deoxyribonuclease (DNase) enzyme.
9. The method of claim 7 or claim 8, wherein the subject has a disease selected from a neurodegenerative disease, a cancer, a chemotherapy-related toxicity, an irradiation induced toxicity, an organ failure, an organ injury, an organ infarct, ischemia, an acute vascular event, a stroke, graft-versus-host-disease (GVHD), graft rejection, sepsis, systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), a traumatic injury, aging, diabetes, atherosclerosis, an autoimmune disorder, eclampsia, infertility, a pregnancy-associated complication, a coagulation disorder, and an infection.
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