WO2020225549A1 - Procédé de prévention de l'inflammation - Google Patents

Procédé de prévention de l'inflammation Download PDF

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Publication number
WO2020225549A1
WO2020225549A1 PCT/GB2020/051100 GB2020051100W WO2020225549A1 WO 2020225549 A1 WO2020225549 A1 WO 2020225549A1 GB 2020051100 W GB2020051100 W GB 2020051100W WO 2020225549 A1 WO2020225549 A1 WO 2020225549A1
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dna
antigen
binding molecule
histone
infection
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PCT/GB2020/051100
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English (en)
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Venizelos PAPAYANNOPOULOS
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The Francis Crick Institute Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to a method for reducing inflammation in a subject using an antigen-binding molecule which is capable of binding to chromatin and preventing a TLR receptor binding to a histone protein, a DNA molecule, and/or a complex thereof or alternatively an endonuclease which prevents DNA-mediated recruitment of the TLR or promotes the proteolytic degradation of proinflam matory histones.
  • the invention also relates to such an antigen-binding molecule or endonuclease and uses thereof in reducing inflammation.
  • Immune cells control infections but can also drive chronic inflammatory disease.
  • Inflammation associated with infection and sterile inflammatory disease depends employs the same array of pro-inflammatory cytokines. During infection, these molecules orchestrate the recruitment of immune cells that kill microbes. In chronic inflammatory diseases, the same molecules recruit immune effector cells that cause tissue pathology. Therefore, while targeting these cytokines therapeutically might reduce pathogenic disease, it also interferes with beneficial immune responses. An increase in serious infections is a prominent side effect of many such cytokine-targeting therapies.
  • Immune cells employ an array of pattern recognition receptors to sense their environment and regulate their activation.
  • Toll-like receptors TLRs
  • PAMPs pathogen-associated molecular patterns
  • NETs neutrophil extracellular traps
  • NETs are composed of highly decondensed chromatin and many other proteins, some of which have been linked to inflammation such as LL-37, S100A8 and HMGB1.
  • NET chromatin is also modified. NETs were first shown to activate plasmacytoid dendritic cells and induce type-l interferons that contribute to autoimmunity. Oxidation of NET DNA during NET formation increases its interferonic capacity via the cytoxolic cGas/STING pathway, although it remains unclear how these extracellular agonists gain access to the cytosol.
  • NETs drive sterile inflammation by priming macrophages to produce I L- 1 b .
  • This priming mechanism has been implicated in the initiation of chronic sterile inflammatory diseases such as atherosclerosis and gout disease.
  • histones are the major proinflammatory constituent in extracellular chromatin as found in NETs and comprise major drivers of sterile inflammatory diseases such as atherosclerosis.
  • histones also exacerbate inflammation during infection despite the presence of PAMPs.
  • histone proteins without complexed DNA lost the capacity to potently activate pro-inflammatory monocytes.
  • the present inventors surprisingly found that histones induce cytokine expression by binding and activating toll-like receptor (TLR) 4. Moreover, the inventors found that DNA was required for the induction of pro-inflammatory cytokines by histones as it promoted the recruitment of TLR4 to histone-containing endosomes.
  • the present invention is predicated on the principle of preventing the activation of monocytes, macrophages, other immune cells and other pro-inflammatory reactions, by chromatin and its derivatives, particularly histones and DNA, based on the principle of prevention of activation of TLRs by chromatin, or prevention of DNA-mediated recruitment of TLRs or promotion of the proteolytic degradation of proinflammatory histones.
  • the invention is based on the surprising finding that it is histones that are the major proinflammatory constituent in extracellular chromatin, and that preventing TLR recruitment and/or activation by a histone protein, a DNA molecule, and/or a complex thereof, may have an advantageous therapeutic effect.
  • the present inventors have surprisingly found that antigen-binding molecules with particular characteristics as described herein are able to reduce inflammation in a subject.
  • the present invention therefore provides in one aspect a method for reducing, preventing or delaying inflammation in a subject, comprising administering to said subject a therapeutically effective amount of an antigen-binding molecule which is capable of preventing a toll-like receptor (TLR) binding to a histone protein, a DNA molecule, and/or a complex thereof, wherein said antigen-binding molecule is not capable of leading to signalling via an Fc receptor.
  • TLR toll-like receptor
  • the said antigen-binding molecule is not capable of leading to signalling and activation of immune effector cells via an Fc receptor.
  • the invention provides an antigen-binding molecule which is capable of preventing a TLR binding to a histone protein, a DNA molecule, and/or a complex thereof, for use in reducing inflammation in a subject, wherein said antigen-binding molecule is not capable of leading to signalling via an Fc receptor. In one aspect the said antigen-binding molecule is not capable of leading to signalling and activation of immune effector cells via an Fc receptor.
  • the invention provides the use of an antigen-binding molecule which is capable of preventing a TLR binding to a histone protein, a DNA molecule, and/or a complex thereof, for use in the manufacture of a medicament for reducing inflammation in a subject, wherein said antigen-binding molecule is not capable of leading to signalling via an Fc receptor.
  • said antigen-binding molecule is not capable of leading to signalling and activation of immune effector cells via an Fc receptor.
  • the invention provides the use of an antigen-binding molecule which is capable of preventing a TLR binding to a histone protein, a DNA molecule, and/or a complex thereof, for reducing inflammation in a subject, wherein said antigen-binding molecule is not capable of leading to signalling via an Fc receptor.
  • said antigen-binding molecule is not capable of leading to signalling and activation of immune effector cells via an Fc receptor.
  • the invention provides an antigen-binding molecule which is capable of preventing a TLR binding to a complex of histone protein and DNA, and which is not capable of leading to signalling via an Fc receptor.
  • the said antigen-binding molecule is not capable of leading to signalling and activation of immune effector cells via an Fc receptor.
  • the invention provides a method of preventing a TLR binding to a complex of histone protein and DNA, whereby histone-bound DNA- mediated TLR recruitment to an endosome is prevented with an endonuclease.
  • a TLR binding to a complex of histone protein and DNA whereby histone-bound DNA- mediated TLR recruitment to an endosome is prevented with an endonuclease.
  • the invention provides a method for reducing, preventing or delaying inflammation in a subject, comprising administering to said subject a therapeutically effective amount of an endonuclease.
  • said endonuclease is capable of preventing TLR recruitment to an endosome, for example an endosome containing an internalised nucleosome.
  • the invention provides an endonuclease for use in reducing inflammation in a subject.
  • the invention provides the use of an endonuclease in the manufacture of a medicament for reducing inflammation in a subject.
  • the invention provides the use of an endonuclease for reducing inflammation in a subject.
  • the endonuclease promotes the proteolytic degradation of pro-inflammatory histones.
  • a method to clear histones from circulation for example to reduce inflammatory cytokines.
  • This is a specific mechanistic endpoint which may be central in the treatment of multiple inflammatory conditions.
  • C Cells from (A) were fixed and immunostained for citrullinated histone H3 (cit-H3, green), myeloperoxidase (MPO, red), neutrophil elastase (NE) and DNA (DAPI, blue). Scale bar: 50 pm.
  • E Representative micrographs of consecutive aortic sections (upper and lower panel) from ApoE/PAD4 deficient animals after 6 weeks on high fat diet, fluorescently immunostained for the neutrophil marker Ly6G (yellow, top panel) which is also found in NETs, the anti chromatin PL2-3 antibody (magenta), antibodies against citrullinated histone H3 (cit-H3, cyan) and MPO (yellow, lower panel), and DAPI (blue). Scale bar: 50 pm.
  • FIG. 1 NET-mediated activation of human primary monocytes depends on citrullinated histones and DNA
  • G I L1 -b protein expression in a bronchial alveolar lavage (BAL) of lungs of WT and transgenic mice expressing H2B-GFP 24 hrs after infection with WT or yeast-locked (YL) Ahgd Candida albicans fungi.
  • ns p>0.05, *: p ⁇ 0.05, **: p ⁇ 0.01 , ***: p ⁇ 0.001.
  • C Aortic root cross-sections from female ApoE or ApoE/PAD4 deficient animals either untreated or treated with vehicle (veh, Fab elution buffer) or an anti-chromatin PL2-3 mouse Fab (a-chro) for 6 weeks on high fat diet (HFD). Sections were stained with the lipid dye Oil Red O (red) and counterstained with haematoxylin. Scale bar: 200pm. Images are representative of >5 animals per group.
  • (C) Total I L-1 b induced in primary human monocytes stimulated with cit-H3 (0.03mM) and NET DNA (20ng) in the presence or absence of anti-TLR2 and anti-TLR4 neutralizing antibodies. Statistical analysis by two tailed student t-test. Data are representative of three independent experiments.
  • (D) Total I L-1 b induced in primary human monocytes either untreated or stimulated with recombinant cit-H3 (0.3mM) alone in the presence of anti-TLR2 and anti-TLR4 neutralizing antibodies or ODNi, or all inhibitors together. Statistical analysis by two tailed student t-test. Data are representative of three independent experiments.
  • TLR4-mediated NF-KB activity assessed in a heterologous HEK-Blue cell system, by NF-KB-driven induction of secreted embryonic alkaline phosphatase (SEAP).
  • SEAP embryonic alkaline phosphatase
  • TLR4 HEK-Blue reporter cells were treated with different fragments of purified HL-60 chromatin or mouse GFP-tagged nucleosomes or LPS (0.5ng/ml).
  • E TLR4 HEK-Blue reporter line stimulated with LPS or human optimized CpG (1 mM) or recombinant citrullinated H3 alone or in complex with NET DNA (20ng). Data are representative of three independent experiments
  • HEK cells expressing TLR4-HA or TLR2-HA were incubated with recombinant H3 (1 mM) fused to maltose-binding protein (MBP-H3) alone or complexed to NET DNA (80ng). Cells were lysed, interacting proteins were pulled down with amylose beads and western immunoblotted with antibodies against HA. The total cell lysate (input) and the pulldown are depicted. Input was diluted 20x more than the pulldown.
  • HEK cells expressing TLR4-HA were incubated alone (-) or with either human mono- nucleosomes (1 mM) or mouse GFP-mono-nucleosomes (1 mM) either unprocessed or citrullinated. Lysates were immunoprecipitated using an antibody against histone H3 and immunoblotted with antibodies against HA.
  • ns p>0.05, *: p ⁇ 0.05, **: p ⁇ 0.01 , ***: p ⁇ 0.001.
  • TLR4 is required for the recognition of histones in mouse macrophages independently of DNA.
  • BMDMs bone marrow derived mouse macrophages
  • C Total I L-1 b induced in bone marrow derived mouse macrophages (BMDMs) from either WT, TLR4, TLR9 or STING deficient mice, incubated with increasing concentrations of cit-H3 alone or in the presence of purified NET-DNA or human-preferred CpG (hCpG).
  • BMDMs bone marrow derived mouse macrophages
  • ns p>0.05, *: p ⁇ 0.05, **: p ⁇ 0.01 , ***: p ⁇ 0.001.
  • TLR4 Analysis of TLR4 localization in human monocytes, human macrophages, or murine BMDMs.
  • TLR4 is intracellular (intraTLR4) whereas in the murine cells TLR4 (extraTLR4) is predominately localized on the plasma membrane.
  • Figure 7 Synergy between histone and DNA is required to ensure monocyte activation below the histone cytotoxicity threshold.
  • mRNA levels were analyzed by RT-PCR, normalized to HPRT1 expression. Statistical analysis by two- tailed t-test.
  • ns p>0.05, *: p ⁇ 0.05, **: p ⁇ 0.01 , ***: p ⁇ 0.001.
  • a and B TLR2-mediated NF-KB activity assessed in a heterologous HEK-Blue cell system, by NF-KB-driven induction of secreted embryonic alkaline phosphatase (SEAP).
  • SEAP secreted embryonic alkaline phosphatase
  • BMDMs from WT, TLR4 or TLR9 deficient mice were plated and incubated with the indicated concentrations of fluorescent human and mouse optimized CpG-FITC alone or in the presence of 10- 7 M histone H3 for 2 hours. Cells were washed, lysed and captured CpG fluorescence was measured using a plate reader.
  • Figure 12 (A) NETs (outlined by dotted lines) in representative micrographs of consecutive aortic sections (left and right panel) from ApoE (upper row) and ApoE/PAD4 (lower row) deficient animals after 16 weeks on high fat diet, fluorescently immunostained for the neutrophil marker Ly6G (yellow, left column) the anti-chromatin PL2-3 antibody (magenta), antibodies against citrullinated histone H3 (cit-H3, cyan) and MPO (yellow, right column), and DAPI (blue). Scale bar: 100 pm.
  • ns p>0.05, *: p ⁇ 0.05, **: p ⁇ 0.01 , ***: p ⁇ 0.001 , ***:p ⁇ 0.0001.
  • Figure 13 (A) Total I L-1 b expression induced in human monocytes with nucleosomes at the indicated chromatin sizes purified from WT C57/BI6 mice or GAG::H2B-EGFP mice or LPS (55pg/ml). (B) IL-1 b in the BAL of WT and transgenic mice expressing an EGFP-tagged H2B fusion protein ( GAG::H2B-EGFP )) infected intratracheally with C. albicans for 24 hours. Statistical analysis by Mann-Whitney test.
  • E IFN-a protein in the supernatant of primary human monocytes stimulated with LPS (0.5ng/ml), or CpG, or citrullinated histone H3 alone or pre-complexed with NET DNA.
  • F IFN-a protein in the supernatant of primary human monocytes stimulated with LPS (0.5ng/ml), or O.dmM mono-nucleosomes alone or treated with proteinase K (PK) or benzonase (Benzo).
  • LPS 0.5ng/ml
  • PK proteinase K
  • Benzo benzonase
  • G IFN-b protein in the supernatant of primary human monocytes stimulated with LPS (0.5ng/ml), or CpG, or citrullinated histone H3 alone or pre-complexed with NET DNA.
  • H IFN-b protein in the supernatant of primary human monocytes stimulated with LPS (0.5ng/ml), or O.dmM mono-nucleosomes alone or treated with proteinase K (PK) or benzonase (Benzo).
  • LPS 0.5ng/ml
  • PK proteinase K
  • Benzo benzonase
  • Figure 14 DNA promotes synergy by promoting intracellular TLR4 translocation to histone- containing endosomes.
  • TLR4 Analysis of TLR4 localization by flow cytometry in human monocytes by flow cytometry depicting cell surface expression of TLR4 (extraTLR4), intracellular (intraTLR4) and total protein (totalTLR4).
  • ns p>0.05, *: p ⁇ 0.05, **: p ⁇ 0.01 , ***: p ⁇ 0.001 , ***:p ⁇ 0.0001.
  • Figure 15 DNase I treatment clears free circulating nucleosomes and blocks inflammation in a murine model of systemic candidiasis.
  • ns p>0.05, *: p ⁇ 0.05, **: p ⁇ 0.01 , ***: p ⁇ 0.001.
  • Figure 17 schematic of mechanisms of inhibition of chromatin-mediated proinflammatory signalling.
  • Chromatin is a complex involving proteins and DNA found in eukaryotic cells. Its primary function is packaging very long DNA molecules into a more compact, denser shape, which prevents the strands from becoming tangled and plays important roles in reinforcing the DNA during cell division, preventing DNA damage, and regulating gene expression and DNA replication.
  • chromatin facilitates proper segregation of the chromosomes in anaphase; the characteristic shapes of chromosomes visible during this stage are the result of DNA being coiled into highly condensed networks of chromatin.
  • the antigen-binding molecule is capable of binding to chromatin or components thereof as described herein.
  • One skilled in the art will be able to determine using routine methods whether antigen-binding molecule is capable of binding to chromatin, histone/DNA complex and/or nucleosome.
  • the level or production of I L-1 b may be assessed.
  • the antigen-binding molecule according to the present invention may lead to a decrease in the level of I L- 1 b , as described in the Examples.
  • the primary protein components of chromatin are histones, which bind to DNA and function as "anchors" around which the DNA strands are wound.
  • Histones are proteins that condense and structure the DNA of eukaryotic cell nuclei into units called nucleosomes. Their main functions are to compact DNA and regulate chromatin, therefore impacting gene regulation.
  • the antigen-binding molecule is capable of binding to a nucleosome.
  • the antigen-binding molecule is capable of binding to a histone protein, DNA molecule, and/or complex thereof.
  • H1/H5 Five major families of histones exist: H1/H5, H2A, H2B, H3, and H4.
  • Histones H2A, H2B, H3 and H4 are known as the core histones, and they come together to form one nucleosome.
  • the nucleosome core is formed of two H2A-H2B dimers and a H3-H4 tetramer.
  • Histones H1/H5 are known as the linker histones.
  • the core histones exist as dimers, which are similar in that they all possess the histone fold domain: three alpha helices linked by two loops. It is this helical structure that allows for interaction between distinct dimers, particularly in a head-tail fashion (also called the handshake motif). The resulting four distinct dimers then come together to form one octameric nucleosome core,
  • the antigen-binding molecule is capable of binding to a complex of DNA and histone H2A, and/or H2B. In one aspect the antigen-binding molecule is capable of binding to a nucleosome which is a complex of DNA and histone H1/H5, H2A, H2B, H3, or H4.
  • the antigen-binding molecule is capable of binding to a complex of DNA and histone H3.
  • the antigen-binding molecule is capable of binding to a complex of DNA and histone H4.
  • the histone protein may be modified.
  • the modification is citrullination.
  • Citrullination or deimination is the conversion of the amino acid arginine in a protein into the amino acid citrulline.
  • TLRs Toll-like receptors
  • the TLRs include TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11 , TLR12, and TLR13.
  • the present inventors have surprisingly found that the interaction of TLRs with complexes of histone proteins and DNA may potentiate pro-inflammatory responses in a subject.
  • the invention relates to preventing the interaction of TLRs with the complex of histone and DNA.
  • the invention relates to an antigen-binding molecule which is capable of preventing TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11 , TLR12 or TLR13 binding to a histone protein, DNA molecule and/or complex thereof.
  • the invention relates to an antigen-binding molecule which is capable of preventing TLR2 or TLR4 binding to a histone protein, DNA molecule, and/or complex thereof. In one aspect the invention relates to an antigen-binding molecule which is capable of preventing TLR2 and TLR4 binding to a histone protein, DNA molecule, and/or complex thereof.
  • an antigen binding molecule can prevent the interaction of a TLR with a histone protein, DNA molecule, and /or complex thereof.
  • one skilled in the art could utilise the methods set out in the present Examples.
  • antigen binding molecule (antibody PL2-3) which binds a complex of the H2A and H2B proteins and DNA is known in the art, see Losman et al. J Immunol. March 1 , 1992, 148 (5) 1561-1569.
  • the present invention involves an antigen-binding molecule.
  • One skilled in the art would be aware of how to design a suitable antigen-binding molecule according to the invention using routine methods, for example as described in the present Examples.
  • the antigen-binding molecule may be an antibody or a fragment thereof.
  • Antibody includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity.
  • immunoglobulin Ig may be used interchangeably with “antibody”.
  • antibody fragment comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • the “Fc” fragment comprises the portion of the antibody carboxy to the hinge sequence of the heavy chain.
  • An alternative definition known to one skilled in the art includes the hinge region of an antibody.
  • the effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
  • the antibody may be a human antibody.
  • a "human antibody” refers to an antibody naturally existing in humans, a functional fragment thereof, or a humanized antibody, i.e., a genetically engineered antibody a portion of which (e.g., Fc region) derives from a naturally- occurring human antibody.
  • a "humanized antibody” is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human.
  • An example of a humanised antibody in which all but the variable domain sequences of a non human antibody are replaced with human sequences is typically reffered to as a chimeric antibody.
  • the amino acids considered to confer the binding properties of a non human antibody are transferred to a human antibody.
  • non-human amino acid residues are often referred to as "import” or“donor” residues, which are typically taken from an “import” or“donor” (non human) variable domain.
  • a humanised antibody typically is comprised of at least one non-human derived hypervariable region sequence replacing for the corresponding sequence of a human antibody in addition to key non human derived framework sequences considered to confer the binding specificity. Accordingly, such "humanized” antibodies are chimeric antibodies wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non human species.
  • humanised antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the antibody is a monoclonal antibody.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations or modifications that may be present in minor amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies may be prepared by the hybridoma methodology (Kohler et al., Nature, 256:495 (1975)), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells. Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581 -597 (1991), for example.
  • Monoclonal antibodies may also be produced by recombinant DNA methods that are known in the art.
  • DNA encoding suitable monoclonal antibodies may be isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies) and inserted into suitable expression vectors.
  • In vitro methods are also suitable for preparing monovalent antibodies.
  • Digestion of antibodies to produce fragments thereof, particularly Fab fragments can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a F(ab')2 fragment and a pFc' fragment.
  • Monoclonal antibodies may include“chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the same biological activity.
  • Antibody fragments may also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the binding activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody fragment must possess a bioactive property, such as binding activity, regulation of binding at the binding domain, etc. Functional or active regions of the antibody may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods will be known to one skilled in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody fragment.
  • Antibodies may be humanized antibodies or human antibodies.
  • Humanized forms of non human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab' or other antigen-binding sub-sequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody non-human species
  • Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody may comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non human. These non-human amino acid residues are often referred to as“import” residues, which are typically taken from an“import” variable domain. Humanization can be essentially performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. As such,“humanized” antibodies are chimeric antibodies wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Transgenic animals may be used to produce a full repertoire of human antibodies in the absence of endogenous immunoglobulin production.
  • endogenous immunoglobulin production e.g., mice
  • the homozygous deletion of the antibody heavy chain joining region gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production.
  • Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge.
  • Human antibodies can also be selected from human antibody phage display libraries.
  • Synthetic antibody as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • the antigen-binding molecule may alternatively be an antibody mimetic, such as a scaffold which can be engineered to have an antigen binding surface. In one aspect this may be a Zn-finger domain. In one aspect the antigen-binding molecule may be a VHH antibody (or nanobody), which is the antigen binding fragment of heavy chain only antibodies.
  • the antigen-binding molecule may be capable of binding multiple antigens. In one aspect the antigen-binding molecule may bind more than one TLR.
  • the antigen-binding molecule may comprise a second moiety, for example albumin or PEG or an alternative half-life-extending moiety.
  • the antigen binding molecule is not capable of leading to signalling and/or activation via an Fc receptor of immune effector cells.
  • One skilled in the art would be able to determine whether an antigen-binding molecule binds to an Fc receptor and leads to signalling and/or activation of immune effector cells using methods known in the art.
  • An Fc receptor is a protein found on the surface of certain cells - including, among others, B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells - that contribute to the protective functions of the immune system.
  • Fc receptors bind to antibodies that are attached to infected cells or invading pathogens. Their activity stimulates phagocytic or cytotoxic cells to destroy microbes, or infected cells by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity.
  • Some viruses such as flaviviruses use Fc receptors to help them infect cells, by a mechanism known as antibody-dependent enhancement of infection.
  • Fc receptors There are several Fc receptors, classified according to the IgG type to which they bind, and also the cells which express them. Fc receptors include FcyRI, FcyRIIA, FcyRIIBI ,
  • FcyRIIB2 FcyRIIIA, FcyRIIIB, FcsRI, FcsRII, FcaRI, and Fca/pR.
  • Fc receptor is FcyRIIIA or FcyRIIIB.
  • the antigen-binding molecule does not comprise a functional Fc region.
  • the antigen-binding molecule does not comprise an Fc region.
  • the antigen binding molecule is a Fab fragment of an antibody.
  • the antigen binding molecule has an Fc region which is not capable of binding to an Fc receptor.
  • the antigen-binding molecule is not capable of binding to any one or more of FcyRI, FcyRIIA, FcyRIIBI , FcyRI I B2, FcyRIIIA, FcyRIIIB, FcsRI, FcsRII, FcaRI, Fca/pR and FcRn.
  • the antigen binding molecule has an Fc region which does not lead to signalling when bound to an Fc receptor.
  • the Fc region may contain mutations which render the Fc region“effector silent”, that is no effector functions are obtained when the antigen-binding molecule binds to an Fc receptor.
  • the antigen-binding molecule does not lead to Fc effector functions.
  • the Fc domain is mutated relative to the wild-type antibody.
  • the mutations lead to a loss of function or loss of binding in respect of the Fc region of the antibody.
  • the effect of mutation may be assessed by known techniques or assays, for example FcgRIII binding.
  • the antigen-binding molecule is not a full-length antibody.
  • the antigen-binding molecule does not lead to signalling via an Fc receptor. In one aspect the antigen-binding molecule does not lead to signalling via any one or more of FcyRI, FcyRIIA, FcyRIIBI , FcyRIIB2, FcyRIIIA, FcyRIIIB, FcsRI, FcsRII, FcaRI, and Fca/pR.
  • said signalling is immune signalling, that is signalling which is involved in, or results in, an immune response.
  • the antigen-binding molecule does not lead to signalling and activation via an Fc receptor of immune effector cells.
  • immune effector cells is meant immune cells, which, once activated, modulate downstream immune and inflammatory signalling, for example monocytes, macrophages and other inflammatory cells.
  • the present invention may be used to reduce inflammation in a subject.
  • the subject may have an inflammatory disease.
  • the inflammation may be chronic inflammation.
  • the inflammation may be sterile inflammation, that is, inflammation not caused by a pathogen.
  • the inflammation may be associated with an inflammatory disease selected from the group consisting of atherosclerosis, cystic fibrosis, fibrosis, inflammatory bowel disease, allergic rhinitis, sinusitis, atopic dermatitis, gout, psoriasis, arthritis, inflammatory lung disease, interstitial lung disease, and sarcoidosis.
  • an inflammatory disease selected from the group consisting of atherosclerosis, cystic fibrosis, fibrosis, inflammatory bowel disease, allergic rhinitis, sinusitis, atopic dermatitis, gout, psoriasis, arthritis, inflammatory lung disease, interstitial lung disease, and sarcoidosis.
  • said inflammatory disease is an autoimmune disease.
  • said subject may have an autoimmune disease. In one aspect said
  • autoimmune disease may be selected from rheumatoid arthritis (RA), myasthenia gravis (MG), multiple sclerosis (MS), systemic lupus erythematosus (SLE), autoimmune thyroiditis (Hashimoto's thyroiditis), Graves' disease, inflammatory bowel disease, autoimmune uveoretinitis, polymyositis and certain types of diabetes, systemic vasculitis, polymyositis- dermatomyositis, systemic sclerosis (scleroderma), Sjogren's Syndrome, ankylosing spondylitis and related spondyloarthropathies, rheumatic fever, hypersensitivity pneumonitis, allergic bronchopulmonary aspergillosis, inorganic dust pneumoconioses, sarcoidosis, autoimmune hemolytic anemia, immunological platelet disorders, cryopathies such as cryofibrinogenemia and autoimmune polyendocrinopathies.
  • RA r
  • the autoimmune disease is rheumatoid arthritis (RA).
  • the inflammatory disease is an allergy.
  • Said allergy may be selected from hay fever, extrinsic asthma, insect bite and sting allergies, food and drug allergies, allergic rhinitis, bronchial asthma chronic bronchitis, anaphylactic syndrome, urticaria, angioedema, atopic dermatitis, allergic contact dermatitis, erythema nodosum, erythema multiforme, Stevens- Johnson Syndrome, rhinoconjunctivitis, conjunctivitis, cutaneous necrotizing venulitis, inflammatory lung disease and bullous skin diseases.
  • the inflammatory disease is cystic fibrosis.
  • the inflammatory disease is bacterial keratitis.
  • the inflammatory disease is septic shock.
  • the inflammatory disease is atherosclerosis.
  • atheroscleoric plaque formation may be reduced or prevented, for example plaque size or area may be reduced.
  • the invention as described herein provides a method for reducing atherosclerotic plaque size and/or formation. In one aspect is provided a method for preventing atherosclerotic plaque formation.
  • the level of I L1 b may be reduced in a subject, that is to say that I L1 b production may be inhibited in a subject.
  • the level of IL6 may be reduced in a subject, that is to say that IL6 production may be inhibited in a subject.
  • the inflammatory disease is due to an infection, such as a fungal infection, for example a pulmonary fungal infection.
  • an infection such as a fungal infection, for example a pulmonary fungal infection.
  • the infection is Candida albicans.
  • the inflammatory disease is sepsis.
  • the disease is pulmonary infection.
  • the disease is a viral infection.
  • the virus may be a coronavirus, for example the COVID-19 coronavirus.
  • the subject has cancer.
  • the cancer may be, for example, bladder cancer, gastric, oesophageal, breast cancer, colorectal cancer, cervical cancer, ovarian cancer, endometrial cancer, kidney cancer (renal cell), lung cancer (small cell, non-small cell and mesothelioma), brain cancer (e.g. gliomas, astrocytomas, glioblastomas), melanoma, lymphoma, small bowel cancers (duodenal and jejunal), leukemia, pancreatic cancer, hepatobiliary tumours, germ cell cancers, prostate cancer, head and neck cancers, thyroid cancer and sarcomas.
  • the subject is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit or guinea pig, but most preferably the subject is a human.
  • the subject is female, for example in the event that the invention is used for treating atherosclerosis.
  • treatment refers to reducing, alleviating or eliminating one or more symptoms of the disease which is being treated, relative to the symptoms prior to treatment.
  • Prevention refers to delaying or preventing the onset of the symptoms of the disease. Prevention may be absolute (such that no disease occurs) or may be effective only in some individuals or for a limited amount of time.
  • the present invention further provides a composition which comprises an antigen-binding molecule as described herein, for example an antigen-binding molecule which is capable of preventing a TLR binding to a histone protein, a DNA molecule, and/or a complex thereof, wherein said antigen-binding molecule is not capable of leading to signalling and activation via an Fc receptor.
  • an antigen-binding molecule as described herein, for example an antigen-binding molecule which is capable of preventing a TLR binding to a histone protein, a DNA molecule, and/or a complex thereof, wherein said antigen-binding molecule is not capable of leading to signalling and activation via an Fc receptor.
  • the composition may be used in any of the methods or uses as described herein.
  • compositions as described herein may be a pharmaceutical composition additionally comprising a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
  • Such a formulation may, for example, be in a form suitable for intravenous infusion.
  • compositions are administered using any amount and by any route of administration effective for preventing or treating a subject for an inflammation or an autoimmune disease.
  • An effective amount refers to a sufficient amount of the composition to beneficially prevent or ameliorate the symptoms of the disease or condition.
  • the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect in a subject. Additional factors which may be taken into account include the severity of the disease state, e.g., liver function, cancer progression, and/or intermediate or advanced stage of macular degeneration; age; weight; gender; diet, time; frequency of administration; route of administration; drug combinations; reaction sensitivities; level of immunosuppression; and
  • compositions are administered, for example, hourly, twice hourly, every three to four hours, daily, twice daily, every three to four days, every week, or once every two weeks depending on half-life and clearance rate of the particular composition.
  • the active agents of the pharmaceutical compositions of embodiments of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage.
  • dosage unit form refers to a physically discrete unit of active agent appropriate for the patient to be treated.
  • the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the therapeutically effective dose is estimated initially either in cell culture assays or in animal models, potentially mice, pigs, goats, rabbits, sheep, primates, monkeys, dogs, camels, or high value animals.
  • the cell-based, animal, and in vivo models provided herein are also used to achieve a desirable concentration, total dosing range, and route of administration. Such information is used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active agent that ameliorates the symptoms or condition or prevents progression of the disease or condition.
  • Therapeutic efficacy and toxicity of active agents are determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 5 o (dose therapeutically effective in 50% of the population) and LD 5 o (dose lethal to 50% of the population).
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which is expressed as the ratio, LD 50/ED 50.
  • Pharmaceutical compositions having large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use.
  • the pharmaceutical composition or methods provided herein is administered to humans and other mammals for example topically for skin tumors (such as by powders, ointments, creams, or drops), orally, rectally, mucosally, sublingually, parenterally, intracisternally, intravaginally, intraperitoneally, intravenously,
  • Injections of the pharmaceutical composition include intravenous, subcutaneous, intra muscular, intraperitoneal, or intra-ocular injection into the inflamed or diseased area directly, for example, for esophageal, breast, brain, head and neck, and prostate inflammation.
  • Liquid dosage forms are, for example, but not limited to, intravenous, ocular, mucosal, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms potentially contain inert diluents commonly used in the art such as, for example, water or other solvents; solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used
  • the ocular, oral, or other systemically-delivered compositions also include adjuvants such as wetting agents, emulsifying agents, and suspending agents.
  • Dosage forms for topical or transdermal administration of the pharmaceutical composition herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches.
  • the active agent is admixed under sterile conditions with a pharmaceutically acceptable carrier. Preservatives or buffers may be required.
  • ocular or cutaneous routes of administration are achieved with aqueous drops, a mist, an emulsion, or a cream.
  • Administration is in a therapeutic or prophylactic form.
  • Certain embodiments of the invention herein contain implantation devices, surgical devices, or products which contain disclosed compositions (e.g., gauze bandages or strips), and methods of making or using such devices or products. These devices may be coated with, impregnated with, bonded to or otherwise treated with the composition herein.
  • Transdermal patches have the added advantage of providing controlled delivery of the active ingredients to the eye and body.
  • dosage forms can be made by dissolving or dispensing the compound in the proper medium.
  • Absorption enhancers are used to increase the flux of the compound across the skin. Rate is controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
  • Injectable preparations of the pharmaceutical composition for example, sterile injectable aqueous or oleaginous suspensions are formulated according to the known art using suitable dispersing agents, wetting agents, and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 ,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or a suspending medium.
  • bland fixed oil including synthetic mono-glycerides or di glycerides is used.
  • fatty acids such as oleic acid are used in the
  • injectable formulations are sterilized prior to use, for example, by filtration through a bacterial-retaining filter, by irradiation, or by
  • Injectable depot forms are made by forming microencapsule matrices of the agent in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of active agent to polymer and the nature of the particular polymer employed, the rate of active agent release is controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the agent in liposomes or microemulsions that are compatible with body tissues.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active agent is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate, dicalcium phosphate, fillers, and/or extenders such as starches, sucrose, glucose, mannitol, and silicic acid; binders such as carboxymethylcellulose, alginates, gelatin,
  • polyvinylpyrrolidinone polyvinylpyrrolidinone, sucrose, and acacia
  • humectants such as glycerol
  • disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents such as paraffin; absorption accelerators such as quaternary ammonium compounds; wetting agents, for example, cetyl alcohol and glycerol monostearate; absorbents such as kaolin and bentonite clay; and lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.
  • solution retarding agents such as paraffin
  • absorption accelerators such as quaternary ammonium compounds
  • wetting agents for example, cetyl alcohol and glycerol monostearate
  • absorbents such as kaolin and bentonite clay
  • lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixture
  • Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using excipients such as milk sugar as well as high molecular weight PEG and the like.
  • excipients such as milk sugar as well as high molecular weight PEG and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules are prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings known in the art of pharmaceutical formulating.
  • the active agent(s) are admixed with at least one inert diluent such as sucrose or starch.
  • inert diluent such as sucrose or starch.
  • Such dosage forms also include, as is standard practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose.
  • the dosage forms may also include buffering agents.
  • the composition optionally contains opacifying agents that release the active agent(s) only, preferably in a certain part of the intestinal tract, and optionally in a delayed manner. Examples of embedding compositions include polymeric substances and waxes.
  • the methods and uses for treating according to the present invention may be performed in combination with additional therapies.
  • “In combination’ may refer to administration of the additional therapy before, at the same time as or after administration of any of the methods or uses according to the present invention.
  • the present invention may be performed in combination with antibiotics, for example in the context of inflammatory diseases where infection may be involved.
  • the invention may be performed in combination with steroid and non-steroid anti-inflammatories.
  • the invention may be performed in combination with steroid and non-steroid anti-inflammatories.
  • the invention may be performed in combination with anti-TNFa therapy, for example in the context of rheumatoid arthritis. In one aspect the invention may be performed in combination with dornase and/or other DNases, for example in the context of cystic fibrosis.
  • the invention may be performed in combination with anti-IL1 b therapy.
  • the invention may be performed in combination with therapies for
  • cardiovascular disease and/or sepsis for example statins, anti-coagulants and/or blood pressure medications.
  • the histone-bound and/or linker DNA can alternatively be targeted by an endonuclease, in order to prevent histone- bound and/or linker DNA-mediated TLR recruitment to an endosome, thereby reducing proinflammatory effects and preventing or treating inflammation caused by circulating histones/DNA.
  • Example 2 and Figure 15 show that circulating histones and DNA play a critical role in inducing systemic IL-6 in models of systemic candidiasis. It was surprisingly found that DNase I administration cleared free circulating histones and DNA from the circulation of infected mice, lowering the
  • DNase I does not degrade histones directly, but by removing the DNA it allows serum proteases to degrade them.
  • the endonuclease promotes the proteolytic degradation of pro- inflammatory histones.
  • the invention provides a method for reducing proinflammatory cytokines in a patient, comprising administering an endonuclease, such as DNase I to said patient.
  • a method to clear histones from circulation for example to reduce inflammatory cytokines.
  • This is a specific mechanistic endpoint which may be central in the treatment of multiple inflammatory conditions.
  • the invention provides in a further aspect a method for promoting histone degradation in a patient, comprising administering an endonuclease, such as DNase I to said patient.
  • the invention also provides a method for blocking histone-mediated signalling in a patient, comprising administering an endonuclease, such as DNase I to said patient.
  • the invention also provides a method for reducing circulating IL-6 in a patient, comprising administering an endonuclease, such as DNase I to said patient.
  • An endonuclease that can degrade histone-bound DNA and/or linker DNA may be suitable for use according to the invention.
  • An endonuclease is an enzyme that breaks down a nucleotide chain into two or more shorter chains by cleaving the internal covalent bonds linking nucleotides.
  • the endonuclease may selected from DNase I, DNase II, DNase III and Benzonase.
  • DNase deoxyribonuclease
  • a deoxyribonuclease is an enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA.
  • the endonuclease may be DNase I (Deoxyribonuclease I).
  • DNase I is an endonuclease encoded in humans by the human gene DNASE1.
  • DNase I is a nuclease that cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding 5'-phosphate-terminated polynucleotides with a free hydroxyl group on position 3', on average producing tetranucleotides. It acts on single-stranded DNA, double-stranded DNA, and chromatin.
  • DNase I for use according to the invention will be known to one of skill in the art.
  • DNase I enzymes are also commercially available, for example Pulmozyme.
  • Benzonase is also commercially available from numerous sources, for example Sigma-Aldrich.
  • the endonuclease may reduce circulating pro-inflammatory cytokines in a patient.
  • the invention provides a method for reducing circulating I L- 1 a , I L- 1 b , IL-18, IL- 5, IL-6, IL-36, TNF-a, G-CSF, GM-CSF, IL-10, MCP-1 (CCL2), CXCL-1 , CXCL-2, CXCL-8, and/or Eotaxin in a patient, comprising administering an endonuclease, such as DNase I, to said patient
  • circulating IL-6 is reduced by at least 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145 or 150 fold, for example compared to a suitable control, e.g. a patient to whom DNase I has not been administered.
  • a suitable control e.g. a patient to whom DNase I has not been administered.
  • the invention also provides a method for reducing circulating I L-1 b in a patient, comprising administering a DNase I to said patient.
  • circulating IL-1 b is reduced by at least 1 , 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145 or 150 fold, for example compared to a suitable control, e.g. a patient to whom DNase I has not been administered.
  • the invention also provides a method for reducing circulating I L-1 a in a patient, comprising administering a DNase I to said patient.
  • circulating IL-1 a is reduced by at least 1 , 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145 or 150 fold, for example compared to a suitable control, e.g. a patient to whom DNase I has not been administered.
  • a suitable control e.g. a patient to whom DNase I has not been administered.
  • the patient may have a viral infection.
  • the patient may have a pulmonary infection.
  • the invention also provides DNase I for use in the treatment or prevention of a fungal infection, and also use of a DNase I in the manufacture of a medicament for the treatment or prevention of a fungal infection.
  • the DNase I may be used for treating or preventing a fungal infection, for example a Candida albicans infection.
  • BIOLOGY 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this disclosure.
  • Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation.
  • protein includes proteins, polypeptides, and peptides.
  • Cholesterol (Sigma-Aldrich) was solubilized in 95% Ethanol by incubating at 65°C, at a concentration of 12.5mg/ml. Cholesterol crystals were formed by 5 consecutive
  • Peripheral blood was isolated from consenting healthy adult volunteers, according to approved protocols of the ethics board of the Francis Crick Institute.
  • Neutrophils were isolated with a two-step purification protocol using histopaque separation followed by a discontinuous Percoll gradient (Aga et al. , 2002).
  • Isolated neutrophils were plated in the presence or absence of 200mM PAD inhibitor Cloride-amidine (Cl-amidine) (Calbiochem). After settling, the neutrophils were stimulated with 0.5mg/ml cholesterol crystals.
  • NET formation was confirmed by visualization using 0.1 mM Sytox green (Invitrogen) and images were captured using a LEICA DMIRB microscope (20x objective) and analyzed using Fiji/lmageJ software.
  • NETs four hours after cholesterol crystal stimulation, cells were fixed for 20 min at RT with 2% paraformaldehyde and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline.
  • samples were blocked with 2% bovine serum albumin (BSA) and 2% donkey serum in phosphate-buffered saline and incubated with anti- histone 3 citrulline R2+R8+R17 (Abeam, ab5103) and anti-human myeloperoxidase (R&D, AF3667) antibodies, followed by Alexa Fluor 488-conjugated donkey anti-rabbit, Alexa Fluor 568-conjugated donkey anti-mouse (Invitrogen).
  • BSA bovine serum albumin
  • R&D anti-human myeloperoxidase
  • NET DNA concentration was determined using Quant-iT PicoGreen dsDNA Reagent (Thermo Fisher Scientific) after a Proteinase K (New England Biolabs) treatment (100ug/ml 2hours at 56°C). Equal amounts of NETs containing 120ng of DNA were analyzed by Western-blot using anti-histone H3 citrulline R2+R8+R17 (Abeam, ab5103), anti-human histone H3 (Millipore, 07-690) and anti-human myeloperoxidase (R&D, AF3667).
  • HL-60 cells or cells from homogenated lung, liver and spleen of H2B-EGFP transgenic mice were lysed on ice in a buffer containing 20mM HEPES pH7.5, 0.25M Sucrose, 3mM MgCI2, 20mM KCI, 0.1 % NP-40, 1 mM DTT, 0.4mM PMSF and 1x complete (protease inhibitor tablet, Sigma-Aldrich).
  • the lysate was then layered over equal volume of lysis buffer containing 2M sucrose and the layers were spun at 800g.
  • Pelleted nuclei were resuspended in 20mM HEPES pH7.5, 3mM MgCI2, 0.2mM EGTA, 1 mM DTT, 0.4mM PMSF and 1x complete. Then, an equal volume of the same buffer supplemented with 0.6M KCI and 10% glucerol was added over gentle vortex. After a short incubation on ice, the preparation was pelleted at 17500g. The pellet was resuspended using a douncer in 20mM HEPES pH7.5, 0.4M NaCI, 1mM EDTA, 5% glycerol, 1 mM DTT, 0.5mM PMSF and 1x complete.
  • pellets were resuspended in a high salt buffer containing 20mM HEPES pH7.5, 0.65M NaCI, 1 mM EDTA, 0.34M sucrose, 1mM DTT, 0.5mM PMSF and 1x complete, and were homogenized using a tight dounce for 40-50 strokes.
  • a final centrifugation at 10000g to remove any unopened nuclei and nuclear debris the supernatant was dialyzed overnight into a low salt buffer of 20mM
  • nucleosomes after MNase digestion to a 0.2kb DNA length, preps were incubated with proteinase K and then heat inactivated at 99°C. Lack of intact proteins was confirmed by electrophoresis and InstantBlue Coomassie Stain (Expedeon Protein Solutions). Any aggregates were removed with high speed centrifugation before use.
  • Histone H3.1 was amplified from a cDNA library derived from mouse splenocytes using a BamHI-H3.1 -Forward primer: 5-CGCGGATCCATGGCTCGTACTAAGCAG-3 and an Xhol- H3.1 -Reverse primer: 5-GAGCTCGAGTTACGCCCTCTCCCCGC-3. It was then inserted into the pMAL-c2x plasmid cleaved with BamHI and Sail (New England Biolabs). Finally a TEV protease cleavage sequence was introduced preceding the H3.1 using Bell (New England biolabs) and BamHI.
  • hPAD4 was amplified from a cDNA library of human neutrophils using the primers BamHI-hPAD4- forward: 5’-CACGGATCCAT GGCCCAGGGGACATT G-3’ and Sall-hPAD4-Reverse: 5’- GCGGTCGACTCAGGGCACCATGTTCC-3’ and subsequently cleaved with BamHI/Sall (New England biolabs).
  • hPAD4 was then ligated to a pBH4 plasmid backbone, containing a 6His-tag on the N-terminal site, previously linearized with BamHI and Sail. 6His-hPAD4 was then recombinantly expressed in BL21(DE3) E.coli cells.
  • Ion exchange chromatography was performed using a Q-Sepharose column (ResourceTM Q, GE lifesciences, 1ml) and the sample was eluted with a 200-1 M NaCI gradient. The fractions containing the enzyme were identified by tandem mass spectrometry and enzymatic activity was confirmed on recombinant histones (Cayman).
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs Peripheral blood mononuclear cells
  • CD14- positive monocytes were isolated using MACS CD14 microbeads (MACS Miltenyi Biotec) according to manufacturer instructions.
  • Cells were plated in plates pre-coated with poly-D- lysine (PDL, Millipore) in HBSS with Ca 2+ and Mg 2+ supplemented with 10% heat inactivated foetal calf serum (Gibco).
  • cells were preincubated with 5ug/ml anti-TLR2 and anti-TLR4 neutralizing antibodies (Invivogen, pab-hstlr2 and pab-hstlr4) or 10mM DNA signalling inhibitory ODN TTAGGG (A151) (Invivogen), before stimulating with the indicated concentrations of NET fragments preparations, Nucleosome preparations (prepared from either HL-60 or H2b-EGFP cells), recombinant human Histone H3 (Cayman), recombinant human Histone H3 citrullinated overnight at 37°C with PAD4 (100pM Histone-3 with 25nM recombinant human PAD4) preincubated with NET DNA (purified from NET fragment preparations cleaned of protein content, using the QIAquick Gel extraction kit by Qiagen following manufacturer’s instructions), CpG ODN 2006 (Invivogen), LPS from Salmonella Minnesota R595 (Invivogen). After overnight incubation, 0.1 % NP
  • cytokine mRNA level assessment monocytes were incubated with the indicated stimulants for 1 hour at 37°C. Total cellular RNA was isolated using TriReagent/Chloroform/lsopropanol (Sigma-Aldrich). 2ug of RNA were then used to perform reverse transcription and generate cDNA using the Transcriptor high fidelity cDNA synthesis kit from Roche using anchored-oligo(dT)18 primer. Gene expression was measured using TaqMan Universal PCR Master Mix with I L- 1 b , I L- 1 a , IL-6 and HPRT-1 specific primers on a 7900HT Fast Real Time PCR System (Applied biosystems). The cycling threshold for each cytokine was analyzed and then normalized to that of HPRT1. Then, the relative gene expression was measured by the change in cyclin threshold (AACT) method.
  • AACT cyclin threshold
  • Bone marrow was isolated from the femur and tibia from WT mice or animals deficient in TLR4 or TLR9 or STING (Hemmi et al., 2000; Hoshino et al. , 1999; Ishikawa and Barber, 2008).
  • Red blood cells were lysed using ACK lysis buffer (Gibco) and the remaining cells were cultured in DMEM supplemented with 20% L929 cell culture supernatant (in-house preparation), 10mM HEPES (Lonza), 1 % L-glutamine (Gibco), 10% FCS (Invitrogen), 100 U/ml penicillin and 100ug/ml streptomycin (Gibco) and 0.05mM 2-mercaptoethanol (Gibco) for 7 days. Cells were washed in PBS and collected using 2.5mM EDTA (Invitrogen) in PBS with 5% FCS.
  • ACK lysis buffer Gibco
  • FCS Invitrogen
  • cells were then incubated with 5ug/ml anti-TLR2 and anti- TLR4 neutralizing antibodies (Invivogen, pab-hstlr2 and pab-hstlr4) or 10mM DNA signalling inhibitory ODN TTAGGG (A151) (Invivogen), before stimulating with the indicated concentrations of Nucleosome preparations (from HL-60 or H2B-GFP expressing mouse cells), recombinant histone H3 (Cayman) with NET DNA, CpG human preferred ODN2006 or mouse preferred ODN1668 (Invivogen) or LPS from Salmonella Minnesota R595
  • BMDMs were plated in a solid black plate (corning) in DMEM with 1 % FCS, 1 % L- glutamine, 100U/ml penicillin and 100ug/ml streptomycin, 10mM HEPES and 0.05mM 2- mercaptoethanol.
  • CD14-positive monocytes were cultured for 6 days in RPMI 1640 supplemented with
  • 293/TLR2-HA and 293/TLR4-HA cells were stimulated for 2 hours with 1 mM MBP-H3 or 1 pM of MBP (preincubated with or without 80ng of NET DNA), 1 pM Nucleosomes from HL-60 or from H2B-EGFP cells (preincubated with or without 480nM recombinant human PAD4). Samples were then lysed in a buffer containing 50mM Tris pH- 8, 100mM NaCI, 1 mM EDTA, 1 % NP-40, 0.1 % sodium deoxycholate, 0.1 % SDS and 1x complete protease inhibitor (Sigma-Aldrich).
  • MBP-H3 and MBP stimulated samples were incubated with Amylose Resin high flow (New England Biolabs) and Nucleosome stimulated lysates were incubated with a rabbit Histone H3 antibody (Millipore, 07-690) and subsequently incubated with Sepharose Fast flow beads (Sigma-Aldrich). Samples were eluted in Laemmli buffer before analyzed on a Western blot using an anti-HA tag antibody.
  • HEK-Blue reporter cell lines overexpressing human TLR2, TLR4 or TLR9 were obtained by Invivogen. These cell lines detect stimulants of the respective overexpressed receptors by induction of secreted embryonic alkaline phosphatase (SEAP).
  • SEAP secreted embryonic alkaline phosphatase
  • Cells were stimulated with recombinant Histone H3 (citrullinated with recombinant PAD4), preincubated with or without 100ng/ml of purified NET DNA, or Nucleosomes purified from HL-60, GFP-Nucleosomes purified from H2B-EGFP expressing mice, LPS (from Salmonella Minnesota R595, Enzo) or CpG ODN2006 (Invivogen) or FSL-1 (Invivogen).
  • the levels of SEAP induction are determined with HEK-Blue Decetion
  • mice All mice were housed in a pathogen free, 12-hour light-dark cycle environment. All experiments were performed under an approved project license and following the Home Office guidelines.
  • ApoE/PAD4 deficient mice were generated by crossing ApoE deficient with PAD4 deficient mice (Hemmers et al. , 2011 ; Piedrahita et al., 1992) on a C57BL/6J background. Male or female mice of 8-10 weeks old were fed for 6 weeks on a high fat diet (60% energy from fat, Testdiet).
  • ApoE -/- were injected once a week for 6 weeks under high fat diet with 0.8mg/kg of the Fab fragment of the mouse anti-chromatin PL-2-3 antibody (Losman et al., 1992), digested using the PierceTM Fab preparation Kit by Thermo Fisher Scientific) or equivalent volume of vehicle (PBS passed through the PierceTM Fab preparation Kit), or anti-histone 3 citrulline R2+R8+R17 (Abeam) or rabbit IgG Isotype (abeam). Mice were sacrificed by terminal anaesthesia and exsanguinated from the jugular vein for a terminal blood plasma sample (collected in heparin containing tubes).
  • a PBS perfusion of the arterial system was then performed via the left ventricle of the heart.
  • the heart was fixed with 4% PFA and then dehydrated overnight with 20% sucrose.
  • Hearts were embedded in Optimum Cutting Temperature compound (OCT, VWR) and frozen in a dry ice cooled slurry of absolute Ethanol.
  • OCT, VWR Optimum Cutting Temperature compound
  • the sections that comprised the aortic root as determined by the presence of the aortic valve leaflets, were serially sectioned on a Leica CM3050 S Cryostat at a thickness of 10pm, collected and stored at -80°C. Sections were then stained with Oil Red O (Sigma-Aldrich) in 60% isopropyl alcohol and haematoxylin (RAL
  • aortic root sections were first dried for at least 30 min at RT followed by incubation with 2% donkey serum in order to block nonspecific binding, before being incubated with anti-histone 3 citrulline R2+R8+R17 (Abeam ab5103) and anti-mouse myeloperoxidase (R&D, AF3667) as well as Alexa 647-conjugated anti-mouse Ly6G (Biolegend, 127610, Clone 1A8).
  • CD14+ human monocytes were stimulated with the indicated concentrations of H2B-EGFP Nucleosomes.
  • samples were stained with anti-mouse CD11b antibody (clone M1/70, Biolegend) and LIVE/DEADTM Fixable blue dead cell stain kit (Thermo Fisher), and then fixed with 4% PFA (Sigma), before acquisition on the cell analyser LSRFortessa (BD).
  • TLR4 staining human monocytes, human macrophages and murine BMDMs were stained for extracellular and intracellular TLR4 with anti-human TLR4 (clone TF901 , BD Biosciences) or anti-mouse TLR4 (clone SA15-21 , BioLegend) by using the Foxp3 staining buffer set (eBioscience). Data were analyzed using FlowJo software v10. Data were analysed using FlowJo software v10.
  • Human monocytes were stimulated with 0.5uM of MBP-Histone 3 preincubated with or without 300ng/ml NET DNA. After 2 hours, cells were fixed in 2% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS.
  • Samples were then blocked with 2% BSA and Donkey serum in PBS and stained with anti-Rab5 (C8B1 , Cell Signaling Technology, 3547) anti-MBP (BioLegend, 906901) and anti-actin (Millipore, MAB1501 R), followed by Alexa Fluor 488-conjugated donkey anti-rabbit, Alexa Fluor 568-conjugated donkey anti-rat and Alexa Fluor 647-conjugated donkey anti-mouse (all Invitrogen). Samples were
  • Citrullination is not required for NETosis in atherosclerosis
  • Histone citrullination by PAD4 has been implicated in NET formation.
  • Cl-amidine a potent inhibitor of PAD4
  • Uncitrullinated NETs contained key NET markers such as neutrophil elastase (NE), myeloperoxidase (MPO), processed histones and S100A8 proteins (Fig. 1C, and D) .
  • NE neutrophil elastase
  • MPO myeloperoxidase
  • S100A8 proteins Fig. 1C, and D
  • nucleosomes are sufficient to activate monocytes.
  • HL-60 granulocytic human leukemia 60
  • Mnase micrococcal nuclease
  • Histone H3 alone could moderately activate monocytes at a concentration of 0.3 mM but adding DNA at sub-threshold concentrations dramatically enhanced monocyte responsiveness to histone H3.
  • monocytes failed to produce I L-1 b in response to concentrations of DNA up to 1.0 pg/mL but we significantly sensitized to DNA stimulation in the presence of a non-activating concentration of H3.
  • citrullination of recombinant histones using a purified recombinant PAD4 enzyme further potentiated monocyte stimulation.
  • these data indicated that compared to synergy induced by DNA, citrullination played a less important role (Fig.2D).
  • chromatin is a key driver of inflammation and atherosclerotic lesions. Targeting chromatin systemically with an antibody against both the histone and DNA component is effective strategy to prevent the development of atherosclerosis, provided that the antibody is missing its Fc effector domains.
  • TLR2/4i Antibodies against TLR2 and TLR4 (abbreviated as TLR2/4i) reduced I L-1 b induction to concentrations that were comparable to blockade of citrullination suggesting that histones are the major pro- inflammatory moieties in NETs by engaging TLR2/4 (Fig. 4A).
  • An inhibitory TLR9 oligonucleotide (ODNi) also blocked cytokine induction by NETs, consistent with a synergistic role between histones and DNA.
  • TLR2/4 neutralization also reduced the pro-inflammatory capacity of mono-nucleosomes, whereas ODNi blocked cytokine induction less efficiently indicating that the activity of mono-nucleosomes indicating a primary role for histone-mediated TLR signaling (Fig. 4B).
  • ODNi blocked cytokine induction less efficiently indicating that the activity of mono-nucleosomes indicating a primary role for histone-mediated TLR signaling (Fig. 4B).
  • TLR2/4 also blocked cytokine induction more efficiently upon activation by 10 8 M nucleosomes than a TLR9 antagonist inhibitory oligonucleotide (ODNi).
  • ODNi inhibitory oligonucleotide
  • TLR4 The critical role of TLR4 was also confirmed when we activated monocytes with recombinant cit-H3 and NET DNA as an agonist (Fig. 4C). However, blockade of both TLR2 and TLR4 receptors leads to more efficient inhibition in human monocytes activated with recombinant citrullinated H3 alone, suggesting that TLR2 may play a secondary role in histone recognition (Fig. 4D). In this experiment we also found unexpectedly that while ODNi was not sufficient to block the ability of recombinant histone H3 to induce I L- 1 b , it increased the blocking capacity of TLR2 and TLR4 blocking antibodies. This finding suggests that TLR4 or an associated co receptor that is implicated in histone sensing may have DNA binding properties.
  • TLR2 or TLR4 mediated NF-KB activation is measured by induction of the secreted embryonic alkaline phosphatase (SEAP).
  • SEAP embryonic alkaline phosphatase
  • Mono-nucleosomes (Fig. 4E) or recombinant citrullinated H3 protein (Fig. 4F) activated HEK-Blue cells expressing TLR4, whereas HEK-Blue cells expressing TLR2 showed very low sensitivity to histones (Fig. 8A and B), suggesting that NET histones engage primarily via TLR4.
  • TLR4 To investigate whether histones physically interact with TLR4, we incubated HEK cells expressing either TLR2-HA or TLR4-HA with recombinant citrullinated H3 or mono- nucleosomes. HA-tagged TLR4, but not TLR2, could be immunoprecipitated with recombinant MBP-H3 protein (Fig. 4G) or antibodies against mono-nucleosomes (Fig. 4H). Therefore, native histones bind and activate TLR4 and this interaction is not affected by their association to DNA. These data confirmed the TLR4 reporter results, collectively suggesting that DNA does not promote activation by potentiating the binding of histones with TLR4.
  • BMDMs bone marrow-derived macrophages
  • TLR4, TLR9 or the intracellular DNA receptor STING we employed bone marrow-derived macrophages (BMDMs) that were deficient in TLR4, TLR9 or the intracellular DNA receptor STING.
  • BMDMs bone marrow-derived macrophages
  • Mnase or chromatin prepared with benzonase to completely remove the DNA, or treated with proteinase K followed by heat inactivation to degrade the histones.
  • Mouse macrophages responded potently to nucleosomes, but their activation dependent only on detection of histones, since activation was sensitive to proteinase K treatment, but not benzonase (Fig. 5A).
  • TLR4 The cellular localization of TLR4 influence the requirement for DNA.
  • TLR4 histone mediate signaling selectively in human monocytes and murine macrophages
  • Sensitization to nucleosomes enables activation below their cytotoxicity threshold.
  • wild type C. albicans SC5314 clinical isolate
  • YEPD yeast extract peptone dextrose
  • mice of 8-12 weeks were infected intratracheally with 1x10 6 C. albicans in PBS.
  • animals were sacrificed 24 hours post infection, lungs were homogenized in PBS and were plated onto sabourad dextrose agar plates.
  • mice were sacrificed 24 hours post infection, lungs were rinsed with PBS and mouse I L-1 b was measured by ELISA (eBioscience, according to manufacturer’s protocol)
  • human monocytes after stimulation with 1 mM of MBP-H3 and 300ng/ml NET DNA were stained for extracellular and intracellular TLR4 with anti-human TLR4 (clone TF901 , BD Biosciences) or anti-mouse TLR4 (clone SA15-21 , BioLegend) by using the Foxp3 staining buffer set (eBioscience). All data were analysed using FlowJo software v10.
  • Wild-type Candida albicans (C. albicans, clinical isolate SC5314) was cultured overnight and subcultered to an optical density (Aboo) of 0.4-0.8 for 4 hours at 37°C in yeast extract peptone dextrose (YEPD) medium with 1 % penicillin-streptomycin. Subcultures were examined for lack of hyphae, and then centrifuged, washed and resuspended in sterile phosphate-buffered saline (PBS) immediately prior to infection. Mice were injected intravenously with 4-5 x 10 5 C. albicans yeast particles per mouse.
  • Aboo optical density
  • YEPD yeast extract peptone dextrose
  • PBS sterile phosphate-buffered saline
  • mice received 2000units deoxyribonuclease from bovine pancreas (DNase, Sigma) in 100ul PBS via intraperitoneal administration.
  • the weight and rectal temperature of the mice were recorded prior to infection and daily over the course of infection to analyse the health status.
  • a body temperature below 32°C, a weight loss superior to 80% of initial weight and slow movement and responsiveness were considered collectively as septic shock and the humane endpoint for the mice.
  • DNA quantitation PicoGreen assay protocol (Quant-iTTM PicoGreenTM dsDNA assay kit, ThermoFisher) was used to measure DNA levels in plasma based on the fluorescent signal (excitation at 488nm), and according to manufacturer’s instructions.
  • Organs were homogenised with a tissue homogenizer in lysis buffer, containing proteinase inhibitors and Nonidet P-40 (NP40). Cytokine and chemokine analysis from organ lysates and plasma were performed with a 36-plex ProcartaPlex cytokine array kit (Thermo Scientific) following the protocol provided by the manufacturer.
  • an antibody against chromatin inhibited the formation of atherosclerotic lesions better than a deficiency in the enzyme protein arginine deiminase 4 (PAD4).
  • the PL2-3 Fab antibody did not reduce the levels of cholesterol and LDL in mice placed on a high fat data, indicating that the mechanism of action did not involve changes in lipid metabolism or affecting systemic levels of circulating I L- 1 b .
  • the PL2-3 antibody blocked the nucleosome dependent induction of I L-1 b by cultured human monocytes, whereas comparable levels of an anti-H3 antibody failed to block cytokine production.
  • EGFP-H2B containing nucleosomes do not activate monocytes for cytokine induction due to inability to bind to TLR4. Moreover, the inventors found that transgenic mice that express EGFP-H2B produce significantly lower levels of the pro- inflammatory cytokine IL-1 b upon pulmonary fungal infection which is consistent with an important role for extracellular host histones in inflammation during infection.
  • the present inventors surprisingly found that DNA synergizes with histones in activating immune cells by regulating the localization of the histone-binding receptor TLR4. They found that in human monocytes TLR4 is intracellular and DNA is required to recruit TLR4 to histone-containing endosomes in order to promote histone-mediated cytokine induction.
  • Figure 15 presents evidence that circulating histones and DNA play a critical role in inducing systemic IL-6 in models of systemic candidiasis.
  • DNase I administration cleared free circulating histones and DNA from the circulation of infected mice, lowering the concentrations of IL-6 by 120-fold and the concentration of IL-1 b by 10-fold.
  • Mice receiving DNase I survived significantly longer than mice treated with PBS control. This finding is surprising because DNase I does not degrade histones directly but by removing the DNA it allows serum proteases to degrade them.
  • An anti-collagen arthritogenic monoclonal antibody cocktail (ArthritoMabTM Arthritis Inducing Antibody Cocktail) (CIA-MAB-2C, Mdbioproducts by Mdbioscience) was used to induce collagen-antibody induced arthritis (CAIA).
  • Animals were injected with 2 to 8 mg of the antibody cocktail via one intravenous (IV) injection on day 0 and subsequent intraperitoneal injections.
  • IV intravenous
  • mice were also injected intravenously or intraperitoneously (IP) on day 0 with vehicle (PBS) or PL2-3 Fab (20 pg).
  • mice were injected intraperitoneously with 100 mg of LPS (MDLPS, Mdbioproducts by Mdbioscience). Mice were treated with the correspondent substance depending on the group, every 3-4 days. Experiment was terminated on day 1 1 at the peak of the arthritis phenotype.
  • LPS LPS
  • mice exhibiting sickness after LPS injection or/and mice that lost more than 15% of their original weight were culled before termination of the experiment.
  • mice were culled using 200pL of pentobarbital (for 20mg mice). Pictures of the most inflamed front and hind paws were taken from three different angles: top, bottom and transversal. Blood was collected by cardiac puncture or jugular exsanguination, collected in EDTA-coated tubes, spin down at 2000g for 20 minutes at 4C, and plasma was used for cytokine analysis by ELISA.
  • the hind limb thickness measurements were analysed and compared between the groups by 2-way ANOVA test using GraphPhad Prism 8.0.0.
  • Sections were first deparaffinized by heating them up for 1 hour at 60C, and rehydration process using a change of buffers starting with Neo-ClearTM (Xylene Substitute) (64741-65-7, Thermo Fisher Scientific) (three passages), to absolute 100% ethanol (two passages), to a lowering percentage of EtOH (96%-80%-70%-50%), to pure miliQ water to end up on PBS. Then, sections were introduced for 10 minutes on Weigert’s Iron hematoxylin working solution (Weigert’s iron hematoxylin working solution A:B (1 : 1)) (HT1079, Sigma Aldrich) and washed in running tap water for 10 minutes.
  • Weigert Iron hematoxylin working solution
  • HT1079 iron hematoxylin working solution A:B (1 : 1)
  • Sections were first deparaffinized by heating them up for 1 hour at 60°C, and rehydration process using a change of buffers starting with Neo-ClearTM (Xylene Substitute) (64741-65-7, Thermo Fisher Scientific) (three passages), to absolute 100% ethanol (two passages), to a lowering percentage of EtOH (96%-80%-70%-50%), to pure miliQ water to end up on PBS.
  • Neo-ClearTM Xylene Substitute
  • mice anti E-cadherin (610181 , BD Bioscience) (1 :500), rabbit anti-Collagen IV (ab6586, Abeam) (1 :500) and goat anti-MPO (AF3667, R&D systems) (1 :40).
  • mouse anti E-cadherin (610181 , BD Bioscience) (1 :500)
  • rabbit anti-citrulinate H3 (ab5103, Abeam) (1 :400)
  • goat anti-MPO AF3667, R&D systems
  • Fluorescence images of the immunostainings were taken using a Leica SP5-lnvert confocal fluorescence microscope. Images were analysed and processed using Fiji-lmage J (Java 8 version) and Adobe Photoshop 2020.
  • Circulating histones are mediators of trauma-associated lung injury.
  • American journal of respiratory and critical care medicine 187, 160-169.
  • AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 458, 509-513.
  • PAD4-mediated neutrophil extracellular trap formation is not required for immunity against influenza infection.
  • AIM2 recognizes cytosolic dsDNA and forms a caspase-1 -activating inflammasome with ASC. Nature 458, 514-518.
  • TLR4 Toll-like receptor 4
  • STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674-678.
  • Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus. Sci Transl Med 3, 73ra19.
  • Plasmacytoid dendritic cells sense self- DNA coupled with antimicrobial peptide. Nature 449, 564-569.
  • CX3CR1 is required for monocyte homeostasis and atherogenesis by promoting cell survival. Blood 113, 963- 972.
  • RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA. J Exp Med 210, 2447-2463.
  • Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786- 791.
  • Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock. Nat Med 13, 1042-1049.

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Abstract

La présente invention concerne un procédé pour réduire une inflammation chez un sujet à l'aide d'une molécule de liaison à l'antigène qui peut se lier à la chromatine et empêcher un récepteur TLR de se lier à une protéine d'histone, une molécule d'ADN et/ou un complexe de celle-ci ou, en variante, d'une endonucléase qui empêche le recrutement induit par l'ADN du TLR ou favorise la dégradation protéolytique d'histones pro-inflammatoires. L'invention concerne également une telle molécule ou endonucléase de liaison à l'antigène et des utilisations associées dans la réduction de l'inflammation.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029348A2 (fr) 1993-06-03 1994-12-22 Therapeutic Antibodies Inc. Production de fragments d'anticorps
WO2015059168A1 (fr) * 2013-10-22 2015-04-30 Novimmune S.A. Procédés et compositions de diagnostic et de traitement de troubles chez des patients présentant des niveaux élevés de ligands de tlr4 et autres biomarqueurs
US20180057574A1 (en) * 2014-06-24 2018-03-01 Immunomedics, Inc. Anti-histone therapy in acute kidney injury
WO2018053080A1 (fr) * 2016-09-14 2018-03-22 Immunomedics, Inc. Thérapie anti-histone utilisée dans l'insuffisance rénale aiguë
US20180298089A1 (en) * 2013-02-15 2018-10-18 Immunomedics, Inc. Chimeric and Humanized Anti-Histone Antibodies

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029348A2 (fr) 1993-06-03 1994-12-22 Therapeutic Antibodies Inc. Production de fragments d'anticorps
US20180298089A1 (en) * 2013-02-15 2018-10-18 Immunomedics, Inc. Chimeric and Humanized Anti-Histone Antibodies
WO2015059168A1 (fr) * 2013-10-22 2015-04-30 Novimmune S.A. Procédés et compositions de diagnostic et de traitement de troubles chez des patients présentant des niveaux élevés de ligands de tlr4 et autres biomarqueurs
US20180057574A1 (en) * 2014-06-24 2018-03-01 Immunomedics, Inc. Anti-histone therapy in acute kidney injury
WO2018053080A1 (fr) * 2016-09-14 2018-03-22 Immunomedics, Inc. Thérapie anti-histone utilisée dans l'insuffisance rénale aiguë

Non-Patent Citations (61)

* Cited by examiner, † Cited by third party
Title
ABRAMS, S.T.ZHANG, N.MANSON, J.LIU, T.DART, C.BALUWA, F.WANG, S.S.BROHI, K.KIPAR, A.YU, W. ET AL.: "Circulating histones are mediators of trauma-associated lung injury", AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, vol. 187, 2013, pages 160 - 169, XP055288777, DOI: 10.1164/rccm.201206-1037OC
AGA, E.KATSCHINSKI, D.M.VAN ZANDBERGEN, G.LAUFS, H.HANSEN, B.MULLER, K.SOLBACH, W.LASKAY, T.: "Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major", J IMMUNOL, vol. 169, 2002, pages 898 - 905
ATIANAND, M.K.FITZGERALD, K.A.: "Molecular basis of DNA recognition in the immune system", J IMMUNOL, vol. 190, 2013, pages 1911 - 1918, XP055635048, DOI: 10.4049/jimmunol.1203162
BERTHELOOT, D.NAUMOVSKI, A.L.LANGHOFF, P.HORVATH, G.L.JIN, T.XIAO, T.S.GARBI, N.AGRAWAL, S.KOLBECK, R.LATZ, E.: "RAGE Enhances TLR Responses through Binding and Internalization of RNA", J IMMUNOL, vol. 197, 2016, pages 4118 - 4126
BRANZK, N.LUBOJEMSKA, A.HARDISON, S.E.WANG, Q.GUTIERREZ, M.G.BROWN, G.D.PAPAYANNOPOULOS, V.: "Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens", NAT IMMUNOL, vol. 15, 2014, pages 1017 - 1025, XP055465493, DOI: 10.1038/ni.2987
BROZ, P.DIXIT, V.M.: "Inflammasomes: mechanism of assembly, regulation and signalling", NATURE REVIEWS. IMMUNOLOGY, vol. 16, 2016, pages 407 - 420
CHAMILOS, G.GREGORIO, J.MELLER, S.LANDE, RKONTOYIANNIS, D.P.MODLIN, R.L.GILLIET, M.: "Cytosolic sensing of extracellular self-DNA transported into monocytes by the antimicrobial peptide LL37", BLOOD, vol. 120, 2012, pages 3699 - 3707
CLACKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628
DE NARDO, D.DE NARDO, C.M.NGUYEN, T.HAMILTON, J.A.SCHOLZ, G.M.: "Signaling crosstalk during sequential TLR4 and TLR9 activation amplifies the inflammatory response of mouse macrophages", J IMMUNOL, vol. 183, 2009, pages 8110 - 8118
FELDMAN, C.H.HIRAKI, L.T.LIU, J.FISCHER, M.A.SOLOMON, D.H.ALARCON, G.S.WINKELMAYER, W.C.COSTENBADER, K.H.: "Epidemiology and sociodemographics of systemic lupus erythematosus and lupus nephritis among US adults with Medicaid coverage, 2000-2004", ARTHRITIS RHEUM, vol. 65, 2013, pages 753 - 763
FERNANDES-ALNEMRI, T.YU, J.W.DATTA, P.WU, J.ALNEMRI, E.S.: "AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA", NATURE, vol. 458, 2009, pages 509 - 513
FRANCK, G.MAWSON, T.L.FOLCO, E.J.MOLINARO, R.RUVKUN, V.ENGELBERTSEN, D.LIU, X.TESMENITSKY, Y.SHVARTZ, E.SUKHOVA, G.K. ET AL.: "Roles of PAD4 and NETosis in Experimental Atherosclerosis and Arterial Injury: Implications for Superficial Erosion", CIRCULATION RESEARCH, vol. 123, 2018, pages 33 - 42
GAIDT, M.M.EBERT, T.S.CHAUHAN, D.RAMSHORN, K.PINCI, F.ZUBER, S.O'DUILL, F.SCHMID-BURGK, J.L.HOSS, F.BUHMANN, R. ET AL.: "The DNA Inflammasome in Human Myeloid Cells Is Initiated by a STING-Cell Death Program Upstream of NLRP3", CELL, vol. 171, 2017, pages 1110 - 1124
GARCIA-ROMO, G.S.CAIELLI, S.VEGA, B.CONNOLLY, J.ALLANTAZ, F.XU, Z.PUNARO, M.BAISCH, J.GUIDUCCI, C.COFFMAN, R.L. ET AL.: "Netting neutrophils are major inducers of type IIFN production in pediatric systemic lupus erythematosus", SCI TRANSL MED, vol. 3, 2011, pages 73ra20
GEHRKE, N.MERTENS, C.ZILLINGER, T.WENZEL, J.BALD, T.ZAHN, S.TUTING, T.HARTMANN, G.BARCHET, W.: "Oxidative damage of DNA confers resistance to cytosolic nuclease TREX1 degradation and potentiates STING-dependent immune sensing", IMMUNITY, vol. 39, 2013, pages 482 - 495
HADJANTONAKIS, A.K.PAPAIOANNOU, V.E.: "Dynamic in vivo imaging and cell tracking using a histone fluorescent protein fusion in mice", BMC BIOTECHNOL, vol. 4, 2004, pages 33, XP021005951, DOI: 10.1186/1472-6750-4-33
HE ZHENGYU ET AL: "Inhibiting toll-like receptor 4 signaling ameliorates pulmonary fibrosis during acute lung injury induced by lipopolysaccharide: an experimental study", RESPIRATORY RESEARCH, BIOMED CENTRAL LTD., LONDON, GB, vol. 10, no. 1, 18 December 2009 (2009-12-18), pages 126, XP021065449, ISSN: 1465-9921 *
HEMMERS, S.TEIJARO, J.R.ARANDJELOVIC, S.MOWEN, K.A.: "PAD4-mediated neutrophil extracellular trap formation is not required for immunity against influenza infection", PLOS ONE, vol. 6, 2011, pages e22043
HEMMI, H.TAKEUCHI, O.KAWAI, T.KAISHO, T.SATO, S.SANJO, H.MATSUMOTO, M.HOSHINO, K.WAGNER, H.TAKEDA, K.: "A Toll-like receptor recognizes bacterial DNA", NATURE, vol. 408, 2000, pages 740 - 745
HORNUNG, V.ABLASSER, A.CHARREL-DENNIS, M.BAUERNFEIND, F.HORVATH, G.CAFFREY, D.R.LATZ, E.FITZGERALD, K.A.: "AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC", NATURE, vol. 458, 2009, pages 514 - 518
HOSHINO, K.TAKEUCHI, O.KAWAI, T.SANJO, H.OGAWA, T.TAKEDA, Y.TAKEDA, K.AKIRA, S.: "Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product", J IMMUNOL, vol. 162, 1999, pages 3749 - 3752
HUANG, H.EVANKOVICH, J.YAN, W.NACE, G.ZHANG, L.ROSS, M.LIAO, X.BILLIAR, T.XU, J.ESMON, C.T.: "Endogenous histones function as alarmins in sterile inflammatory liver injury through Toll-like receptor 9 in mice", HEPATOLOGY, vol. 54, 2011, pages 999 - 1008, XP055194650, DOI: 10.1002/hep.24501
ISHIKAWA, H.BARBER, G.N.: "STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling", NATURE, vol. 455, 2008, pages 674 - 678, XP055021546, DOI: 10.1038/nature07317
KAYAGAKI, N.STOWE, I.B.LEE, B.L.O'ROURKE, K.ANDERSON, K.WARMING, S.CUELLAR, T.HALEY, B.ROOSE-GIRMA, M.PHUNG, Q.T. ET AL.: "Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling", NATURE, vol. 526, 2015, pages 666 - 671, XP055609281, DOI: 10.1038/nature15541
KOHLER ET AL., NATURE, vol. 256, 1975, pages 495
KUMAR, S.V.KULKARNI, O.P.MULAY, S.R.DARISIPUDI, M.N.ROMOLI, S.THOMASOVA, D.SCHERBAUM, C.R.HOHENSTEIN, B.HUGO, C.MULLER, S. ET AL.: "Neutrophil Extracellular Trap-Related Extracellular Histones Cause Vascular Necrosis in Severe GN", J AM SOC NEPHROL, vol. 26, 2015, pages 2399 - 2413
LANDE, R.GANGULY, D.FACCHINETTI, V.FRASCA, L.CONRAD, C.GREGORIO, J.MELLER, S.CHAMILOS, G.SEBASIGARI, R.RICCIERI, V. ET AL.: "Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus", SCI TRANSL MED, vol. 3, 2011, pages 73ra19
LANDE, R.GREGORIO, J.FACCHINETTI, V.CHATTERJEE, B.WANG, Y.H.HOMEY, B.CAO, W.WANG, Y.H.SU, B.NESTLE, F.O. ET AL.: "Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide", NATURE, vol. 449, 2007, pages 564 - 569
LANDSMAN, L.BAR-ON, L.ZERNECKE, A.KIM, K.W.KRAUTHGAMER, R.SHAGDARSUREN, E.LIRA, S.A.WEISSMAN, I.L.WEBER, C.JUNG, S.: "CX3CR1 is required for monocyte homeostasis and atherogenesis by promoting cell survival", BLOOD, vol. 113, 2009, pages 963 - 972
LESHNER, M.WANG, S.LEWIS, C.ZHENG, H.CHEN, X.A.SANTY, L.WANG, Y.: "PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures", FRONT IMMUNOL, vol. 3, 2012, pages 307
LOOD, C.BLANCO, L.P.PURMALEK, M.M.CARMONA-RIVERA, C.DE RAVIN, S.S.SMITH, C.K.MALECH, H.L.LEDBETTER, J.A.ELKON, K.B.KAPLAN, M.J.: "Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease", NAT MED, vol. 22, 2016, pages 146 - 153
LOSMAN ET AL., J IMMUNOL., vol. 148, no. 5, 1 March 1992 (1992-03-01), pages 1561 - 1569
LOSMAN, M.J.FASY, T.M.NOVICK, K.E.MONESTIER, M.: "Monoclonal autoantibodies to subnucleosomes from a MRL/Mp(-)+/+ mouse. Oligoclonality of the antibody response and recognition of a determinant composed of histones H2A, H2B, and DNA", J IMMUNOL, vol. 148, 1992, pages 1561 - 1569
MARKS ET AL., J. MOL. BIOL., vol. 222, 1991, pages 581 - 597
MUNOZ, L.E.BILYY, R.BIERMANN, M.H.KIENHOFER, D.MAUERODER, C.HAHN, J.BRAUNER, J.M.WEIDNER, D.CHEN, J.SCHARIN-MEHLMANN, M. ET AL.: "Nanoparticles size-dependently initiate self-limiting NETosis-driven inflammation", PROC NATL ACAD SCI U S A, vol. 113, 2016, pages E5856 - E5865
NAPOLITANI, G.RINALDI, A.BERTONI, F.SALLUSTO, F.LANZAVECCHIA, A.: "Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells", NAT IMMUNOL, vol. 6, 2005, pages 769 - 776, XP055403614, DOI: 10.1038/ni1223
NEGISHI, H.YANAI, H.NAKAJIMA, A.KOSHIBA, R.ATARASHI, K.MATSUDA, A.MATSUKI, K.MIKI, S.DOI, T.ADEREM, A. ET AL.: "Cross-interference of RLR and TLR signaling pathways modulates antibacterial T cell responses", NAT IMMUNOL, vol. 13, 2012, pages 659 - 666
PALUDAN, S.R.BOWIE, A.G.: "Immune sensing of DNA", IMMUNITY, vol. 38, 2013, pages 870 - 880, XP055491055, DOI: 10.1016/j.immuni.2013.05.004
PANDEY, S.KAWAI, T.AKIRA, S.: "Microbial sensing by Toll-like receptors and intracellular nucleic acid sensors", COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY, vol. 7, 2014, pages a016246
PAPAYANNOPOULOS, V.: "Neutrophil extracellular traps in immunity and disease", NATURE REVIEWS. IMMUNOLOGY, vol. 18, 2018, pages 134 - 147
PAPAYANNOPOULOS, V.METZLER, K.D.HAKKIM, A.ZYCHLINSKY, A.: "Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps", JOURNAL OF CELL BIOLOGY, vol. 191, 2010, pages 677 - 691, XP055625824, DOI: 10.1083/jcb.201006052
PAPAYANNOPOULOS, V.STAAB, D.ZYCHLINSKY, A.: "Neutrophil Elastase Enhances Sputum Solubilization in Cystic Fibrosis Patients Receiving DNase Therapy", PLOS ONE, 2011, pages 6
PIEDRAHITA, J.A.ZHANG, S.H.HAGAMAN, J.R.OLIVER, P.M.MAEDA, N.: "Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells", PROC NATL ACAD SCI U S A, vol. 89, 1992, pages 4471 - 4475, XP002290504, DOI: 10.1073/pnas.89.10.4471
SCHOENFELD, S.R.KASTURI, S.COSTENBADER, K.H.: "The epidemiology of atherosclerotic cardiovascular disease among patients with SLE: a systematic review", SEMIN ARTHRITIS RHEUM, vol. 43, 2013, pages 77 - 95, XP028690813, DOI: 10.1016/j.semarthrit.2012.12.002
SIROIS, C.M.JIN, T.MILLER, A.L.BERTHELOOT, D.NAKAMURA, H.HORVATH, G.L.MIAN, A.JIANG, J.SCHRUM, J.BOSSALLER, L. ET AL.: "RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA", J EXP MED, vol. 210, 2013, pages 2447 - 2463
SMITH, C.K.KAPLAN, M.J.: "The role of neutrophils in the pathogenesis of systemic lupus erythematosus", CURR OPIN RHEUMATOL, vol. 27, 2015, pages 448 - 453
SUN, L.WU, J.DU, F.CHEN, X.CHEN, Z.J.: "Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway", SCIENCE, vol. 339, 2013, pages 786 - 791, XP055294100, DOI: 10.1126/science.1232458
TALUKDAR, S.OH DA, Y.BANDYOPADHYAY, G.LI, D.XU, J.MCNELIS, J.LU, M.LI, P.YAN, Q.ZHU, Y. ET AL.: "Neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase", NAT MED, vol. 18, 2012, pages 1407 - 1412
TAN, R.S.HO, B.LEUNG, B.P.DING, J.L.: "TLR cross-talk confers specificity to innate immunity", INT REV IMMUNOL, vol. 33, 2014, pages 443 - 453
TIAN, J.AVALOS, A.M.MAO, S.Y.CHEN, B.SENTHIL, K.WU, H.PARROCHE, P.DRABIC, S.GOLENBOCK, D.SIROIS, C. ET AL.: "Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE", NAT IMMUNOL, vol. 8, 2007, pages 487 - 496, XP002554808, DOI: 10.1038/ni1457
ULAS, T.PIRR, S.FEHLHABER, B.BICKES, M.S.LOOF, T.G.VOGL, T.MELLINGER, L.HEINEMANN, A.S.BURGMANN, J.SCHONING, J. ET AL.: "S100-alarmin-induced innate immune programming protects newborn infants from sepsis", NAT IMMUNOL, vol. 18, 2017, pages 622 - 632, XP055380998, DOI: 10.1038/ni.3745
URBAN, C.F.ERMERT, D.SCHMID, M.ABU-ABED, U.GOOSMANN, C.NACKEN, W.BRINKMANN, V.JUNGBLUT, P.R.ZYCHLINSKY, A.: "Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans", PLOS PATHOGENS, 2009, pages 5
URBONAVICIUTE, V.FURNROHR, B.G.MEISTER, S.MUNOZ, L.HEYDER, P.DE MARCHIS, F.BIANCHI, M.E.KIRSCHNING, C.WAGNER, H.MANFREDI, A.A. ET : "Induction of inflammatory and immune responses by HMGB1-nucleosome complexes: implications for the pathogenesis of SLE", J EXP MED, vol. 205, 2008, pages 3007 - 3018, XP055053902, DOI: 10.1084/jem.20081165
VILLANUEVA, E.YALAVARTHI, S.BERTHIER, C.C.HODGIN, J.B.KHANDPUR, R.LIN, A.M.RUBIN, C.J.ZHAO, W.OLSEN, S.H.KLINKER, M. ET AL.: "Netting Neutrophils Induce Endothelial Damage, Infiltrate Tissues, and Expose Immunostimulatory Molecules in Systemic Lupus Erythematosus", JOURNAL OF IMMUNOLOGY, 2011
VOGL, T.TENBROCK, K.LUDWIG, S.LEUKERT, N.EHRHARDT, C.VAN ZOELEN, M.A.NACKEN, W.FOELL, D.VAN DER POLL, T.SORG, C.: "Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock", NAT MED, vol. 13, 2007, pages 1042 - 1049, XP002568723, DOI: 10.1038/nm1638
WAGSTAFF, K.M.GLOVER, D.J.TREMETHICK, D.J.JANS, D.A.: "Histone-mediated transduction as an efficient means for gene delivery", MOL THER, vol. 15, 2007, pages 721 - 731
WANG, Y.LI, M.STADLER, S.CORRELL, S.LI, P.WANG, D.HAYAMA, R.LEONELLI, L.HAN, H.GRIGORYEV, S.A. ET AL.: "Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation", J CELL BIOL, vol. 184, 2009, pages 205 - 213, XP055499919, DOI: 10.1083/jcb.200806072
WARNATSCH, A.LOANNOU, M.WANG, Q.PAPAYANNOPOULOS, V.: "Inflammation. Neutrophil extracellular traps license macrophages for cytokine production in atherosclerosis", SCIENCE, vol. 349, 2015, pages 316 - 320
WESTMAN, J.PAPAREDDY, P.DAHLGREN, M.W.CHAKRAKODI, B.NORRBY-TEGLUND, A.SMEDS, E.LINDER, A.MORGELIN, M.JOHANSSON-LINDBOM, B.EGESTEN,: "Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo", PLOS PATHOG, vol. 11, 2015, pages e1005319
XU, J.ZHANG, X.MONESTIER, M.ESMON, N.L.ESMON, C.T.: "Extracellular histones are mediators of death through TLR2 and TLR4 in mouse fatal liver injury", J IMMUNOL, vol. 187, 2011, pages 2626 - 2631, XP055082161, DOI: 10.4049/jimmunol.1003930
XU, J.ZHANG, X.PELAYO, R.MONESTIER, M.AMMOLLO, C.T.SEMERARO, F.TAYLOR, F.B.ESMON, N.L.LUPU, F.ESMON, C.T.: "Extracellular histones are major mediators of death in sepsis", NATURE MEDICINE, vol. 15, 2009, pages 1318 - U1117

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