WO2016177898A1 - Agents immunosuppresseurs et leur utilisation en thérapie - Google Patents

Agents immunosuppresseurs et leur utilisation en thérapie Download PDF

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Publication number
WO2016177898A1
WO2016177898A1 PCT/EP2016/060231 EP2016060231W WO2016177898A1 WO 2016177898 A1 WO2016177898 A1 WO 2016177898A1 EP 2016060231 W EP2016060231 W EP 2016060231W WO 2016177898 A1 WO2016177898 A1 WO 2016177898A1
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amino acid
agent
seq
composition
kit
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PCT/EP2016/060231
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WO2016177898A9 (fr
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Marit Otterlei
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Norwegian University Of Science And Technology (Ntnu)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to novel agents, particularly peptides or mimetics thereof and their encoding nucleic acids, pharmaceutical compositions comprising at least one of said agents, and their use in the suppression of cellular immune system responses and/or the treatment of infectious diseases.
  • the agents find particular use in the treatment of disorders or conditions associated with cytokine release from non-proliferating immune cells, e.g. unwanted, undesirable or excessive cytokine release.
  • the agents may be useful alone or in combination with other therapeutically active compounds, such as
  • immunosuppressive compounds anti-inflammatory compounds, anti-microbial compounds etc.
  • Such compounds may be, for instance, antagonists or inhibitors of signal transduction pathways, particularly toll-like receptor signalling pathways, such as toll-receptor antagonists and/or protein kinase inhibitors.
  • therapeutic methods which comprise the use of said agents for the aforementioned uses.
  • the agents may also be used in the manufacture or preparation of medicaments for the aforementioned therapies.
  • APIM peptides are a group of peptides that interact with PCNA (proliferating cell nuclear antigen) via a novel PCNA interacting motif (Gilljam et al., 2009. Identification of a novel, widespread, and functionally important PCNA-binding motif, J. Cell Biol. 186(5), pp. 645-654).
  • the motif has been termed APIM (AlkB homologue 2 (hABH2) PCNA- interacting motif) since it was first identified as mediating the interaction between hABH2 and PCNA, but as will be clear from the disclosure below, APIM motifs have now been identified in a wide range of proteins.
  • PCNA binding motif found in APIM peptides typically is defined using the consensus sequence, [R/K]-[F/W/Y]-[L/IA /A]-[L/IA /A]-[K/R] (SEQ ID NO:1297) and it has been determined that a more diverse motif, [R/K/H]-[W/F/Y]- [L/IA /A/M/S/T/N/Q/C]-[L/IA /A/M/G/S/T/N/Q/R/H/K/C]-[K/R/H/P] (SEQ ID NO: 1298), is present in various proteins that interact with PCNA (see International application No.
  • PCNA is a member of the sliding clamp family of proteins, which is known to be involved in both DNA replication and DNA repair.
  • the main function of PCNA is to provide replicative polymerases with the high processivity needed for duplication of the genome.
  • PCNA tagged with green fluorescent protein (GFP) forms distinct foci representing sites of replication. It can therefore be used as an S-phase marker.
  • Peptides that contain a PIP-box typically are extremely cytotoxic and therefore unsuitable for use in therapy.
  • APIM peptides have been shown to be useful in therapy. Specifically APIM peptides have been shown to be effective in sensitizing cells to cytotoxic and cytostatic agents, particularly DNA-damaging agents (WO 2009/104001 ) and indeed as an apoptosis-inducing cytotoxic agent in its own right (MGIIer et al., 2013. Targeting
  • APIM peptides have been shown to be useful in combination with cytotoxic and/or cytostatic agents in the treatment of a disorder or condition where it is desirable to inhibit the growth of cells, or in a treatment which involves cytostatic therapy, i.e. to prevent or inhibit the unwanted proliferation of cells.
  • PCNA is present in the cytosol in some cell types, particularly terminally differentiated cells, where it is thought it interacts with a variety of cytosolic polypeptides in the regulation of apoptosis, e.g. via caspases. Accordingly, PCNA was not expected to be present in the cytosol of non-proliferating cells, particularly cells that are not terminally differentiated.
  • APIM peptides have an effect on non-proliferating cells. Additionally, APIM peptides have been shown by the inventors to interfere with protein interactions between proteins involved in various signal transduction pathways, particularly toll-like receptor signal transduction pathways. As shown in more detail in the Examples, the inventors determined that APIM peptides can reduce the release of pro-inflammatory cytokines from non-proliferating immune cells, e.g. non-terminally differentiated immune cells, such as monocytes, without inducing apoptosis.
  • non-proliferating immune cells e.g. non-terminally differentiated immune cells, such as monocytes
  • TLRs Toll-like receptors form one of the major classes of transmembrane pattern- recognition receptors (PRRs).
  • PRRs transmembrane pattern- recognition receptors
  • PAMPs pathogen associated molecular patterns
  • TLR1 , 2, 4, 5, and 6 are localized at the cell surface and detect mainly bacterial cell wall components, such as lipopolysaccharide (LPS) from gram-negative bacteria.
  • LPS lipopolysaccharide
  • TLR3, 7, 8, and 9 localize to intracellular membrane-bound compartments, including endolysosomes, where they recognize viral and bacterial nucleic acids.
  • TLR3 recognizes double-stranded RNA (dsRNA) from viruses and the synthetic dsRNA analog polyinosinic-polycytidylic acid (polylC).
  • PRRs Once activated, PRRs induce intracellular signal transduction pathways that result in the expression and secretion of cytokines and chemokines, which function to coordinate the immune response in-host defence against microbial pathogens.
  • TLRs Upon ligand binding, TLRs recruit the main adaptor proteins MyD88 and/or TRIF and activate signal transduction pathways that trigger the expression and secretion of cytokines needed to induce an immune response.
  • the MyD88-dependent pathway is used by all TLRs except TLR3. Activation of the MyD88 pathway induces the production of inflammatory cytokines via the transcription factor NF- ⁇ and mitogen-activated protein kinases (MAPKs).
  • MPKs mitogen-activated protein kinases
  • TLR3 and TLR4 both recruit the adaptor TRIF and induce type I interferons (IFNs) via the transcription factor IRF3.
  • IFNs type I interferons
  • TRIF activates NF- ⁇ and MAPKs.
  • TLR signalling also activates the phosphatidylinositide 3-kinase (PI3K)/Akt pathway. It has been suggested that PI3K Akt activity is required for full activation of IRF3 in the TRIF-dependent pathway during TLR3 and 4 signalling.
  • PI3K Akt activity is required for full activation of IRF3 in the TRIF-dependent pathway during TLR3 and 4 signalling.
  • cytosolic receptors including RIG-1 , MDA5 and PKR also recognize dsRNA and mediate antiviral responses via signalling pathways similar to TLRs.
  • cytokines and chemokines are involved in coordinating the responses of the innate cellular immune defence system, which is central to surviving microbial and parasitic challenges.
  • This system is also relevant for managing deviant cells after viral infections and in autoimmune diseases.
  • the system uses cytokines and chemokines to communicate and coordinate immune responses and these signals are enormous important in controlling local and systemic responses.
  • cytokines and chemokines are widely important in controlling local and systemic responses.
  • chemokines have been linked to a variety of diseases and disorders, typically as a result of abnormal levels of these molecules, which can trigger undesirable responses in vivo. For instance, the over-secretion of cytokines, such as in a so-called “cytokine storm", can have severe adverse effects on health.
  • Cytokines and chemokines are peptide, protein or glycoprotein signalling molecules that are used extensively in cellular communication.
  • the terms cytokine and chemokine are used interchangeably herein and encompass large and diverse families of molecules that are produced widely throughout the body. Whilst virtually all nucleated cells are capable of producing cytokines and chemokines, immune cells are a particularly important source of cytokines and chemokines. Immune cells may be identified and characterised, at least to some extent, by the mixture of cytokines and chemokines that they secrete, as this forms a crucial part of their role in the immune system.
  • Monocytes are a type of immune cell (leukocyte or white blood cell) that are produced by the bone marrow from hematopoietic stem cell precursors called monoblasts. Monocytes are non-proliferating cells that are not terminally differentiated. Accordingly, monocytes are primarily found in blood and the spleen, and typically constitute between about 3-8% of the leukocytes in the blood. These cells tend to circulate in the bloodstream for about one to three days before moving into tissues throughout the body, where they terminally differentiate into different types of macrophages, depending on the anatomical location.
  • Monocytes characteristically display the cell surface receptor CD14 and may also express and display the cell surface receptor CD16. These cells are particularly associated with the release of pro-inflammatory cytokines and chemokines such as TNFa, IL1 RA, ⁇ _-1 ⁇ , IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12, IL-13, IL-15, IL- 17, CCL1 1 , BasicFGF, G-CSF, GM-CSF, INFv, CXCL10, CCL2, CCL3, CCL4, PDGF- ⁇ , CCL5 and VEGF, particularly following stimulation by components of microbial cells (such as LPS) and/or viruses (such as double stranded nucleic acids).
  • microbial cells such as LPS
  • viruses such as double stranded nucleic acids
  • Monocytes are often found at very high levels in subjects with severe infections, e.g. in cases of sepsis and septicaemia, where it is thought that overproduction of cytokines and chemokines contributes to symptoms and complications associated with these conditions, e.g. multiple organ dysfunction syndrome (i.e. multiple organ failure).
  • hypercytokinemia from immune cells in blood, such as monocytes
  • monocytes are associated with a variety of disorders.
  • pro-inflammatory cytokines and/or chemokines is the primary driving force of disease and death caused by Plasmodium falciparum infections, i.e. malaria.
  • Other infectious diseases including bacterial, fungal and some viral infections (e.g. influenza, such as swine influenza, avian influenza), may result in excessive pro-inflammatory cytokine and/or chemokine release in blood.
  • influenza such as swine influenza, avian influenza
  • allogenic tissue transplants can result in graft versus host disease, in which immune cells produce excess levels of cytokines and chemokines, such as TNFa.
  • therapies for disorders caused by, or associated with, excessive release of cytokines and chemokines have typically focussed on the inhibition of particular cytokines and chemokines or small groups of related cytokines and chemokines.
  • therapies may utilize specific binding proteins, such as antibodies or soluble receptors, to neutralise the released and undesirable cytokines and chemokines.
  • binding proteins or small molecule antagonists may be used to block cytokine and chemokine receptors to prevent downstream signalling.
  • a combination of such therapies may be required to deal with an excess of several cytokines and chemokines, and combining multiple therapeutic agents can be expensive and may have undesirable and unpredictable side effects.
  • corticosteroids Whilst the use of corticosteroids has been suggested for use in subjects suffering from hypercytokinemia, there is limited evidence that such treatments are effective. Furthermore, therapies to date have centred on the treatment of particular tissues, such as the lungs, e.g. the treatment of COPD (chronic obstructive pulmonary disease) by, for instance, inhibiting G-CSF (Granulocyte colony-stimulating factor).
  • COPD chronic obstructive pulmonary disease
  • G-CSF Gramulocyte colony-stimulating factor
  • APIM peptides can reduce the release of pro-inflammatory cytokines and chemokines from non-proliferating immune cells, e.g. non-terminally differentiated immune cells, such as monocytes, without inducing apoptosis.
  • non-proliferating immune cells e.g. non-terminally differentiated immune cells, such as monocytes
  • APIM peptides may function via a general mechanism on non-proliferating immune cells to inhibit (e.g. reduce, diminish, lessen etc.) the release of cytokines and/or chemokines from said cells, e.g. when the cells have been stimulated with any one of a variety of components, e.g.
  • infectious entities microbial, viral and parasitic
  • LPS double stranded nucleic acid
  • the inventors have also determined that the PCNA binding motif found in APIM peptides may be modified substantially (primarily by the internal insertion of an additional aromatic amino acid or "large” hydrophobic amino acid (having an R group with at least 3 carbon atoms)) without reducing the capacity of the peptide to bind to PCNA, i.e. the "conventional" APIM sequence that is found in naturally- occurring proteins that interacts with PCNA may be modified. Indeed, as shown in the Examples below, it can be seen that these "longer” or “extended” APIM peptides (i.e.
  • peptides containing a longer or extended APIM sequence may in some instances have improved affinity for PCNA relative to peptides containing the conventional or naturally- occurring APIM sequence. Nevertheless, the longer APIM peptides are not cytotoxic to normal cells. Accordingly, the longer or extended APIM peptides are expected to be at least as effective at inhibiting protein interactions with PCNA as peptides containing the conventional or naturally-occurring APIM sequence discussed above (SEQ ID NOs: 1297 and 1298).
  • APIM peptides i.e. peptides comprising a PCNA binding motif (particularly peptides containing a longer or extended APIM sequence), namely for use in treating a condition or disorder associated with cytokine release from non-proliferating immune cells in blood, particularly conditions or disorders associated with excessive, undesirable or uncontrolled pro-inflammatory cytokine release from non-proliferating immune cells in blood, e.g. an infectious disease or infection or a disease or condition exacerbated or caused by an infection.
  • APIM peptides for treating or preventing excessive, undesirable or uncontrolled pro-inflammatory cytokine release from non- proliferating immune cells in blood in a subject, e.g. a subject with an infectious disease or infection or a disease or condition exacerbated or caused by an infection.
  • the invention can be seen to provide a method of treating or preventing a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition which results in or from, or is associated with, hypercytokinemia, said method comprising administering (particularly administering an effective amount of) an agent comprising or encoding a peptide comprising an APIM sequence (particularly a peptide containing a longer or extended APIM sequence) or a composition (e.g. a pharmaceutical composition) containing an agent comprising or encoding a peptide comprising an APIM motif (particularly a peptide containing a longer or extended APIM sequence) to a subject in need thereof.
  • the invention provides an agent comprising or encoding a peptide comprising an APIM sequence (particularly a peptide containing a longer or extended APIM sequence) or a composition (e.g. a pharmaceutical composition) containing an agent comprising or encoding a peptide comprising an APIM sequence (particularly a peptide containing a longer or extended APIM sequence), for use in treating or preventing a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition resulting in or from, or associated with, hypercytokinemia.
  • an agent comprising or encoding a peptide comprising an APIM sequence (particularly a peptide containing a longer or extended APIM sequence) in the manufacture of a medicament for the treatment or prevention of a disorder or condition associated with cytokine release from non- proliferating immune cells in blood, particularly a disorder or condition resulting in or from, or associated with, hypercytokinemia.
  • the invention provides a method of treating or preventing a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition which results in or from, or is associated with, hypercytokinemia, said method comprising administering (particularly administering an effective amount of) an agent or a composition (e.g. a pharmaceutical composition) containing an agent to a subject in need thereof, wherein said agent comprises:
  • an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound
  • PCNA interacting motif is X 1 X 2 X 3 X 4 X 5 X 6 (SEQ ID NO: 1 ) and
  • X 1 is a basic amino acid
  • X 2 is an aromatic amino acid
  • X 3 is an aromatic amino acid or a hydrophobic amino acid that has an R group comprising at least three carbon atoms;
  • X 4 is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);
  • X 5 is any amino acid other than an acidic amino acid or an aromatic amino acid
  • X 6 is any amino acid other than an acidic amino acid or an aromatic amino acid, preferably a basic amino acid or Proline (P),
  • X 3 is not an aromatic amino acid
  • X 5 is not lysine (K)
  • X 6 is a basic amino acid or Proline (P);
  • an agent or a composition e.g. a
  • composition containing an agent for use in treating or preventing a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, particularly a disorder or condition resulting in or from, or associated with, hypercytokinemia, wherein said agent comprises:
  • an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound
  • PCNA interacting motif is X 1 X 2 X 3 X 4 X 5 X 6 (SEQ ID NO: 1 ) and
  • X 1 is a basic amino acid
  • X 2 is an aromatic amino acid
  • X 3 is an aromatic amino acid or a hydrophobic amino acid that has an R group comprising at least three carbon atoms;
  • X 4 is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);
  • X 5 is any amino acid other than an acidic amino acid or an aromatic amino acid
  • X 6 is any amino acid other than an acidic amino acid or an aromatic amino acid, preferably a basic amino acid or Proline (P),
  • X 3 is not an aromatic amino acid
  • X 5 is not lysine (K)
  • X 6 is a basic amino acid or Proline (P);
  • an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound
  • PCNA interacting motif is X 1 X 2 X 3 X 4 X 5 X 6 (SEQ ID NO: 1 ) and
  • X 1 is a basic amino acid
  • X 2 is an aromatic amino acid
  • X 3 is an aromatic amino acid or a hydrophobic amino acid that has an R group comprising at least three carbon atoms;
  • X 4 is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);
  • X 5 is any amino acid other than an acidic amino acid or an aromatic amino acid;
  • X 6 is any amino acid other than an acidic amino acid or an aromatic amino acid, preferably a basic amino acid or Proline (P),
  • X 3 is not an aromatic amino acid
  • X 5 is not lysine (K)
  • X 6 is a basic amino acid or Proline (P);
  • the invention provides a method of treating or preventing hypercytokinemia resulting from cytokine release from non-proliferating immune cells in blood (e.g. monocytes), said method comprising administering an agent, or a composition containing an agent, to a subject in need thereof, wherein said agent comprises:
  • an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound
  • PCNA interacting motif is X 1 X 2 X 3 X 4 X 5 X 6 (SEQ ID NO: 1 ) and
  • X 1 is a basic amino acid
  • X 2 is an aromatic amino acid
  • X 3 is an aromatic amino acid or a hydrophobic amino acid that has an R group comprising at least three carbon atoms;
  • X 4 is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);
  • X 5 is any amino acid other than an acidic amino acid or an aromatic amino acid
  • X 6 is any amino acid other than an acidic amino acid or an aromatic amino acid, preferably a basic amino acid or Proline (P),
  • X 3 is not an aromatic amino acid
  • X 5 is not lysine (K)
  • X 6 is a basic amino acid or Proline (P);
  • the invention provides an agent, or composition containing an agent, for use in treating or preventing hypercytokinemia in a subject resulting from cytokine release from non-proliferating immune cells in blood (e.g.
  • said agent comprises: (i) an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound,
  • PCNA interacting motif is X 1 X 2 X 3 X 4 X 5 X 6 (SEQ ID NO: 1 ) and
  • X 1 is a basic amino acid
  • X 2 is an aromatic amino acid
  • X 3 is an aromatic amino acid or a hydrophobic amino acid that has an R group comprising at least three carbon atoms;
  • X 4 is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);
  • X 5 is any amino acid other than an acidic amino acid or an aromatic amino acid
  • X 6 is any amino acid other than an acidic amino acid or an aromatic amino acid, preferably a basic amino acid or Proline (P),
  • X 3 is not an aromatic amino acid
  • X 5 is not lysine (K)
  • X 6 is a basic amino acid or Proline (P);
  • the invention provides the use of an agent in the manufacture of a medicament for the treatment or prevention of hypercytokinemia in a subject resulting from cytokine release from non-proliferating immune cells in blood (e.g. monocytes), wherein said agent comprises:
  • an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound
  • PCNA interacting motif is X 1 X 2 X 3 X 4 X 5 X 6 (SEQ ID NO: 1 ) and
  • X 1 is a basic amino acid
  • X 2 is an aromatic amino acid
  • X 3 is an aromatic amino acid or a hydrophobic amino acid that has an R group comprising at least three carbon atoms;
  • X 4 is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);
  • X 5 is any amino acid other than an acidic amino acid or an aromatic amino acid
  • X 6 is any amino acid other than an acidic amino acid or an aromatic amino acid, preferably a basic amino acid or Proline (P)
  • X 3 is not an aromatic amino acid
  • X 5 is not lysine (K)
  • X 6 is a basic amino acid or Proline (P);
  • the agent as defined herein may be used in combination with one or more additional active agents, e.g. other therapeutically active agents, such as immunosuppressive compounds, anti-inflammatory compounds, anti-microbial compounds, steroids (e.g. corticosteroid), kinase inhibitors (such as p38 MAPK inhibitors, class I PI3K inhibitors) etc., in order to enhance the effect of the agent.
  • additional active agents e.g. other therapeutically active agents, such as immunosuppressive compounds, anti-inflammatory compounds, anti-microbial compounds, steroids (e.g. corticosteroid), kinase inhibitors (such as p38 MAPK inhibitors, class I PI3K inhibitors) etc.
  • additional active agents e.g. other therapeutically active agents, such as immunosuppressive compounds, anti-inflammatory compounds, anti-microbial compounds, steroids (e.g. corticosteroid), kinase inhibitors (such as p38 MAPK inhibitors, class I PI3K inhibitors) etc.
  • a method of treating or preventing a disorder or condition associated with cytokine release from non-proliferating immune cells in blood e.g. monocytes
  • a disorder or condition which results in or from, or is associated with, hypercytokinemia comprising administering (particularly administering an effective amount of) an agent or a composition (e.g. a pharmaceutical composition) containing an agent and separately, simultaneously or sequentially administering of one or more additional active agents, e.g. a therapeutically active agent, to a subject in need thereof, wherein said agent comprises:
  • an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound
  • PCNA interacting motif is X 1 X 2 X 3 X 4 X 5 X 6 (SEQ ID NO: 1 ) and
  • X 1 is a basic amino acid
  • X 2 is an aromatic amino acid
  • X 3 is an aromatic amino acid or a hydrophobic amino acid that has an R group comprising at least three carbon atoms;
  • X 4 is an uncharged amino acid other than an aromatic amino acid, Glycine
  • X 5 is any amino acid other than an acidic amino acid or an aromatic amino acid
  • X 6 is any amino acid other than an acidic amino acid or an aromatic amino acid, preferably a basic amino acid or Proline (P), wherein when X 3 is not an aromatic amino acid, X 5 is not lysine (K) and X 6 is a basic amino acid or Proline (P); or
  • the invention provides a method of treating or preventing hypercytokinemia resulting from cytokine release from non-proliferating immune cells in blood (e.g. monocytes), said method comprising administering
  • an agent as defined herein or a composition e.g. a pharmaceutical composition
  • a composition e.g. a pharmaceutical composition
  • additional active agents e.g. a therapeutically active agent
  • an agent or composition e.g. a
  • compositions as defined herein for use in combination with one or more additional active agents, e.g. a therapeutically active agent, in the treatment or prevention of a disorder or condition associated with cytokine release from non-proliferating immune cells in blood (e.g. monocytes), particularly a disorder or condition which results in or from, or is associated with, hypercytokinemia.
  • additional active agents e.g. a therapeutically active agent
  • an agent or composition e.g. a pharmaceutical composition
  • one or more additional active agents e.g. a therapeutically active agent
  • hypercytokinemia in a subject resulting from cytokine release from non-proliferating immune cells in blood e.g. monocytes.
  • an agent as defined herein in the manufacture of a medicament for use in combination with one or more additional active agents, e.g. a therapeutically active agent, in the treatment or prevention of a disorder or condition associated with cytokine release from non-proliferating immune cells in blood (e.g.
  • monocytes particularly a disorder or condition which results in or from, or is associated with, hypercytokinemia.
  • the invention provides the use of an agent as defined herein in the manufacture of a medicament for use in combination with one or more additional active agents, e.g. a therapeutically active agent, in the treatment or prevention of hypercytokinemia in a subject resulting from cytokine release from non- proliferating immune cells in blood (e.g. monocytes).
  • additional active agents e.g. a therapeutically active agent
  • the medicament may further comprise one or more additional active agents, such as a therapeutically active agent that is useful in treating or preventing the disease or condition (or the symptoms of, or associated with the disease or condition, e.g. hypercytokinemia), associated with cytokine release from non-proliferating immune cells in blood (e.g. monocytes).
  • a therapeutically active agent may be, but is not limited to, an immunosuppressive compound, an anti-inflammatory compound, antimicrobial compound, a steroid (e.g. a corticosteroid) or a kinase inhibitor (such as a p38 MAPK inhibitor or a class I PI3K inhibitor).
  • the medicament may be in the form of a single composition (e.g. a pharmaceutical composition) comprising both the agent as defined herein and the one or more additional active agents, e.g. therapeutically active agent, or it may be in the form of a kit or product containing them for separate (e.g. simultaneous or sequential) administration.
  • a single composition e.g. a pharmaceutical composition
  • additional active agents e.g. therapeutically active agent
  • manufacture of a medicament for the treatment or prevention of a disorder or condition associated with cytokine release from non-proliferating immune cells in blood e.g.
  • monocytes particularly a disorder or condition which results in or from, or is associated with, hypercytokinemia, wherein the medicament is administered separately,
  • hypercytokinemia in a subject resulting from cytokine release from non-proliferating immune cells in blood e.g. monocytes
  • the medicament is administered separately, simultaneously or sequentially with one or more additional active agents, e.g. a therapeutically active agent.
  • the invention provides a product or kit containing an agent as defined herein together with one or more additional active agents, e.g. a therapeutically active agent, as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of a disorder or condition associated with cytokine release from non-proliferating immune cells in blood (e.g. monocytes), particularly a disorder or condition which results in or from, or is associated with, hypercytokinemia.
  • additional active agents e.g. a therapeutically active agent
  • the invention provides a product or kit containing an agent as defined herein together with one or more additional active agents, e.g. a therapeutically active agent, as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of hypercytokinemia in a subject resulting from cytokine release from non-proliferating immune cells in blood.
  • additional active agents e.g. a therapeutically active agent
  • the invention provides an oligopeptidic compound (an agent) (e.g. a peptide) comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound,
  • X 1 is a basic amino acid
  • X 2 is an aromatic amino acid
  • X 3 is an aromatic amino acid or a hydrophobic amino acid that has an R group comprising at least three carbon atoms;
  • X 4 is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P);
  • X 5 is any amino acid other than an acidic amino acid or an aromatic amino acid
  • X 6 is any amino acid other than an acidic amino acid or an aromatic amino acid, preferably a basic amino acid or Proline (P),
  • X 3 is not an aromatic amino acid
  • X 5 is not lysine (K)
  • X 6 is a basic amino acid or Proline (P).
  • the invention provides a nucleic acid molecule encoding an oligopeptidic compound (e.g. a peptide) as defined above. Also provided is the complement of such a nucleic acid molecule.
  • a further aspect relates to a vector comprising said nucleic acid molecule or complement, which is defined further below.
  • the oligopeptidic compound which is capable of interacting with PCNA, or its encoding nucleic acid, may function in the methods and uses of the invention, the compound must be capable of entering the cell, i.e. crossing the cell membrane into the cytosol (cytoplasm), and optionally into one or more other cellular locations, e.g. the nucleus. This may be achieved using any convenient mechanism, e.g. associating directly or indirectly the oligopeptidic compound or its encoding nucleic acid molecule with one or more molecules capable of facilitating the uptake of said molecule into a cell, such as with a liposome or import peptide.
  • a particularly advantageous mechanism may be to generate an oligopeptidic compound that comprises a domain that assists the transit of the compound across the cell membrane, i.e. to generate a fusion peptide or chimeric peptide (a peptide formed from two or more domains that are not normally found together in nature).
  • a peptide comprising a cell membrane permeable motif e.g. a cell penetrating peptide (an uptake or import peptide, or a peptide transduction domain).
  • the fusion peptide (an oligopeptidic compound) may optionally comprise further sequences to facilitate the targeting of the peptide (i.e. to direct the peptide) to a particular sub-cellular location, e.g. a target peptide, signal peptide or transit peptide.
  • the oligopeptidic compound comprises a PCNA interacting motif and a domain that facilitates its uptake
  • the compound comprises at least 6 residues and the final size of the compound will be dependent on the size and number of the domains that make up said compound, i.e. the PCNA interacting motif and uptake (import) peptide may be viewed as domains of the oligopeptidic compound.
  • a domain may be viewed as a distinct portion (i.e. a sequence within the full-length peptidic sequence) of the oligopeptidic compound that can be assigned or ascribed a particular function or property.
  • the oligopeptidic compound for use in the methods and uses of the invention comprises at least 2 domains, i.e. the PCNA interacting motif domain and the domain that facilitates the cellular uptake of said compound, e.g. uptake (import) peptide sequence domain.
  • the oligopeptidic compound may comprise additional domains that may facilitate its function and/or stability, e.g. the capacity of the peptide to interact with its target, i.e. PCNA or an equivalent protein.
  • the oligopeptidic compound may comprise at least 2, 3, 4 or 5 domains, e.g. 6, 7, 8, 9, 10, 12, 15 or more domains.
  • the oligopeptidic compound may comprise one or more linker domains, i.e. a domain that interspaces between two other domains, i.e. occupies the space in between and connects two domains of the oligopeptidic compound.
  • the oligopeptidic compound may comprise a domain that directs the oligopeptidic compound to a cellular or subcellular location, e.g. a signal peptide (also known as a target or transit peptide), such as a nuclear localization signal (NLS) sequence.
  • the one or more linker domains may have an additional function, i.e. a linker domain may also function as a signal peptide, e.g. a NLS. Alternatively put, a signal peptide domain may function as a linker domain in some embodiments.
  • the oligopeptidic compound may comprise a PCNA interacting motif domain, a domain that facilitates its cellular uptake (e.g. an uptake (import) peptide sequence domain) and a linker domain.
  • a PCNA interacting motif domain e.g. an uptake (import) peptide sequence domain
  • a linker domain e.g. an uptake (import) peptide sequence domain
  • the oligopeptidic compound may also comprise a nuclear localisation signal sequence domain.
  • the nuclear localization signal sequence domain may function as a linker domain.
  • the agent of the invention may take the form of a construct containing (i.e. comprising) an oligopeptidic compound which comprises a PCNA interacting motif as defined herein, together with a domain that facilitates its cellular uptake (e.g. an uptake peptide sequence) and optionally additional domains.
  • the invention may accordingly be seen to provide a construct comprising an oligopeptidic compound which is capable of interacting with PCNA.
  • the longer or extended PCNA motif of the invention has been determined to mediate the interaction of an oligopeptidic compound (e.g. peptide) or protein containing such a motif with PCNA.
  • the compound may be characterised insofar as it must be capable of interacting with PCNA, i.e. the oligopeptidic compounds of the invention and for use in the methods and uses of the invention must be competent and/or proficient PCNA interacting molecules.
  • the PCNA protein used to determine the capacity and/or affinity of the oligopeptidic compound:PCNA interaction may be from any suitable source, e.g. a PCNA from any animal, particularly a mammal such as a human, rodent (e.g. mouse, rat), canine or any other non-human animal.
  • a PCNA from any animal, particularly a mammal such as a human, rodent (e.g. mouse, rat), canine or any other non-human animal.
  • rodent e.g. mouse, rat
  • the oligopeptidic compound:PCNA interaction is determined, characterised or assessed using human PCNA protein.
  • the interaction may be direct or indirect, and may involve direct binding of the motif to the PCNA protein, or the motif may bind indirectly, for example binding may be mediated by another molecule.
  • This reference to "PCNA-interacting" or “PCNA-binding” can thus include any form of interaction, and both direct and indirect binding.
  • the interaction is direct binding.
  • X 1 is preferably selected from lysine (K), arginine (R), histidine (H), ornithine (Orn), methyllysine (MeK), diaminobutyric acid (Dbu), citrulline (Cit), acetyllysine (AcK), and any suitable basic amino acid selected from the non-conventional amino acids in Table 2. Whilst the standard or conventional basic amino acids are preferred, e.g. K, R and H, particularly K and R, these may be substituted by any functionally equivalent non- conventional basic amino acid.
  • X 2 is preferably selected from phenylalanine (F), tryptophan (W), tyrosine (Y), tert.- butylglycine, cyclohexylalanine, ie f.-butylphenylalanine, biphenylalanine and tri tert.- butyltryptophan (in certain embodiments this list may exclude W).
  • the standard or conventional aromatic amino acids are preferred, e.g. F, W and Y, these may be substituted by any functionally equivalent non-conventional aromatic amino acid, e.g. from Table 2.
  • X 2 may be selected from W and Y, F and Y, or F and W or in specific embodiments X 2 may be F, or W or Y, or functionally equivalent non- conventional aromatic amino acids.
  • the binding of the motif to PCNA may in certain embodiments be improved when X 2 is W or Y.
  • X 2 is not F. However, as indicated above, in other embodiments it may be F.
  • X 3 is preferably selected from phenylalanine (F), tryptophan (W), tyrosine (Y), tert.- butylglycine, cyclohexylalanine, ie f.-butylphenylalanine, biphenylalanine and tri tert.- butyltryptophan, leucine (L), isoleucine (I), valine (V), methionine (M), norleucine (Nor) (in certain embodiments this list may exclude W or L).
  • X 3 is preferably an aromatic amino acid selected from phenylalanine (F), tryptophan (W), tyrosine (Y), ie f.-butylglycine, cyclohexylalanine, ie f.- butylphenylalanine, biphenylalanine and tri ie f.-butyltryptophan (in certain embodiments this list may exclude W).
  • the standard or conventional aromatic amino acids are preferred, e.g. F, W and Y, these may be substituted by any functionally equivalent non- conventional aromatic amino acid, e.g. from Table 2.
  • X 3 may be selected from W and Y, F and Y, or F and W or in specific embodiments X 3 may be F, or W or Y, or functionally equivalent non-conventional aromatic amino acids.
  • X 3 is preferably a hydrophobic amino acid with an R group containing at least 3 carbon atoms, particularly an aliphatic amino acid with an R group containing at least 3 carbon atoms.
  • X 3 may be selected from leucine (L), isoleucine (I), valine (V), methionine (M) or any suitable hydrophobic amino acid with an R group containing at least 3 carbon atoms selected from the non- conventional amino acids in Table 2. More particularly, X 3 may be selected from L, I, V, M or Nor and any suitable hydrophobic (preferably aliphatic) amino acid with an R group containing at least 3 carbon atoms selected from the non-conventional amino acids in Table 2.
  • X 3 may be selected from L, I, V and M and preferably from L, I and V or I, V and M and optionally non-conventional functional equivalents thereof.
  • X 4 is preferably a hydrophobic or polar amino acid, particularly an aliphatic amino acid or polar amino acid.
  • X 4 may be selected from leucine (L), isoleucine (I), valine (V), alanine (A) methionine (M), norleucine (Nor), serine (S), threonine (T), glutamine (Q), aspargine (N) or cysteine (C) or any suitable hydrophobic or polar amino acid selected from the non-conventional amino acids in Table 2. More particularly, X 4 may be selected from L, I , V, A, M, Nor, S or T and any suitable
  • X 4 is not N or Q or a non-conventional functional equivalent thereof and/or in certain embodiments X 4 is not M, S and/or T or a non-conventional functional equivalent thereof.
  • X 4 may not be glycine (G) or proline (P) and this limitation is also intended to exclude non-conventional functional equivalents thereof.
  • X 4 may be selected from L, I , A, V, M, S and T, and preferably from L, I, A, V, S and T and optionally non-conventional functional equivalents thereof.
  • X 4 may be a hydrophobic, and more preferably an aliphatic amino acid.
  • X 4 may be selected from L, I, A, V, M, and preferably from L, I, V and A and optionally non-conventional functional equivalents thereof.
  • X 5 is preferably a hydrophobic, polar, basic or thiol-containing amino acid or proline.
  • X 5 an aliphatic amino acid or a polar amino acid.
  • the polar amino acid does not contain an amine group (NH2) in the R-group.
  • X 5 preferably may be selected from L, I, V, A, M, Nor, S, T, Q, N, H, K, R, G, C or P and any suitable hydrophobic (preferably aliphatic) or polar
  • X 5 is an uncharged amino acid other than an aromatic amino acid, as defined in X 4 .
  • the basic amino acid may be selected from the amino acids as defined in X 1 although in some embodiments, e.g. where X 3 is not an aromatic amino acid, e.g. when X 3 is L, X 5 is not K.
  • X 5 is not C or a non-conventional functional equivalent thereof and/or N or Q or a non- conventional functional equivalent thereof.
  • X 5 is not H and preferably X 5 is not R, K or H or a non-conventional functional equivalent thereof. In still further embodiments X 5 is not S or T or a non-conventional functional equivalent thereof. In some embodiments X 5 is not P. X 5 may not be an aromatic amino acid (as defined in X 2 ) or acidic amino acid, e.g. aspartic acid (D) or glutamic acid (E), and this limitation is also intended to exclude non-conventional functional equivalents thereof. Thus, in some embodiments, X 5 may be selected from L, V, I, A, M, S, T and G, and preferably from L, V, A, I, S and T and optionally non-conventional functional equivalents thereof.
  • X 5 may be a hydrophobic amino acid, and more preferably an aliphatic amino acid or G.
  • X 5 may be selected from L, I, A, V, M, and G and preferably from L, V, I and A and optionally non-conventional functional equivalents thereof.
  • X 6 may be a hydrophobic, polar, basic or thiol-containing amino acid or proline as defined above with respect to X 5 . However, as noted above, X 6 may not be a hydrophobic, polar or thiol-containing amino acid when X 3 is not an aromatic amino acid. Alternatively viewed, X 6 may be a hydrophobic, polar or thiol-containing amino acid only when X 3 is an aromatic amino acid. Thus, in some embodiments, X 6 is preferably a basic amino acid or proline and may be selected from K, R, H, Orn, MeK, Dbu, Cit, AcK, P and any functionally equivalent amino acid selected from the non-conventional amino acids in Table 2. Whilst the standard or conventional amino acids are preferred, e.g. K, R, H and P, particularly K, R and H, e.g. K and R, these may be substituted by any functionally equivalent non- conventional basic amino acid.
  • X 6 is a basic amino acid, preferably selected from K, R and H and optionally non-conventional functional equivalents thereof.
  • X 4 and/or X 5 is a polar amino acid. Accordingly, in certain embodiments only one of X 4 and X 5 is a polar amino acid.
  • X 5 and/or X 6 is a basic amino acid. Accordingly, in certain embodiments X 6 is a basic amino acid.
  • X 4 and X 5 are both uncharged amino acids other than an aromatic amino acid.
  • X 2 and X 3 are both aromatic amino acids, but preferably are not both W, as described further below.
  • a functionally equivalent amino acid may be defined as an amino acid that may be used as a substitute in a peptide or protein for a conventional amino acid without affecting significantly the function of the peptide or protein (or an amino acid that would not be expected to affect or alter significantly the function of the peptide or protein), e.g. an amino acid that has similar structural and/or chemical properties to the conventional amino acid.
  • a functionally equivalent amino acid may be viewed as having the base structure of a standard amino acid, with a non-standard or non-conventional R-group that is structurally and/or chemically similar to the standard R-group.
  • the R-group is structurally similar to the standard R-group of the amino acid being substituted.
  • a conventional or standard amino acid is an amino acid that is used in vivo to produce a polypeptide or protein molecule, i.e. a proteinogenic amino acid.
  • the invention may provide an oligopeptidic compound comprising the motif [R/K/H]-[W/F/Y]-[W/FmL/IM/M]-[L/IMA/M/S/T/N/Q/C]-[L/IMA/M/G/S/T/N/Q/R/H/K/C/P]- [K/R/H/P/L/IA//A/M/G/S/T/N/Q/C] (SEQ ID NO: 2), wherein when the third amino acid (X 3 ) is L (e.g.
  • X 3 is not an aromatic amino acid
  • the fifth amino acid (X 5 ) is not K
  • the sixth amino acid (X 6 ) is a K, R, H or P and wherein said oligopeptidic compound is capable of interacting with PCNA.
  • the motif may be defined as:
  • the motif may be defined as: [R/K]-[W/F]-[L/IA//M]-[L/IA//A/M/S/T]-[L/IA//A/M/G]-[K/R] (SEQ ID NO: 12). In another embodiment the motif may be defined as:
  • the motif may be defined as: [R/K/H]-[W/F/Y]-[W/F/Y]-[L/IA//A/M/S/T/N/Q]-[L/IA//A/M/G/S/T/N/Q/R/H/K]-
  • the motif may be defined as:
  • X a 1 nd X 2 are RW, RF, KF, KW, RY or KY.
  • X 2 and X 3 are not both W.
  • the second and third amino acids in the sequences above are selected from the group consisting of WF, WY, WL, Wl, WV, WM, FW, FF, FY, FL, Fl, FV, FM, YW, YF, YY, YL, Yl, YV and YM.
  • X 4 and X 5 are LL, LA, LV, AL, VL, VI, LI, IL, W, VA, IV, II, AV, IA, Al, AM, LM, LS, LT, IS, MV, TV, AA, IM, LN, LQ, VM, TL, SL, IT, VT, LG, MA, ML, NL, QL, Ql, Tl, SI, AS, VS, SV, CA, IG, LR, VR, TK or IR.
  • X 4 and X 5 are LL, LA, LV, AL, VL, VI, LI, IL, W, VA, IV, II, AV, IA, Al, AM, LM, LS, LT, IS, MV, TV, AA, IM, LN, LQ or VM.
  • X 4 and X 5 are LV, IV, SV, LS, AV, LG, LA, IR, LR, VR, AR, IK, LK, VK or AK.
  • X 4 and X 5 are LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA, Al, AM, LM, LS or LT, preferably LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, lA or AI.
  • X 4 and X 5 are not AG, AC, CC, NN, QQ, NQ, QN, TS, SS, ST or TT.
  • X 3 and X 4 are not FS, FT, WA or WS.
  • X 6 is K.
  • the oligopeptidic compound has or comprises the sequence RWXLVK (SEQ ID NO: 28).
  • the oligopeptidic compound has or comprises a sequence selected from any one or more of: RWXLLK (SEQ ID NO: 22); RFXLLK (SEQ ID NO: 23); RYXLLK (SEQ ID NO: 24); RWXLLR (SEQ ID NO: 25); RFXLLR (SEQ ID NO: 26); RYXLLR (SEQ ID NO: 27); RWXLVK (SEQ ID NO: 28); RFXLVK (SEQ ID NO: 29); RYXLVK (SEQ ID NO: 30); RWXLVR (SEQ ID NO: 31 ); RFXLVR (SEQ ID NO: 32); RYXLVR (SEQ ID NO: 33); RWXIVK (SEQ ID NO: 34);
  • RFXIVK (SEQ ID NO: 35); RYXIVK (SEQ ID NO: 36); RWXIVR (SEQ ID NO: 37); RFXIVR (SEQ ID NO: 38); RYXIVR (SEQ ID NO: 39); RWXLSK (SEQ ID NO: 40); RFXLSK (SEQ ID NO: 41 ); RYXLSK (SEQ ID NO: 42); RWXLSR (SEQ ID NO: 43); RFXLSR (SEQ ID NO: 44); RYXLSR (SEQ ID NO: 45); RWXISK (SEQ ID NO: 46); RFXISK (SEQ ID NO: 47); RYXISK (SEQ ID NO: 48); RWXISR (SEQ ID NO: 49); RFXISR (SEQ ID NO: 50); RYXISR (SEQ ID NO: 51 ); RWXSVK (SEQ ID NO: 52); RFXSVK (SEQ ID NO: 53);
  • RYXSVK (SEQ ID NO: 54); RWXSVR (SEQ ID NO: 55); RFXSVR (SEQ ID NO: 56);
  • RYXSVR (SEQ ID NO: 57); RWXAVK (SEQ ID NO: 58); RFXAVK (SEQ ID NO: 59);
  • RYXAVK (SEQ ID NO: 60); RWXAVR (SEQ ID NO: 61 ); RFXAVR (SEQ ID NO: 62);
  • RYXAVR (SEQ ID NO: 63); RWXLGR (SEQ ID NO: 64); RFXLGR (SEQ ID NO: 65); RYXLGR (SEQ ID NO: 66); RWXLGK (SEQ ID NO: 67); RFXLGK (SEQ ID NO: 68);
  • RYXLGK (SEQ ID NO: 69); RWXLAR (SEQ ID NO: 70); RFXLAR (SEQ ID NO: 71 );
  • RYXLAR (SEQ ID NO: 72); RWXLAK (SEQ ID NO: 73); RFXLAK (SEQ ID NO: 74); RYXLAK (SEQ ID NO: 75); RWXLTK (SEQ ID NO: 76); RFXLTK (SEQ ID NO: 77);
  • RYXLTK (SEQ ID NO: 78); RWXLTR (SEQ ID NO: 79); RFXLTR (SEQ ID NO: 80);
  • RYXLTR (SEQ ID NO: 81 ); RWXITK (SEQ ID NO: 82); RFXITK (SEQ ID NO: 83); RYXITK (SEQ ID NO: 84); RWXITR (SEQ ID NO: 85); RFXITR (SEQ ID NO: 86); RYXITR (SEQ ID NO: 87); RWXTVK (SEQ ID NO: 88); RFXTVK (SEQ ID NO: 89); RYXTVK (SEQ ID NO: 90); RWXTVR (SEQ ID NO: 91 ); RFXTVR (SEQ ID NO: 92); RYXTVR (SEQ ID NO: 93); RWXIRK (SEQ ID NO: 94); RFXIRK (SEQ ID NO: 95); RYXIRK (SEQ ID NO: 96);
  • RWXIRR (SEQ ID NO: 97); RFXIRR (SEQ ID NO: 98); RYXIRR (SEQ ID NO: 99);
  • RWXLRK (SEQ ID NO: 100); RFXLRK (SEQ ID NO: 101 ); RYXLRK (SEQ ID NO: 102); RWXLRR (SEQ ID NO: 103); RFXLRR (SEQ ID NO: 104); RYXLRR (SEQ ID NO: 105); KWXLLK (SEQ ID NO: 106); KFXLLK (SEQ ID NO: 107); KYXLLK (SEQ ID NO: 108); KWXLLR (SEQ ID NO: 109); KFXLLR (SEQ ID NO: 1 10); KYXLLR (SEQ ID NO: 1 1 1 ); KWXLVK (SEQ ID NO: 1 12); KFXLVK (SEQ ID NO: 1 13); KYXLVK (SEQ ID NO: 1 14); KWXLVR (SEQ ID NO: 1 15); KFXLVR (SEQ ID NO: 1 16); KYXLVR (SEQ ID NO: 1 17); KWXIVK
  • KWXIIR (SEQ ID NO: 259); KFXIIR (SEQ ID NO: 260); KYXIIR (SEQ ID NO: 261 ); RWXLIK (SEQ ID NO: 262); RFXLIK (SEQ ID NO: 263); RYXLIK (SEQ ID NO: 264); RWXLIR (SEQ ID NO: 265); RFXLIR (SEQ ID NO: 266); RYXLIR (SEQ ID NO: 267); KWXLIK (SEQ ID NO: 268); KFXLIK (SEQ ID NO: 269); KYXLIK (SEQ ID NO: 270); KWXLIR (SEQ ID NO: 271 ); KFXLIR (SEQ ID NO: 272); KYXLIR (SEQ ID NO: 273); RWXIAK (SEQ ID NO: 274); RFXIAK (SEQ ID NO: 275); RYXIAK (SEQ ID NO: 276); RWX
  • the sequences do not contain two adjacent tryptophan residues, i.e. the sequences do not contain WW.
  • X (X 3 ) is selected from the group consisting of F, Y, L, I, V and M.
  • the second and third amino acids in the sequences above are selected from the group consisting of WF, WY, WL, Wl, WV, WM, FW, FF, FY, FL, Fl, FV, FM, YW, YF, YY, YL, Yl, YV and YM.
  • X 2 and X 3 are selected from WF, WY, FF, FY, FW, YF, YY and YW.
  • the sequences may contain two adjacent tryptophan residues, i.e. the sequences contain WW.
  • the second and third amino acids in the sequences above are selected from the group consisting of WW, WF, WY, WL, Wl, WV, WM, FW, FF, FY, FL, Fl, FV, FM, YW, YF, YY, YL, Yl, YV and YM.
  • X 2 and X 3 are selected from WW, WF, WY, FF, FY, FW, YF, YY and YW
  • the oligopeptidic compound has or comprises the sequence RWXLVK (SEQ ID NO: 28) or RFXLVK (SEQ ID NO: 29) or RWXVIK (SEQ ID NO: 238) or RFXVIK (SEQ ID NO: 239).
  • the oligopeptidic compound has or comprises the sequence RWFLVK (SEQ ID NO: 1258), RWYLVK (SEQ ID NO: 1259), RWLLVK (SEQ ID NO: 1260), RWILVK (SEQ ID NO: 1261 ), RWVLVK (SEQ ID NO: 1262), RWMLVK (SEQ ID NO: 1263), RFWLVK (SEQ ID NO: 1264), RFFLVK (SEQ ID NO: 1265), RFYLVK (SEQ ID NO: 1266), RFLLVK (SEQ ID NO: 1267), RFILVK (SEQ ID NO: 1268), RFVLVK (SEQ ID NO: 1269), RFMLVK (SEQ ID NO: 1270), RYWLVK (SEQ ID NO: 1271 ), RYFLVK (SEQ ID NO: 1272), RYYLVK (SEQ ID NO: 1273), RYLLVK (SEQ ID NO: 1274), RYILVK (SEQ ID NO
  • the PCNA interacting motifs listed above are preferred motifs of the invention, in some embodiments any one or more of these motifs may be excluded, e.g. any 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more motifs may be excluded, such as any 25, 30, 40, 50 or more motifs or any integer in this range.
  • the oligopeptidic compound does not have or comprise a sequence selected from any one or more of SEQ ID NOs: 22-297, 300, 301 , 1209 and 1258-1288.
  • the oligopeptidic compound is preferably an isolated compound, e.g. an isolated peptide and most preferably the oligopeptidic compound is a synthetic compound, e.g. a synthetic peptide.
  • the nucleic acid molecule encoding the oligopeptidic compound is preferably an isolated nucleic acid molecule and most preferably the nucleic acid molecule is a synthetic nucleic acid molecule. In other words, the oligopeptidic compound and its encoding nucleic acid molecule are non-native, i.e. non-naturally occurring, molecules.
  • the domain that facilitates the uptake of the oligopeptidic compound may be an uptake (import) peptide sequence, which may be a sequence which acts to transport the oligopeptidic compound into a cell, or across a cell membrane (i.e. into the interior of a cell). It may thus be a so-called “cell penetrating” sequence (or more particularly “cell penetrating peptide”) also known in the art as a protein transduction domain (PTD) or protein transduction sequence.
  • uptake (import) peptide sequence which may be a sequence which acts to transport the oligopeptidic compound into a cell, or across a cell membrane (i.e. into the interior of a cell). It may thus be a so-called “cell penetrating” sequence (or more particularly "cell penetrating peptide”) also known in the art as a protein transduction domain (PTD) or protein transduction sequence.
  • PTD protein transduction domain
  • the invention may provide an agent or construct comprising (i) an oligopeptidic compound comprising an APIM motif (i.e. a PCNA- interacting motif) as defined herein, and (ii) a cell penetrating sequence (more particularly a cell penetrating peptide).
  • an APIM motif i.e. a PCNA- interacting motif
  • a cell penetrating sequence more particularly a cell penetrating peptide
  • CPP Cell penetrating peptide
  • CPPs are not characterized by a single structural or functional motif
  • tools to identify CPPs are available and the skilled person can readily determine whether a peptide sequence may function to facilitate the uptake of the peptide of which it forms a domain, i.e. whether a domain may function as an uptake (import) peptide, e.g. a CPP.
  • a peptide sequence may function to facilitate the uptake of the peptide of which it forms a domain, i.e. whether a domain may function as an uptake (import) peptide, e.g. a CPP.
  • Hansen et al Predicting cell-penetrating peptides, Advanced Drug Delivery Reviews, 2008, 60, pp.
  • any suitable CPP may find utility in the invention and, as discussed below, a variety of CPPs have already been identified and tested and could form the basis for determining and identifying new CPPs.
  • CPPs may be derived from naturally-occurring proteins which are able to translocate across cell membranes such as the Drosophila homeobox protein
  • Antennapedia (a transcriptional factor), viral proteins such as the HIV-1 transcriptional factor TAT and the capsid protein VP22 from HSV-1 , and/or they may be synthetically- derived, e.g. from chimeric proteins or synthetic polypeptides such as polyarginine.
  • chimeric proteins such as polyarginine.
  • polyarginine synthetic polypeptides
  • Cell penetrating peptides are also reviewed in Jarver et al. 2006 (Biochimica et Biophysica Acta 1758, pages 260-263) and Table 1 below lists various representative peptides. US 6,645,501 (herein incorporated by reference) further describes various cell penetrating peptides which might be used.
  • Antennapedia-derived CPPs represent a class of particular interest, based around the 16 amino acid Penetratin sequence as shown in Table 1 , which corresponds to the third loop of antennapedia protein and was shown to be responsible for translocation of the protein.
  • Penetratin has been extensively developed as a delivery vehicle, including particularly for pharmaceutical use, and a wide range of Penetratin derivatives and modified sequences have been proposed and described. Reference may be made in particular to WO 91/1891 , WO 00/1417, WO 00/29427, WO 2004/069279 and US 6,080,724 (herein incorporated by reference).
  • the 16 amino acid sequence of Penetratin may be modified and/or truncated, or the peptide may be chemically-modified or retro-, inverso- or retro-inverso analogues may be made whilst retaining cell-penetrating activity.
  • HIV-TAT sequence Another group of cell penetrating peptides which may advantageously be used are based on the HIV-TAT sequence and HIV-TAT and fragments thereof represent a preferred class of CPPs for use according to the present invention.
  • TAT-based CPPs are described in US 5,656,122 (herein incorporated by reference).
  • An exemplary HIV-TAT peptide as used in the Examples below is RKKRRQRRR (SEQ ID NO: 335) but it will readily be appreciated that longer or shorter TAT fragments may be used.
  • CPPs may be identified by particular features, such as for example peptides which are amphipathic and net positively charged.
  • Other groups of CPPs may have a structure exhibiting high ohelical content.
  • Another group may be peptides characterised by a high content of basic amino acids.
  • CPPs may thus be or may comprise oligomers of basic amino acids such as arginine e.g. 5 to 20, 6 to 15 or 6 to 12 R residues e.g. R 7 (SEQ ID NO: 334) , R 8 (SEQ ID NO: 336) or Rn (SEQ ID NO: 337) or QSR 8 (SEQ ID NO: 338).
  • the domain that facilitates the uptake of the oligopeptidic compound may be defined as a peptide of 4-30 amino acids (e.g. 5-29, 6-28, 7-27, 8-26, 9-25 etc. amino acids), wherein at least 4 amino acids (e.g. at least 5, 6, 7, 8, 9, 10 or 1 1 amino acids, e.g. 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, 1 1 -13 amino acids) are positively charged amino acids, preferably selected from K, R or H.
  • 4 amino acids e.g. at least 5, 6, 7, 8, 9, 10 or 1 1 amino acids, e.g. 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, 1 1 -13 amino acids
  • Proline-rich amphipathic peptides are another class of CPP and such peptides characterised by the presence of pyrrolidine rings from prolines are described in Pujals et al. 2008 Advanced Drug Delivery Reviews 60, pages 473-484 (herein incorporated by reference).
  • CPPs include pVEC (Elmquist et al. 2003 Biol.
  • CPPs include Chariot, based on the Pep-1 peptide (Active Motif, France), the Syn-B vectors based on the protegrin peptide PG-1 (Syntem, France), and Express-si Delivery based on the MPG peptide from Genospectra, USA.
  • CPPs include the R41 , R8, M918 and YTA-4 peptides (SEQ ID NOs: 1210- 1213, respectively) disclosed in Eriksson et al. 2013, Antimicrobial Agents and
  • the CPPs may be cyclic peptides, such as those disclosed in Oh et al., 2014, Mol. Pharmaceutics, vol. 1 1 , pp. 3528-3536 (incorporated herein by reference).
  • the CPPs may be amphiphilic cyclic CPPs, particularly containing tryptophan and arginine residues.
  • the CPPs may be cyclic polyarginine peptides and may be modified by the addition of a fatty acyl moiety, e.g.
  • Suitable cyclic CPPs for use in the invention are presented in SEQ ID NOs: 1214-1220.
  • novel or derivative CPP peptides may be designed and synthesized based on known or reported criteria (e.g. known CPP sequences or features such as basic amino acid content, ohelical content etc. as discussed above). Additionally, randomly-designed or other peptides may be screened for CPP activity, for example by coupling or attaching such a peptide containing a reporter molecule, e.g. a detectable label or tag such as a fluorescent tag to the desired cargo (e.g. an oligopeptidic compound as described herein) and testing to see if the construct is translocated across the cell membrane, for example by adding these peptides to live cells followed by examination of cellular import e.g. using confocal microscopy.
  • a reporter molecule e.g. a detectable label or tag such as a fluorescent tag
  • desired cargo e.g. an oligopeptidic compound as described herein
  • the skilled person will be aware of suitable peptide sequences that may facilitate the uptake of the oligopeptidic compound, but by way of example the sequences may include PenetratinTM, a 16-amino acid peptide corresponding to the third helix of the homeodomain of Antennapedia protein, R rich tags such as R6- Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) and derivatives of the HIV Tat protein such as GRKKRRQRRRPPQQ (SEQ ID NO: 339).
  • PenetratinTM a 16-amino acid peptide corresponding to the third helix of the homeodomain of Antennapedia protein
  • R rich tags such as R6- Penetratin (in which arginine-residues were added to the N-terminus of Penetratin)
  • derivatives of the HIV Tat protein such as GRKKRRQRRRPPQQ (SEQ ID NO: 339
  • the domain that facilitates the cellular uptake of the oligopeptidic compound is a CPP and may be selected from any one of:
  • an amphipathic class peptide selected from an amphipathic and net positively charged peptide, a proline-rich amphipathic peptide, a peptide based on the Pep-1 peptide and a peptide based on the MPG peptide;
  • the domain that facilitates the cellular uptake of the oligopeptidic compound is a CPP and may be selected from a sequence selected from any one of SEQ ID NOs: 302-1 162 or a fragment and/or derivative thereof.
  • the details and properties of the CPPs identified in SEQ ID NOs: 340-1 162 can be found at http://crdd.osdd.net/raghava/cppsite/index.php, CPPSite: A database of cell penetrating peptides (herein incorporated by reference).
  • the domain that facilitates the cellular uptake of the oligopeptidic compound is SEQ ID NO: 337.
  • the oligopeptidic compound also comprises one or more domains that provide a signal (target or transit) sequence.
  • the signal sequence may target the oligopeptidic compound to a specific cell type.
  • the oligopeptidic compound may comprise a signal peptide that localises the compound to a specific intracellular compartment, e.g. the nucleus.
  • the oligopeptidic compound is targeted to the cytosol, which may be achieved without an additional signal peptide, i.e. the uptake (import) peptide, e.g. CPP, may be sufficient to direct or localise the oligopeptidic compound to the cytosol of a cell.
  • the signal sequence or signal sequence domain may thus be viewed as any sequence which acts to localise, or alternatively put, to direct, translocate or transport, the oligopeptidic compound to any desired location e.g. to any desired cell type or subcellular location, e.g. nucleus.
  • the oligopeptidic compound (or constructs) of the invention and for use in the use and methods of the invention may comprise one or more signal sequences (i.e. one or more domains that function as signal sequences), e.g. a signal peptide which directs the compound (or construct) into a particular sub-cellular compartment, such as the nucleus.
  • Nuclear localisation signals are again well known in the art and widely described in the literature. For instance, a searchable database of known and predicted NLSs is available, see e.g. Cokol et al (Finding nuclear localization signals, EMBO Reports, 2000, 1 (5), pp. 41 1 -415, herein incorporated by reference).
  • the PSORT II database, http://psort.hgc.jp/ (herein incorporated by reference) can be used for the prediction of nuclear localization of proteins based on NLSs.
  • any known or functional NLS may find utility in the invention.
  • An NLS may vary in length and/or sequence and a wide range of specific NLS sequences have been described. In general, however, it has been found that peptides comprising positively charged amino acids (notably lysine (K), arginine (R) and/or histidine (H)) may function as an NLS.
  • An exemplary NLS may thus be a peptide of e.g. 4-20, more particularly 4-15, 4-12, 4-10 or 4-8 amino acids, wherein at least 4 amino acids (and more particularly at least 60, 70, 75, 80, 85, or 90% of the amino acid residues in the NLS peptide) are positively charged amino acids, preferably selected from K, R or H.
  • Such an exemplary NLS may for example have or comprise the sequence RKRH (SEQ ID NO: 1 163).
  • Nuclear localisation signals including both actual experimentally-determined and predicted or proposed NLS sequences, and strategies for identifying NLSs are also described in Lange et al., J. Biol. Chem. 2007, 282(8), 5101 -5105; Makkerh et al., Current Biology 1996, 6(8), 1025-1027; Leslie et al., Methods 2006, 39, 291 -308; and Lusk et al. Nature Reviews MCB 2007, 8, 414-420 (all herein incorporated by reference).
  • a classical NLS consists of either one (monopartite) or two (bipartite) stretches of basic amino acids.
  • a monopartite NLS may be exemplified by the SV40 large T antigen NLS ( 126 PKKKRKV 132 [SEQ ID NO: 1 164]) and a bipartite NLS by the nucleoplasmin NLS ( 155 KRPAATKKAGQAKKKK 170 [SEQ ID NO: 1 165]).
  • the monopartite NLS consensus sequence K-[K/R]-X-[K/R] (SEQ ID NO: 1 166) has been proposed and accordingly an NLS according to the present invention may in one embodiment comprise or consist of such a consensus sequence (where X is any amino acid).
  • a representative bipartite NLS according to the invention may have the sequence KR-[X] 5 -2o-KKKK (SEQ ID NO: 1 167), e.g. KR-X 10 -KKKK (SEQ ID NO: 1 168) (where X is any amino acid).
  • An alternative exemplary bipartite NLS may take the form RKRH-[X] 2-10 - K (SEQ ID NO: 1 169) e.g. RKRH-X 2 -KK (SEQ ID NO: 1 170), for example RKRH-II-KK (SEQ ID NO: 1 171 ).
  • the oncoprotein c-myc NLS differs from classical NLSs in that only 3 of 9 amino acid residues are basic (PAAKRVKLD [SEQ ID NO: 1 172]), indicating that an NLS need not necessarily conform to the consensus or classical sequences given above.
  • Makkerh et al describe NLS sequences in which a cluster of basic amino acids (e.g. KKKK [SEQ ID NO: 1 173]) is flanked by neutral and acidic residues, for example PAAKKKKLD (SEQ ID NO: 1 174).
  • NLS sequences which may be given by way of example include: PKKKRKVL (SEQ ID NO: 1 175), KKKRK (SEQ ID NO: 1 176), KKKRVK (SEQ ID NO: 1 177), KKKRKVL (SEQ ID NO: 1 178) and RKKRKVL (SEQ ID NO: 1 179).
  • Any NLS which is a derivative of a known NLS e.g. the SV40, nucleoplasmin, UNG2 or c-myc NLS may be used.
  • a putative, proposed or predicted NLS sequence can be tested for NLS activity using principles and assays known and described in the art.
  • a candidate NLS sequence may be attached to the desired cargo (in this case an oligopeptidic compound as defined herein) and the construct may be provided with a detectable reporter molecule (e.g. a tag or label which may be visualised, for example a fluorescent label) and contacted with a test cell. Distribution of the construct in the cell may then be determined.
  • a detectable reporter molecule e.g. a tag or label which may be visualised, for example a fluorescent label
  • nuclear localisation sequences include the SV40 protein derivative KKKRK (SEQ ID NO: 1 176).
  • the oligopeptidic compound comprises a signal sequence (i.e. a domain comprising a signal peptide) that localizes or directs the oligopeptidic compound to a sub-cellular location, such as a NLS and may be selected from any one of:
  • the nuclear localisation signal sequence comprises a sequence selected from any one of SEQ ID NOs: 1 163-1 179 or a fragment and/or derivative thereof, preferably wherein said fragment and/or derivative comprises at least 4 positively charged amino acids, preferably selected from any of K, R or H.
  • an oligopeptidic compound or construct according to the present invention may comprise at least three domains, including (i) an APIM motif domain as defined herein, (ii) a linker domain, which may in some embodiments comprise a nuclear localisation signal sequence, and (iii) a peptide sequence domain that facilitates the cellular uptake of said compound or construct (i.e. an uptake/import peptide sequence domain, e.g. cell penetrating signal sequence domain).
  • the separate elements or components (domains) of a construct according to the present invention may be contained or presented in any order, but preferably in the orders indicated above (e.g. APIM oligopeptidic compound-CPP or APIM oligopeptidic compound-linker-CPP).
  • the APIM motif is located at or towards the N-terminus of the peptide.
  • the APIM motif may be described as being N-terminal to the peptide sequence domain that facilitates the cellular uptake of said compound (e.g. the CPP) and optionally N-terminal to the linker sequence, if present.
  • the oligopeptidic compound comprises a PCNA interacting motif as set forth in any one of SEQ ID NOs: 28-30 (particularly SEQ ID NOs: 28 or 29), a nuclear localisation signal sequence as set forth in SEQ ID NO: 1 176 and a cell penetrating signal sequence as set forth in SEQ ID NO: 337.
  • the oligopeptidic compound comprises a PCNA interacting motif as set forth in any one of SEQ ID NOs: 1258, 1259, 1265-1269 or 1280, a nuclear localisation signal sequence as set forth in SEQ ID NO: 1 176 and a cell penetrating signal sequence as set forth in SEQ ID NO: 337.
  • an oligopeptidic compound or construct according to the invention may contain more than one PCNA-interacting motif.
  • an agent for use in the uses and methods of the present invention may contain or encode an oligopeptidic compound comprising more than one PCNA-interacting motif, preferably more than one longer or extended PCNA-interacting motif (APIM) as defined herein.
  • a construct or oligopeptidic compound may for example contain 1 -10, e.g.
  • motifs may be spaced or located according to choice, e.g. they may be grouped together, or they may be separated by signal sequence elements e.g. motif-motif- CPP, motif-linker-motif-CPP; or motif-linker-motif-motif-CPP; or motif-motif-linker-CPP etc.
  • a “fragment” may comprise at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% of the amino acids of the sequence from which it is derived. Said fragment may be obtained from a central or N-terminal or C-terminal portions of the sequence. Whilst the size of the fragment will depend on the size of the original sequence, in some embodiments the fragments may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or more amino acid residues shorter than the sequence from which it is derived, e.g. 1 -10,
  • a "derivative" of a sequence is at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the sequence to which it is compared.
  • Sequence identity may be determined by, e.g. using the SWISS-PROT protein sequence databank using FASTA pep-cmp with a variable pamfactor, and gap creation penalty set at 12.0 and gap extension penalty set at 4.0, and a window of 2 amino acids. Preferably said comparison is made over the full length of the sequence, but may be made over a smaller window of comparison, e.g. less than 200, 100, 50, 20 or 10 contiguous amino acids.
  • sequence identity related polypeptides i.e. derivatives
  • sequence identity related polypeptides are functionally equivalent to the peptides which are set forth in the recited SEQ ID NOs.
  • the peptides with sequences as set forth in the SEQ ID NOs. may be modified without affecting the sequence of the polypeptide as described below.
  • fragments as described herein may be functional equivalents. Preferably these fragments satisfy the identity (relative to a comparable region) conditions mentioned herein.
  • the peptide may show some reduced efficacy in performing the function relative to the parent molecule (i.e. the molecule from which it was derived, e.g. by amino acid substitution), but preferably is as efficient or is more efficient.
  • functional equivalence may relate to a peptide which is effective in localizing or directing the oligopeptidic compound to the cell type or cellular location, e.g. to facilitate to the uptake of the peptide as described above. This may be tested by comparison of the effects of the derivative peptide relative to the peptide from which it is derived in a qualitative or quantitative manner, e.g. by performing the in vitro analyses described above. Where quantitative results are possible, the derivative is at least 30, 50, 70 or 90% as effective as the parent peptide.
  • Functionally-equivalent peptides which are related to or derived from the parent peptide, may be obtained by modifying the parent amino acid sequence by single or multiple amino acid substitution, addition and/or deletion (providing they satisfy the above- mentioned sequence identity requirements), but without destroying the molecule's function.
  • the parent sequence has less than 20 substitutions, additions or deletions, e.g. less than 10, 5, 4, 3, 2, or 1 such modifications.
  • Such peptides may be encoded by "functionally-equivalent nucleic acid molecules" which may be generated by appropriate substitution, addition and/or deletion of one or more bases.
  • the domains (which may be viewed as components, elements or separate parts) of an oligopeptidic compound or construct of the invention as described herein may be attached or linked to one another in any desired or convenient way according to techniques well known in the art.
  • the domains may be linked or conjugated chemically, e.g. using known chemical coupling technologies or the compound or constructs may be formed as a single whole using genetic engineering techniques e.g. techniques for forming fusion proteins, or they may simply be synthesized as a whole, e.g. using peptide synthesis techniques.
  • the domains may be linked directly to each other or they may be linked indirectly by means of one or more linker (or spacer) sequences.
  • a linker sequence may interspace or separate two or more individual domains (i.e. parts, e.g. or separate motif elements) in an oligopeptidic construct or compound.
  • the precise nature of the linker sequence is not critical and it may be of variable length and/or sequence, for example it may have 0-40, more particularly 0-20, 0-15, 0-12, 0-10, 0-8, or 0-6, 0-4 or 0-3 residues e.g. 1 , 2 or 3 or more residues.
  • the linker sequence may have 1 -15, 1 -12, 1 -10, 1 -8, 1 -6 or 1 -4 residues etc.
  • the nature of the residues is not critical and they may for example be any amino acid, e.g. a neutral amino acid, or an aliphatic amino acid, or alternatively they may be hydrophobic, or polar or charged or structure-forming e.g. proline.
  • a range of different linker sequences have been shown to be of use, including short (e.g. 1 -6) sequences of neutral and/or aliphatic amino acids.
  • Exemplary linker sequences thus include any single amino acid residue, e.g. A, I, L, V, G, R, Q, T, or W, or a di-, tri- tetra- penta- or hexa-peptide composed of such residues.
  • linkers may be mentioned I, II, IL, R, W, WW, WWW, RIL, RIW, GAQ, GAW, VAT, IILVI (SEQ ID NO: 1 180), IILVIII (SEQ ID NO: 1 181 ) etc.
  • linkers between different domains may be the same or different.
  • the linker may comprise or consist of an NLS.
  • an NLS when present, may function both as a signal peptide and a linker.
  • the oligopeptidic compound may comprise a signal peptide (e.g. an NLS) and a linker.
  • Representative compounds (or more particularly constructs) of the invention and for use in the methods and uses of the invention include:
  • MDRYILVKWKKKRKIRRRRRRRRRRRRRRR (SEQ ID NO: 1 196), MDRYLLVKWKKKRKIRRRRRRRRRRRRR (SEQ ID NO: 1 197),
  • the oligopeptidic compound comprises a sequence as set forth in SEQ ID NO: 1 182, 1 183, 1 184, 1 185, 1 186 or 1301.
  • the oligopeptidic compounds shown above comprise N-terminal amino acids that do not form part of the domains that are essential for the compounds to have activity in the methods and uses of the invention, i.e. an "MD" sequence. Some of the peptides may also comprise N-terminal modification, e.g. acetyl groups. These additional amino acids and modifications may facilitate the production of the oligopeptidic compounds, e.g. in vitro or in vivo, and/or help to protect the compounds from degradation in vivo. It will be evident that the oligopeptidic compounds do not require these additional amino acids or modifications for their activity.
  • oligopeptidic compound may comprise an N-terminal sequence, e.g. a sequence at the N- terminus that does not comprise a domain defined above, e.g. a so-called N-terminal flanking sequence.
  • the oligopeptidic compound may comprise a C- terminal sequence, e.g.
  • the oligopeptidic compound may comprise an N-terminal and C-terminal flanking sequence.
  • the oligopeptidic compound of the invention may be in the form of a salt.
  • the oligopeptidic compound may be in the form of an acidic or basic salt.
  • the oligopeptidic compound is in a neutral salt form.
  • the oligopeptidic compound may be in the form of an acetate salt or derivative thereof, e.g. trichloroacetate (TCA), trifluoroacetate (TFA) etc.
  • TCA trichloroacetate
  • TFA trifluoroacetate
  • the oligopeptidic compound may be stabilized by preparing it in the form of a salt, e.g. an acetate salt.
  • a flanking sequence may comprise from about 1 -150 amino acids, such as 1 -120, 1 -100, 1 -90, 1 -80, 1 -70, 1 -60, 1 -50, 1 -40, 1 -35, 1 -30 etc.
  • a flanking sequence may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 amino acids, e.g. 1 -40, 2-39, 3-38, 4-37, 5-36, 6-35, 7-34, 8-33, 9-32, 10-31 , 1 1 -30, 12- 29, 13-28, 14-27, 15-26 amino acids or any combination thereof.
  • Oligopeptidic compounds having sequences as set out in SEQ ID NOs. 1 182-1204 and 1208 comprise separate domains (i.e. components) making up the constructs (i.e. motif-containing sequence, linker/NLS, CPP, etc.)
  • constructs i.e. motif-containing sequence, linker/NLS, CPP, etc.
  • SEQ ID NOs. 1 182-1204 and 1208 represent constructs comprising at least one motif-containing sequence, a linker/NLS and a CPP.
  • NLS sequences based on the SV40 sequence is used, and the CPP sequences are based on an R-rich peptide.
  • K stands for lysine (Lys)
  • I stands for isoleucine (lie) and so on.
  • the oligopeptidic compound may comprise non-conventional or non-standard amino acids. Other domains in the oligopeptidic compound may also incorporate non-standard amino acids.
  • the oligopeptidic compound may comprise one or more, e.g. at least 1 , 2, 3, 4 or 5 non-conventional amino acids, i.e. amino acids which possess a side chain that is not coded for by the standard genetic code, termed herein "non-coded amino acids" (see e.g. Table 2).
  • amino acids which are formed through metabolic processes such as ornithine or taurine, and/or artificially modified amino acids such as 9/-/-fluoren-9-ylmethoxycarbonyl (Fmoc), (tert)-(B)utyl (o)xy (c)arbonyl (Boc), 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc) protected amino acids, or amino acids having the benzyloxy-carbonyl (Z) group.
  • non-coded amino acids are present, they are not located within the motif, but in some embodiments one or more non-coded amino acids are present within the motif.
  • non-coded amino acids are present in more than one domain of the oligopeptidic compound.
  • In vitro and/or in vivo stability of the oligopeptidic compound may be improved or enhanced through the use of stabilising or protecting means known in the art, for example the addition of protecting or stabilising groups, incorporation of amino acid derivatives or analogues or chemical modification of amino acids.
  • Such protecting or stabilising groups may for example be added at the N and/or C-terminus.
  • An example of such a group is an acetyl group and other protecting groups or groups which might stabilise a peptide are known in the art.
  • the oligopeptidic compounds of the invention will typically comprise only amino acids having the L-configuration, but one or more amino acids having the D configuration may be present.
  • the oligopeptidic compound contains at least 1 , 2, 3, 4 or 5 D-amino acids and they are preferably found in the motif, but in another embodiment, D-amino acids are present only outside of the motif. In still further embodiments, D-amino acids may be found in more than one domain of the oligopeptidic compound.
  • the oligopeptidic compound may be linear or cyclic, preferably linear.
  • Retro-inverso oligopeptidic compounds of the oligopeptidic compounds of the invention comprise D-amino acids in reverse (opposite) order to the parental or reference compound sequence.
  • a retro-inverso analogue thus has reversed termini and reversed order of e.g. peptide bonds, while approximately maintaining the topology of the side chains as in the parental or reference sequence.
  • the oligopeptidic compound may include partial retro-inverso sequences, i.e. a domain or part of a domain may comprise a retro-inverso sequence.
  • oligopeptidic compound is meant a compound which is composed of amino acids or equivalent subunits, which are linked together by peptide or equivalent bonds.
  • oligopeptidic compound includes peptides and peptidomimetics.
  • equivalent subunit is meant a subunit which is structurally and functionally similar to an amino acid.
  • the backbone moiety of the subunit may differ from a standard amino acid, e.g. it may incorporate one or more nitrogen atoms instead of one or more carbon atoms.
  • the subunit comprises a standard amino acid backbone, i.e. the backbone of a standard or coded amino acid.
  • the subunit is an amino acid.
  • the amino acid subunit may comprise a nonstandard (non-coded) R-group.
  • peptidomimetic is meant a compound which is functionally equivalent or similar to a peptide and which can adopt a three-dimensional structure similar to its peptide counterparts, but which is not solely composed of amino acids linked by peptide bonds.
  • a preferred class of peptidomimetics are peptoids, i.e. /V-substituted glycines. Peptoids are closely related to their natural peptide counterparts, but they differ chemically in that their side chains are appended to nitrogen atoms along the molecule's backbone, rather than to the ocarbons as they are in amino acids.
  • Peptidomimetics particularly non-peptidic molecules may be generated through various processes, including conformational-based drug design, screening, focused library design and classical medicinal chemistry. Not only may oligomers of unnatural amino acids or other organic building blocks be used, but also carbohydrates, heterocyclic or macrocyclic compounds or any organic molecule that comprises structural elements and conformation that provides a molecular electrostatic surface that mimics the same properties of the 3-dimensional conformation of the peptide may be used by methods known in the art.
  • peptidomimetics may bear little or no resemblance to a peptide backbone.
  • Peptidomimetics may comprise an entirely synthetic non-peptide form (e.g. based on a carbohydrate backbone with appropriate substituents) or may retain one or more elements of the peptide on which it is based, e.g. by derivatizing one or more amino acids or replacing one or more amino acids with alternative non-peptide components.
  • Peptide-like templates include pseudopeptides and cyclic peptides. Structural elements considered redundant for the function of the peptide may be minimized to retain a scaffold function only or removed where appropriate.
  • peptidomimetics retain one or more peptide elements, i.e. more than one amino acid, although such amino acids may be replaced with a nonstandard or structural analogue thereof.
  • Amino acids retained in the sequences may also be derivatised or modified (e.g. labelled, glycosylated or methylated) as long as the functional properties of the oligopeptidic compound are retained.
  • the peptidomimetics are referred to as being "derivable from" a certain polypeptide sequence. By this it is meant that the peptidomimetic is designed with reference to the peptide sequence defined above, such that it retains the structural features of the peptide which are essential for its function.
  • This may be the particular side chains of the peptide, or hydrogen bonding potential of the structure.
  • Such features may be provided by non-peptide components or one or more of the amino acid residues or the bonds linking said amino acid residues of the polypeptide may be modified so as to improve certain functions of the peptide such as stability or protease resistance, while retaining the structural features of the peptide which are essential for its function.
  • non-standard or structural analogue amino acids which may be used are D amino acids, amide isosteres (such as N-methyl amide, retro-inverse amide, thioamide, thioester, phosphonate, ketomethylene, hydroxymethylene, fluorovinyl, (E)- vinyl, methyleneamino, methylenethio or alkane), L-N methylamino acids, D-a methylamino acids, D-N-methylamino acids.
  • Examples of non-conventional, coded, amino acids are listed in Table 2.
  • the oligopeptidic compound is a peptide.
  • the oligopeptidic compound is a peptide consisting of L-amino acids.
  • the oligopeptidic compound is a peptide consisting of standard or coded L-amino acids.
  • the oligopeptidic compound may comprise nonstandard amino acids.
  • the oligopeptidic compound may incorporate di-amino acids and/or ⁇ -amino acids.
  • at least the APIM motif domain consists of oarmino acids.
  • the oligopeptidic compound i.e. all domains and optionally all flanking sequences, consists of a-amino acids.
  • the oligopeptidic compound defined herein comprises more than 6 subunits, but the length of the construct will depend on the size of the uptake peptide sequence and on the number and size of other domains, e.g. linker domains, flanking sequences etc, if present.
  • the prefix "oligo” is used to designate a relatively small number of subunits such as amino acids, i.e. less than 200, preferably less than 150, 100, 90, 80, 70, 60 or 50 subunits.
  • the oligopeptidic compound of the invention may thus comprise more than 5 but no more than 200 subunits. Preferably, it comprises at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 subunits.
  • subunit ranges thus include 12- 50, 12-45, 12-40, 12-35, 12-30, 12-25, 12-22, 12-20, 12-18 etc, 12-30 and 12-40 being preferred. Further representative subunit ranges include 20-50, 21 -45, 22-40, 23-35, 24-30, e.g. 25, 26, 27, 28, 29 or 30.
  • the nature of the subunits of the oligopeptidic compound outside of the APIM motif domain and the uptake peptide sequence is not critical, so the subunits outside of the motif may for example be alanine residues or any other suitable residues.
  • Peptidomimetics typically have a longer half life within a patient's body, so they may be preferred in embodiments where a longer lasting effect is desired. This can help reduce the frequency at which the composition has to be re- administered. However, for bio-safety reasons a shorter half life may be preferred in other embodiments; in those embodiments peptides are preferred.
  • the oligopeptidic compound may form part of a larger unit, e.g. it may be fused to a polypeptide to form a recombinant fusion protein or attached to a scaffold to form a peptide aptamer.
  • fusion proteins or aptamers incorporating the oligopeptidic compound may also find utility in the uses and methods of the invention, i.e. in some embodiments the agent may be a fusion protein or aptamer comprising the oligopeptidic compound defined above.
  • compositions comprising the agent defined herein, e.g. comprising the oligopeptidic compound, fusion protein or aptamer, together with at least one pharmacologically acceptable carrier or excipient.
  • Said composition may be provided for use in the uses and methods of the invention defined below.
  • a nucleic acid molecule encoding a peptide having or comprising (e.g. of) SEQ ID NO: 1 , as defined above is provided for use in the methods and uses of the invention.
  • the agent for use in the uses and methods of the invention may be a nucleic acid molecule encoding a peptide having or comprising (e.g. of) SEQ ID NO: 1 , as defined above.
  • the nucleic acid molecule may not need to encode all of the domains of the oligopeptidic compound described above, e.g. the domain that facilitates the cellular uptake of the peptide.
  • the nucleic acid molecule may be delivered into the cell by another mechanism, e.g. via a liposome.
  • the invention provides a nucleic acid molecule encoding an oligopeptidic compound or construct (e.g. a peptide) as defined above, comprising a PCNA interacting motif (APIM motif) domain and a peptide sequence (domain) that facilitates the uptake of said peptide.
  • an oligopeptidic compound or construct e.g. a peptide
  • APIM motif PCNA interacting motif
  • domain a peptide sequence
  • the nucleic acid molecule may also encode one or more linker and/or signal sequences, as defined above.
  • the nucleic acid molecule of the invention comprises at least 18
  • nucleotides preferably at least 36 nucleotides, and preferably no more than 800 nucleotides, more preferably no more than 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 75 or 50 nucleotides.
  • the nucleic acid molecule is preferably an isolated or synthetic molecule.
  • a further embodiment of the invention relates to a vector comprising a nucleic acid molecule as defined herein and its use in the uses and methods defined below.
  • the vector comprises a promoter sequence operably linked to the sequence encoding a peptide as defined above.
  • the vector may also contain further elements typically found in a vector such as an origin of replication, a selectable marker such as antibiotic resistance, and/or a multiple cloning site.
  • the vector may further be an expression vector, and may comprise further elements, e.g. transcriptional and/or translational control or regulatory elements for expression of the nucleic acid molecules.
  • control elements e.g. promoters, ribosome binding sites, enhancers, terminators etc. are well known and widely described in the art.
  • the vector may for example be a plasmid or a viral genome (or part thereof), preferably the viral gemone is from a virus selected from a retrovirus, an adenovirus and an adenovirus-associated virus.
  • the vector may be administered in the form of a virus comprising a vector containing a nucleic acid molecule encoding an oligopeptidic compound described above.
  • the vector may be a virus.
  • the invention provides a composition (e.g. a pharmaceutical composition) comprising an agent as defined herein and its use in the methods and uses of the invention.
  • said composition e.g. a pharmaceutical composition
  • said composition may comprise an oligopeptidic compound (including a fusion protein or aptamer) and/or nucleic acid molecule as defined herein and/or a vector as defined herein, together with at least one pharmacologically (or pharmaceutically) acceptable carrier or excipient.
  • the excipient may include any excipients known in the art, for example any carrier or diluent or any other ingredient or agent such as buffer, antioxidant, chelator, binder, coating, disintegrant, filler, flavour, colour, glidant, lubricant, preservative, sorbent and/or sweetener etc.
  • any carrier or diluent or any other ingredient or agent such as buffer, antioxidant, chelator, binder, coating, disintegrant, filler, flavour, colour, glidant, lubricant, preservative, sorbent and/or sweetener etc.
  • the excipient may be selected from, for example, lactic acid, dextrose, sodium metabisulfate, benzyl alcohol, polyethylene glycol, propylene glycol, microcrystalline cellulose, lactose, starch, chitosan, pregelatinized starch, calcium carbonate, calcium sulfate, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, powdered cellulose, sodium chloride, sorbitol and/or talc.
  • the pharmaceutical composition may be provided in any form known in the art, for example as a tablet, capsule, coated tablet, liquid, suspension, tab, sachet, implant, inhalant, powder, pellet, emulsion, lyophilisate, effervescent, spray, salve, emulsion, balm, plaster or any mixtures thereof. It may be provided e.g. as a gastric fluid-resistant preparation and/or in sustained action form. It may be a form suitable for oral, parenteral, topical, rectal, genital, subcutaneous, transurethral, transdermal, intranasal, intraperitoneal, intramuscular and/or intravenous administration and/or for administration by inhalation.
  • the pharmaceutical composition may be in a form suitable for liposomal administration, so preferably liposomes containing the pharmaceutical composition are provided.
  • liposomes it may not be necessary to include a further excipient, so in a further embodiment the invention also provides liposomes containing an agent, e.g. oligopeptidic compound, as defined herein, and their use in the methods and uses of the invention.
  • the agents defined herein i.e. oligopeptidic compounds and nucleic acid molecules, and pharmaceutical compositions comprising said agents exhibit therapeutic properties in the treatment of various conditions or disorders, particularly disorders associated with cytokine release from non-proliferating immune cells in blood and infectious diseases or disorders or conditions exacerbated or caused by an infection.
  • a "disorder”, “disease” or “condition” refers to an underlying pathological disturbance in a symptomatic or asymptomatic organism (subject) relative to a normal organism (subject), which may result, for example, from infection and/or an acquired or congenital genetic imperfection.
  • oligopeptidic compounds comprising a PCNA interacting motif can reduce cytokine release in blood, particularly from non-proliferating immune cells in the blood, e.g. monocytes.
  • Cytokines may be released into blood on stimulation with a variety of substances and artificial/synthetic substances. For instance, LPS and poly l:C can be used to simulate or represent bacterial and viral infections, respectively. Excessive levels of pro-inflammatory cytokines are associated with a variety of diseases, e.g. infectious diseases, and it is expected that the agents defined herein may be effective in the treatment or prevention of said disorders. For example, the symptoms and complications associated with sepsis are thought to arise from the overproduction of pro-inflammatory cytokines in blood.
  • the introduction or administration of the agents of the invention e.g.
  • the agents described herein may be expected to find utility in the treatment or prevention of any disease or disorder associated with uncontrolled, undesirable or excessive cytokine release (e.g. hypercytokinemia) from immune cells in blood, e.g. particularly uncontrolled, undesirable or excessive release of one or more pro-inflammatory cytokines.
  • cytokine release e.g. hypercytokinemia
  • Hypercytokinemia also known as a cytokine storm or cytokine cascade
  • cytokines also known as a cytokine storm or cytokine cascade
  • hypercytokinemia is a disease or condition resulting from a potentially fatal immune reaction consisting of a positive feedback loop between cytokines and white blood cells (e.g. monocytes) with highly elevated (hyperexcessive) levels of various cytokines.
  • Hypercytokinemia may be viewed as the systemic expression of a healthy and vigorous immune system resulting in the release of various inflammatory mediators, particularly cytokines. Both pro-inflammatory cytokines and anti-inflammatory cytokines are elevated in the serum of patients with hypercytokinemia and, as discussed below, hypercytokinemia may result from a number of infectious and non-infectious diseases.
  • the inventors have demonstrated that some of the proteins involved in the toll-like receptor signal transduction pathways, which as discussed above are involved in the regulation of immune responses, comprise a "conventional" PCNA interacting motif (e.g. SEQ ID NO: 1297 or 1298). Accordingly, it is thought that the oligopeptidic compounds disclosed herein are able to interfere with the interaction of said proteins with PCNA, e.g. in non-proliferating cells in blood, thereby inhibiting the release of pro-inflammatory cytokines into blood.
  • a "conventional" PCNA interacting motif e.g. SEQ ID NO: 1297 or 1298.
  • the oligopeptidic compounds disclosed herein find particular utility in the treatment or prevention of a condition or disorder associated with cytokine release from non-proliferating immune cells in blood, particularly conditions or disorders associated with excessive, undesirable or uncontrolled pro-inflammatory cytokine release from non- proliferating immune cells in blood, e.g. an infectious disease or infection or a disease or condition exacerbated or caused by an infection.
  • the oligopeptidic compounds disclosed herein find particular utility in the treatment or prevention of excessive, undesirable or uncontrolled proinflammatory cytokine from non-proliferating immune cells in blood in a subject, e.g. a subject with an infectious disease or infection or a disease or condition exacerbated or caused by an infection, particularly hypercytokinemia.
  • API M peptides Whilst not wishing to be bound by theory, it is thought that the effect of API M peptides on cytokine release may be dependent on the strength of the interaction between the APIM peptide and its polypeptide target(s). Thus, as different APIM peptides may interact with target polypeptides with different affinities, it is thought that the dose of any one APIM peptide may determined independently. However, the dose for any APIM peptide may be determined as a matter of routine. In general, a suitable dose may be defined as a dose that does not induce apoptosis, i.e.
  • an apoptosis non-inducing dose which may be deduced by analysing the minimum concentration of oligopeptidic compound required to induce apopotosis in a cell, e.g. using a standard in vitro assay.
  • the oligopeptidic compounds of the invention do not readily induce apoptosis in normal, healthy cells, i.e. the oligopeptidic compounds of the invention are not cytotoxic and accordingly, a wide variety of doses may be suitable in the methods and uses of the present invention.
  • any non-proliferating immune cells in blood may be targeted in the therapies and utilities disclosed and encompassed herein.
  • Such non-proliferating immune cells may include healthy or diseased cells, and particularly cells that are involved in cytokine release.
  • such cells may include in particular monocytes, e.g. cells that display the cell surface receptor CD14 and/or the cell surface receptor CD16.
  • the non-proliferating immune cells e.g. monocytes
  • the non-proliferating immune cells may be characterised or defined by their capacity to release (e.g.
  • cytokines or chemokines selected from TNFa, I LI RA, ⁇ _-1 ⁇ , IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, CCL1 1 , BasicFGF, G-CSF, GM-CSF, INFv, CXCL10, CCL2, CCL3, CCL4, PDGF- ⁇ , CCL5 and VEGF.
  • cytokines or chemokines selected from TNFa, I LI RA, ⁇ _-1 ⁇ , IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, CCL1 1 , BasicFGF, G-CSF, GM-CSF, INFv, CXCL10, CCL2, CCL3, CCL4,
  • the non-proliferating immune cells are monocytes, particularly monocytes that display the cell surface receptor CD14 and/or the cell surface receptor CD16.
  • the non-proliferating immune cells are monocytes that are capable of releasing (e.g.
  • monocytes that secrete or release) any one or more of the cytokines or chemokines selected from TNFa, I LI RA, IL- ⁇ ⁇ , IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12, IL- 13, IL-15, IL-17, CCL1 1 , BasicFGF, G-CSF, GM-CSF, INFv, CXCL10, CCL2, CCL3, CCL4, PDGF- ⁇ , CCL5 and VEGF.
  • treatment refers broadly to any effect or step (or intervention) beneficial in the management of a clinical condition or disorder and thus includes both therapeutic and prophylactic treatments.
  • Treatment may include reducing, alleviating, ameliorating, slowing the development of, or eliminating the condition or one or more symptoms thereof, which is being treated, relative to the condition or symptom prior to the treatment, or in any way improving the clinical status of the subject.
  • a treatment may include any clinical step or intervention which contributes to, or is a part of, a treatment programme or regimen.
  • a prophylactic treatment may include delaying, limiting, reducing or preventing the condition or the onset of the condition, or one or more symptoms thereof, for example relative to the condition or symptom prior to the prophylactic treatment.
  • Prophylaxis thus explicitly includes both absolute prevention of occurrence or development of the condition, or symptom thereof, and any delay in the onset or development of the condition or symptom, or reduction or limitation on the development or progression of the condition or symptom.
  • Treatment thus includes reducing or lowering the levels or amounts of one or more cytokines released into the blood from non-proliferating immune cells, e.g. in response to an infection, such as a microbial, viral or parasitic infection. Treatment also includes the prevention or inhibition of the release into the blood of one or more cytokines from non- proliferating immune cells, e.g. the prevention or inhibition of the release of cytokines above a level or amount considered to be within a normal range. Thus, in some aspects of the invention, treatment may be viewed as maintaining the level or amount of one or more cytokines in blood within a normal range. A normal range may depend on the subject being treated. Thus, for instance, a normal range may be the amount or level of said one or more cytokines in a healthy subject.
  • a normal range may be the average or typical level or amount of said one or more cytokines in subjects suffering from a particular disease or condition as defined herein, e.g. an infection.
  • the treatment may involve reducing or lowering an excessive, unwanted or above normal level or amount of one or more cytokines that have been released into blood from non-proliferating immune cells.
  • Treatment may also encompass the prevention or inhibition of the release of unwanted, excessive or above normal amounts or levels of one of more cytokines from non-proliferating immune cells in blood.
  • the term "treatment” does not necessarily imply cure or complete abolition or elimination of unwanted, excessive or above normal cytokine release from non-proliferating immune cells in blood.
  • disorder or condition associated with cytokine release from non- proliferating immune cells in blood is used broadly herein to include any disorder or condition which involves increased, undesired, unwanted or excessive amounts or levels of cytokines in blood as a result of the release of cytokines from non- proliferating immune cells.
  • disorder or condition associated with cytokine release from non- proliferating immune cells in blood is used broadly herein to include any disorder or condition which involves increased, undesired, unwanted or excessive amounts or levels of cytokines in blood as a result of the release of cytokines from non- proliferating immune cells.
  • cytokines in blood, for example relative to normal or healthy cytokine levels, or cytokine levels in the absence of the condition in question (e.g.
  • cytokine levels taken from healthy or unaffected tissue in the same subject
  • level of cytokines is not increased (or not greatly or significantly increased) over normal, but in which the cytokine level that occurs is unwanted or undesired, whether generally or in a particular context.
  • This may include for example an unwanted or undesired release of cytokines which may occur in a "normal” response, e.g. an immune response or an inflammatory response etc. (in other words a "normal” response which may occur in a particular (e.g. normal) context, but which may nonetheless be unwanted).
  • Such an unwanted cytokine release response may for example be the release of cytokines resulting in an unwanted inflammatory response, or an unwanted immune response such as an autoimmune response, an allergic reaction, rejection of a transplanted organ or tissue etc.
  • the disorder or condition associated with cytokine release from non-proliferating immune cells in blood may be characterised as hypercytokinemia as discussed above, which may be viewed as an unwanted or excessive release (particularly a hyperexcessive release), or increase (e.g. severe increase) in the level or amount, of one or more cytokines in blood.
  • hypercytokinemia may be a global cytokine release, comprising the unwanted (e.g. uncontrolled, excessive or above normal) release or increase of multiple cytokines, e.g. an increase in the level of at least 2 cytokines defined herein, such as an increase of at least 3, 4, 5, 6, 7, 8, 9, 10 or more cytokines as defined herein.
  • a release or increase in multiple cytokines may be termed a
  • the disorder or condition to be treated may be a condition that results in, e.g. can lead to, hypercytokinemia, such as a cytokine storm, e.g. sepsis or malaria.
  • hypercytokinemia may be associated with the release of specific cytokines in blood, e.g. an increase in a particular cytokine.
  • INF- ⁇ interferon- ⁇
  • a viral infection e.g. an acute or persistent (chronic) virus infection
  • a viral infection e.g. an acute or persistent (chronic) virus infection
  • the agent disclosed herein may be used as a preventative, prophylactic or protective agent against an unwanted or excessive (above normal, e.g. a hyperexcessive) cytokine release (e.g. hypercytokinemia) or to maintain cytokine levels or amounts within a normal range, e.g. may be useful in treatment via the prevention of disorder or condition (or the exacerbation of a disorder or condition) associated with cytokine release from non-proliferating immune cells in blood.
  • the agent described herein may be used as a direct therapeutic agent, e.g. to treat a disorder or condition associated with hypercytokinemia, i.e.
  • the disorder or condition associated with cytokine release from non- proliferating immune cells in blood may be viewed as a disorder or condition that results in (may lead to) hypercytokinemia or a disorder or condition that is associated with hypercytokinemia.
  • the disorder or condition associated with cytokine release from non-proliferating immune cells in blood may be viewed as hypercytokinemia resulting from an associated disorder or condition, e.g. hypercytokinemia resulting from an infection.
  • An unwanted or excessive release (e.g. uncontrolled or above normal or hyperexcessive release) or increase in the level or amount of one or more cytokines in blood may be defined as an increase in one or more cytokines by at least 10% relative to normal levels.
  • normal levels may be defined, e.g. relative to the levels in the blood of a healthy subject or the levels in another tissue of the subject to be treated.
  • the normal levels may be defined relative to the average or typical levels in a subject with the disorder or condition to be treated or prevented, e.g. the average or typical levels in a subject at an early stage of the disorder or condition, i.e. a condition where progression of the condition will result in an undesirable release of one or more cytokines.
  • the unwanted or excessive release or increase may be an increase by at least 15, 20, 25, 30, 35, 40, 45 or 50%, e.g. at least 60, 70, 80, 90 or 100%. In still further embodiments, it may be an increase of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 fold of normal levels, such as at least 15, 20, 30, 40, 50 or 100 fold normal levels. It will be evident that the range of the increase will, to some extent, be dependent on the nature of the one or more cytokines in question. Hence, the above values may be applied to a single cytokine or the increase may be measured as an average of the total increase, i.e. where more than one cytokine is increased.
  • a reduction of unwanted or excessive (e.g. uncontrolled or above normal or hyperexcessive) levels or amounts of one or more cytokines in blood may be defined as a decrease in one or more cytokines by at least 10%, e.g. relative to the levels prior to treatment.
  • the unwanted or excessive levels or amounts may be decreased by at least 15, 20, 25, 30, 35, 40, 45 or 50%, e.g. at least 60, 70, 80, 90 or 100%.
  • it may be a decrease of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 fold of the amounts or levels prior to treatment, such as at least 15, 20, 30, 40, 50 or 100 fold decrease.
  • the decrease may be a reduction of the amount or level to within about 25% or less of the normal level, wherein the normal level is as defined above.
  • the decrease may be a reduction to within about 20, 15, 10, 5% or less of the normal level. It will be evident that the range of the decrease or reduction will, to some extent, be dependent on the nature of the one or more cytokines in question. Hence, the above values may be applied to a single cytokine or the decrease or reduction may be measured as an average of the total decrease, i.e. where the level or amount of more than one cytokine is decreased.
  • the agent or composition results in a reduction of cytokines and/or chemokines selected from any one or more of TNFa, IL1 RA, IL- 1 ⁇ , IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, CCL1 1 , BasicFGF, G-CSF, GM-CSF, INFv, CXCL10, CCL2, CCL3, CCL4, PDGF- ⁇ , CCL5 and VEGF.
  • cytokines and/or chemokines selected from any one or more of TNFa, IL1 RA, IL- 1 ⁇ , IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, CCL1 1 , BasicFGF, G-
  • the agent or composition results in a reduction of one or more of TNFa, IL1 RA, ⁇ _-1 ⁇ , IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-9, IL-10, IL-17, G-CSF, INFv, CXCL10, CCL2, CCL3 and CCL4 (e.g. in a disease or condition associated with a bacterial infection).
  • the agent or composition results in a reduction of one or more of TNFa, IL1 RA, IL- ⁇ ⁇ , IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-9, IL-17, G-CSF and INFy (e.g.
  • the agent or composition results in a reduction of one or more of IFNy, IL-1 RA, IL-2, 4, and 9 (e.g. in a disease or condition associated with a bacterial infection). In yet further embodiments, the agent or composition results in a reduction of one or more of TNFa, ⁇ _-1 ⁇ , IL-8 (CXCL8), CXCL10, CCL2, CCL3 and CCL4 (e.g. in a disease or condition associated with a viral infection).
  • the agent or composition results in a reduction of one or more of TNFa, IL-8 (CXCL8), CXCL10, CCL2, CCL3 and CCL4 (e.g. in a disease or condition associated with a viral infection). In some more particular embodiments the agent or composition results in a reduction of one or more of IL-1 RA, CCL2, 3, and 4 (e.g. in a disease or condition associated with a viral infection).
  • the identification, characterisation, diagnosis and/or progression of the disorder or condition to be treated and/or the efficacy of the treatment may be facilitated by the measurement of one or more cytokines in the blood.
  • cytokines Various assays for measuring cytokines are well known in the art and standard kits are commercially available. Typical methods rely on immunoassays, e.g. ELISAs, RIAs, etc., and may be performed using a variety of blood samples, such as serum, plasma or whole blood.
  • the disorder or condition associated with cytokine release from non-proliferating immune cells in blood is an inflammatory disease (e.g. inflammatory arthritis, psoriatic arthritis, rheumatoid arthritis, inflammatory bowel disease) or an autoimmune disease (e.g. systemic lupus erythematosus).
  • an inflammatory disease e.g. inflammatory arthritis, psoriatic arthritis, rheumatoid arthritis, inflammatory bowel disease
  • an autoimmune disease e.g. systemic lupus erythematosus
  • the invention may be seen to provide an agent or composition as defined herein for use in the treatment or prevention of an inflammatory or autoimmune disease or a method for treating or preventing an inflammatory or autoimmune disease comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.
  • the invention may be seen to provide an agent or composition as defined herein for use in the treatment or prevention of hypercytokinemia in a subject with an inflammatory or autoimmune disease or a method of treating or preventing hypercytokinemia in a subject with an inflammatory or autoimmune disease comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.
  • the disorder or condition associated with cytokine release from non-proliferating immune cells in blood is an infection or infectious disease
  • the agent described herein may be for use in treating an infection or infectious disease or the invention may be seen to provide a method of treating a subject with an infection or infectious disease comprising administering (particularly administering an effective amount of) an agent as described herein to a subject in need thereof.
  • the invention may provide an agent or composition as described herein for use in the treatment or prevention of hypercytokinemia in a subject with an infection or infectious disease or a method of treating or preventing hypercytokinemia in a subject with an infection or infectious disease comprising administering (particularly administering an effective amount of) an agent or composition as described herein to a subject in need thereof.
  • the invention also extends to the use of the agent in the manufacture of a medicament for the above utilities.
  • an “infection” or “infectious disease” may be defined as a disease, condition or disorder caused by the invasion of a subject, e.g. one or more organs or tissues of said subject, by one or more disease-causing organisms and their subsequent multiplication.
  • an infection or infectious disease may be characterised by the reaction of the subject (e.g. organ or tissues of said subject) to said organisms and, in some cases, to the toxins produced by said organisms.
  • An infection or infectious disease may be a microbial, viral or parasitic infection and may be local or systemic.
  • a microbial infection may be any bacterial or fungal infection, i.e. caused by a bacterium or fungus. In particularly preferred
  • the disease, condition or disorder caused by a viral infection is not a viral-induced hyperproliferative disease, such as warts and EBV-induced disease (e.g. infectious mononucleosis), scar formation and the like.
  • a viral-induced hyperproliferative disease such as warts and EBV-induced disease (e.g. infectious mononucleosis), scar formation and the like.
  • the infectious disease or infection may be a bacterial infection or disease.
  • bacteria that cause infections or infectious diseases that may be treated or prevented by the agents or compositions described herein may be gram positive or gram negative, or gram test non-responsive. They may be aerobic or anaerobic bacteria.
  • the bacteria may be from any of the genus Achromobacter, Acinetobacter, Actinobacillus, Aeromonas,
  • Agrobacterium Alcaligenes, Alteromonas, Bacillus, Bacteroides, Bartonella, Borrelia, Bordetella, Brucella, Burkholderia, Campylobacter, Cardiobacterium, Chlamydia, Chlamydophila, Chromobacterium, Chyseobacterium, Chryseomonas, Citrobacter, Clostridium, Comamonas, Corynebacterium, Coxiella,
  • Cryptobacterium Edwardsiella, Eikenella, Enterobacter, Enterococcus, Erwinia, Helicobacter, Kingella, Klebsiella, Lactobacillus, Lactococcus, Legionella, Leptospira, Leptotrichia, Leuconostoc, Listeria, Listonella, Mobiluncus, Moraxella, Morganella, Mycobacterium, Mycoplasma, Neisseria, Nocardia, Nocardiopsis, Pantoea, Parachlamydia, Pasteurella, Peptococcus, Peptostreptococcus,
  • Propionibacterium, Proteus Providencia, Pseudomonas, Ralstonia, Rickettsia, Salmonella, Shewenella, Shigella, Sphingobacterium, Sphingomonas, Staphylococcus, Stenotrophomonas, Streptobacillus, Streptococcus, Streptomyces, Treponem and Yersinia, such as Acinetobacter, Bacillus, Burkholderia, Chlamydia, Clostridium, Helicobacter, Staphylococcus, Streptococcus, Pseudomonas,
  • Legionella Listeria, Mycobacterium, Proteus, Klebsiella, Fusobacterium or other enteric or coliform bacteria.
  • the infection or infectious disease may be caused by a gram-positive bacteria such as, M. tuberculosis, M. bovis, M. typhimurium, M. bovis strain BCG, BCG substrains, M. avium, M. intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans, M.
  • a gram-positive bacteria such as, M. tuberculosis, M. bovis, M. typhimurium, M. bovis strain BCG, BCG substrains, M. avium, M. intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans, M.
  • Propionibacterium acnes, and Enterococcus species are Propionibacterium acnes, and Enterococcus species.
  • the infection or infectious disease may be caused by a gram-negative bacteria such as Clostridium tetani, Clostridium perfringens,
  • Chlamydia psittaci Coxiella burnetii, Escherichia coli, Neiserria meningitidis, Neiserria gonorrhea, Haemophilus influenzae, Haemophilus ducreyi, Yersinia pestis, Yersinia enterolitica, Escherichia coli, E. hirae, Burkholderia cepacia, Burkholderia pseudomallei, Francisella tularensis, Bacteroides fragilis,
  • Fusobascterium nucleatum, and Cowdria ruminantium Fusobascterium nucleatum, and Cowdria ruminantium.
  • a disorder or condition associated with cytokine release from non- proliferating immune cells in blood may be the result of, e.g. caused by, a bacterial infection or infectious disease, such as sepsis.
  • a bacterial infection may be associated with a particular disorder or condition, i.e. subjects suffering from a particular disorder or condition may be particularly susceptible to one or more bacterial infections. For instance, it is common for subjects with cystic fibrosis to suffer from chronic Pseudomonas infections.
  • invention may be seen to extend to an agent or composition as defined herein for use in treating a disease or condition (e.g.
  • hypercytokinemia exacerbated or caused by an infection, e.g. bacterial pneumonia, cystic fibrosis, gastric ulcers, bacterial meningitis, Legionellosis (Legionnaires' disease), Legionellosis (Pontiac fever), Pertussis (Whooping cough), Salmonellosis, Tuberculosis etc.
  • an infection e.g. bacterial pneumonia, cystic fibrosis, gastric ulcers, bacterial meningitis, Legionellosis (Legionnaires' disease), Legionellosis (Pontiac fever), Pertussis (Whooping cough), Salmonellosis, Tuberculosis etc.
  • a condition or disease caused by an infection is sepsis, which is a condition that results from a severe infection.
  • the invention may be seen to provide an agent or composition as defined herein for use in the treatment or prevention of sepsis or a method for treating or preventing sepsis comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.
  • the invention may be seen to provide an agent or composition as defined herein for use in the treatment or prevention of hypercytokinemia in a subject with sepsis or a method of treating or preventing hypercytokinemia in a subject with sepsis comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.
  • cystic fibrosis which is a condition that is commonly associated with a chronic Pseudomonas infection.
  • the invention may be seen to provide an agent or composition as defined herein for use in the treatment of cystic fibrosis (or more particularly, treatment of a subject with cystic fibrosis, e.g. suffering from an infection, i.e. alleviating the one or more symptoms of a subject suffering from cystic fibrosis).
  • the invention provides a method for treating cystic fibrosis (or more particularly, treating a subject with cystic fibrosis, e.g. suffering from an infection, i.e. alleviating the one or more symptoms of a subject suffering from cystic fibrosis) comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.
  • the infectious disease or infection may be a fungal infection or disease.
  • the fungus may be a mold or yeast, preferably a yeast.
  • the fungus may be selected from any one or more of a Dermatophyte, Aspergillus sp. (such as Aspergillus fumigatus, Aspergillus nigricans or flavescens), Zygomycota sp., Fusarium sp., Trichophyton sp,. Basidiobolus ranarum, Piedraia sp. (such as Piedraia hortae), Blastomyces dermatitidis, Candida sp.
  • Aspergillus sp. such as Aspergillus fumigatus, Aspergillus nigricans or flavescens
  • Zygomycota sp. Fusarium sp.
  • Trichophyton sp Trichophyton sp
  • Basidiobolus ranarum Piedra
  • Coccidioides sp. such as Coccidioides immitis and Coccidioides posadasii
  • Conidiobolus sp. such as Conidiobolus coronatus and Conidiobolus incongruus
  • Cryptococcus sp. such as Cryptococcus gattii and Cryptococcus neoformans
  • fungi that cause infections or infectious diseases that may be treated or prevented by the agents and compositions described herein include fungi from the genera Candida, Aspergillus,
  • Candida albicans Candida dubliniensis
  • Cryptococcus neoformans Histoplama capsulatum
  • Aspergillus fumigatus Coccidiodes immitis
  • Paracoccidiodes brasiliensis Coccidiodes immitis
  • Blastomyces dermitidis Pneomocystis carnii
  • Penicillium marneffi and Alternaria alternate, etc.
  • a fungal infection commonly start in the lungs or on the skin, e.g. a fungal infection (a mycosis) may result from the inhalation of fungal spores or localized colonization of the skin may initiate persistent infections.
  • a disease or condition exacerbated or caused by a fungal infection (mycosis) to be treated or prevented by the agent or composition of the invention may be in any tissue or organ of the subject to be treated, such as the lungs (including the respiratory tract), skin (including wounds), mouth, ear, eye etc.
  • the fungal infection may be a respiratory infection, skin infection, ear infection, eye infection etc.
  • a disorder or condition associated with cytokine release from non- proliferating immune cells in blood may be the result of, e.g. caused by, a fungal infection or infectious disease, caused by any one or more of the fungi (or fungi from one or more the genera) described above.
  • a disorder or condition associated with cytokine release from non-proliferating immune cells in blood may be the result of, or exacerbated by, a microbial infection, such as a microbe selected from any one or more of the genera Actinebacter, Citrobacter, Enterobacter, Escherichia, Hafnia, Serratia, Yersinia, Peptostreptococcus, Bacteriodes, Pseudomonas, Legionella, Staphylococcus, Enterococcus, Streptococcus, Klebsiella, Candida, Proteus, Burkholderia, Fusobacterium and Mycobacterium, for instance, Staphylococcus aureus, Staphylococcus epidermidis, Legionella pneumophila, Candida albicans, Pseudomonas aeruginosa, Burkholderia cepacia and Streptococcus Pyogenes.
  • a microbial infection such as a
  • the infectious disease or infection may be a viral infection or disease.
  • viruses that cause infections or infectious diseases that may be treated or prevented by the agents described herein include Australian bat lyssavirus, Banna virus, Barmah forest virus, Bunyamwera virus, Bunyavirus La Crosse, Bunyavirus snowshoe hare, Cercopithecine herpesvirus, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhori virus, Dugbe virus, Duvenhage virus, Eastern equine encephalitis virus, Ebolavirus, Echovirus, Encephalomyocarditis virus, European bat lyssavirus, Hantaan virus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis delta virus, Human herpesvirus 1 , Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalo
  • Japanese encephalitis virus Junin arenavirus, Kl Polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoria marburgvirus, Langat virus, Lassa virus, Lordsdale virus, Louping ill virus, Lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, Measles virus, Mengo encephalomyocarditis virus, Mokola virus, Molluscum contagiosum virus, Monkeypox virus, Mumps virus, Murray valley encephalitis virus, New York virus, Nipah virus, Norwalk virus, O'nyong-nyong virus, Orf virus,
  • Oropouche virus Pichinde virus, Poliovirus, Punta toro phlebovirus, Puumala virus, Rabies virus, Rift valley fever virus, Ross river virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Sandfly fever Sicilian virus, Sapporo virus, Semliki forest virus, Seoul virus, Sindbis virus, Victoria virus, St.
  • louis encephalitis virus Tick-borne powassan virus, Toscana virus, Uukuniemi virus, Varicella-zoster virus, Variola virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, West Nile virus and Yellow fever virus.
  • viruses that cause infections or infectious diseases that may be treated or prevented by the agents and
  • compositions described herein may be particularly selected from any one or more of Dengue virus, Ebolavirus, Echovirus, Encephalomyocarditis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis delta virus, Human herpesvirus 1 , Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus 68-70, Human herpesvirus 2, Human herpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, Human
  • immunodeficiency virus Human parainfluenza, Human parvovirus B19, Human respiratory syncytial virus, Human rhinovirus, Human SARS coronavirus, Human T- lymphotropic virus, Human torovirus, Influenza A virus, Influenza B virus, Influenza C virus, Japanese encephalitis virus, Measles virus, Mumps virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, West Nile virus and Yellow fever virus.
  • the viral infection is not an infection that causes or results in viral-induced
  • hyperproliferative disease such as warts and EBV-induced disease (e.g. infectious mononucleosis), scar formation and the like.
  • a disorder or condition associated with cytokine release from non- proliferating immune cells in blood may be the result of, e.g. caused by, a viral infection or infectious disease, such as AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza and hepatitis.
  • a viral infection or infectious disease such as AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza and hepatitis.
  • the invention may be seen to provide an agent or composition as defined herein for use in the treatment of a viral infection, e.g. AIDS/HIV,
  • Dengue fever, measles, mumps, rubella, influenza or hepatitis or the treatment of the symptoms of a viral infection, e.g. AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza or hepatitis) or a method for treating a viral infection, e.g.
  • AIDS/HIV Dengue fever, measles, mumps, rubella, influenza or hepatitis
  • treating a subject suffering from a viral infection e.g. AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza or hepatitis comprising administering
  • the invention may be seen to provide an agent or composition as defined herein for use in the treatment or prevention of hypercytokinemia in a subject with a viral infection, e.g. AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza or hepatitis, or a method of treating or preventing hypercytokinemia in a subject with a viral infection, e.g. AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza or hepatitis, comprising administering (particularly administering an effective amount of) an agent as defined herein to a subject in need thereof.
  • a viral infection e.g. AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza or hepatitis
  • a method of treating or preventing hypercytokinemia in a subject with a viral infection e.g. AIDS/HIV, Dengue fever, measles, mumps, rubella, influenza or hepatitis
  • the infectious disease or infection may be a parasitic infection or disease.
  • parasites that cause infections or infectious diseases that may be treated or prevented by the agents and compositions described herein include Plasmodium sp., such as Plasmodium falciparum, Plasmodium knowlesi, Plasmodium vivax, Plasmodium berghei and Plasmodium yoelii infections, protozoa such as Toxoplasma species e.g. Toxoplasma gondii, Trypanosoma brucei, Trypanosoma cruzi, Leishmania species such as Leishmania major, Schistosoma such as Schistosoma mansoni and Entamoeba histolytica.
  • Plasmodium sp. such as Plasmodium falciparum, Plasmodium knowlesi, Plasmodium vivax, Plasmodium berghei and Plasmodium yoelii infections
  • protozoa such as Toxo
  • a disorder or condition associated with cytokine release from non- proliferating immune cells in blood may be the result of, e.g. caused by, a parasitic infection or infectious disease, such as malaria, toxoplasmosis, trypanosomiasis and schistosomiasis.
  • a parasitic infection or infectious disease such as malaria, toxoplasmosis, trypanosomiasis and schistosomiasis.
  • a condition or disease caused by an infection is a parasitic infection, such as malaria, which is a condition that results from a parasitic infection, e.g. Plasmodium infection, such as Plasmodium falciparum, Plasmodium knowlesi, Plasmodium vivax, Plasmodium berghei or Plasmodium yoelii infection.
  • Plasmodium infection such as Plasmodium falciparum, Plasmodium knowlesi, Plasmodium vivax, Plasmodium berghei or Plasmodium yoelii infection.
  • the invention may be seen to provide an agent or composition as defined herein for use in the treatment of a parasitic infection, e.g. malaria, toxoplasmosis, trypanosomiasis or schistosomiasis, (or the treatment of the symptoms of a parasitic infection, e.g.
  • malaria toxoplasmosis, trypanosomiasis or schistosomiasis
  • a method for treating a parasitic infection e.g. malaria, toxoplasmosis, trypanosomiasis or schistosomiasis, (or treating a subject suffering from a parasitic infection, e.g. malaria, toxoplasmosis, trypanosomiasis or schistosomiasis) comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.
  • the invention may be seen to provide an agent or composition as defined herein for use in the treatment or prevention of hypercytokinemia in a subject with a parasitic infection, e.g. malaria, toxoplasmosis, trypanosomiasis or schistosomiasis, or a method of treating or preventing hypercytokinemia in a subject with a parasitic infection, e.g. malaria, toxoplasmosis, trypanosomiasis or schistosomiasis, comprising administering (particularly administering an effective amount of) an agent as defined herein to a subject in need thereof.
  • a parasitic infection e.g. malaria, toxoplasmosis, trypanosomiasis or schistosomiasis
  • the disorder or condition associated with cytokine release from non-proliferating immune cells in blood is graft versus host disease (GVHD), i.e. an immune response associated with the transplantation of cells, tissues or organs to a recipient from a genetically non-identical donor of the same species.
  • GVHD graft versus host disease
  • the transplant is may be viewed as an allograft, allogeneic transplant or homograft.
  • the transplant may be a stem cell and/or bone marrow transplant.
  • the agent or composition may be for use in treating or preventing graft versus host disease or the invention may be seen to provide a method of treating a subject with graft versus host disease comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.
  • the invention may provide an agent or composition as defined herein for use in the treatment or prevention of hypercytokinemia in a subject with a graft versus host disease or a method of treating or preventing hypercytokinemia in a subject with a graft versus host disease comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.
  • the invention also extends to the use of the agent in the manufacture of a medicament for the above utilities.
  • the disorder or condition associated with cytokine release from non-proliferating immune cells in blood is trauma, e.g. trauma caused by injury (i.e. a physical wound or injury, such as a fracture or blow, e.g. a polytrauma (affecting multiple sites), a head trauma, chest trauma, abdominal trauma or extremity trauma) or surgery.
  • injury or wound resulting from trauma may be a burn.
  • a burn may be caused by heat, electricity, chemicals, friction or radiation.
  • the burn is a partial- thickness or second-degree burn (comprising damage to some of the underlying skin layers).
  • the burn is a full-thickness or third-degree burn (comprising damage that extends to all layers of the skin). In some embodiments, the burn is a fourth-degree burn, which additionally involves injury to deeper tissues, such as muscle or bone.
  • the agent or composition may be for use in treating trauma (e.g. caused by injury or surgery) or the invention may be seen to provide a method of treating a subject with trauma (e.g. caused by injury or surgery) comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.
  • the invention may provide an agent or composition as defined herein for use in the treatment or prevention of hypercytokinemia in a subject with trauma (e.g. caused by injury or surgery) or a method of treating or preventing hypercytokinemia in a subject with trauma (e.g. caused by injury or surgery) comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.
  • the invention also extends to the use of the agent in the manufacture of a medicament for the above utilities.
  • hypercytokinemia which may be a result of a disease or condition as described herein, such as sepsis, malaria, graft versus host disease (GVHD) etc. can result in, or increase the risk of, multiple organ dysfunction syndrome (MODS), which is also known as multiple organ failure (MOF) or multisystem organ failure (MSOF).
  • MODS multiple organ dysfunction syndrome
  • MOF multiple organ failure
  • MSOF multisystem organ failure
  • the invention provides an agent or composition as defined herein for use in the treatment or prevention of MODS or a method for treating or preventing MODS comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.
  • the invention may be seen to provide an agent or composition as defined herein for use in the treatment or prevention of hypercytokinemia in a subject with MODS or a method of treating or preventing hypercytokinemia in a subject with MODS comprising administering (particularly administering an effective amount of) an agent or composition as defined herein to a subject in need thereof.
  • the agent or composition as defined herein may be useful for the prevention of hypercytokinemia in a subject suffering from, or at risk from, any of the conditions mentioned above.
  • the agent or composition as defined herein is used in combination with one or more additional active agents, e.g. an immunosuppressive compound, an anti-inflammatory compound, anti-microbial compound, or steroid (e.g. a corticosteroid) or a kinase inhibitor (such as a p38 MAPK inhibitor or a class I PI3K inhibitor), in order to enhance or complement the effect of the agent or composition defined herein, e.g. to treat symptoms of the disease or condition that are not directly affected by the agent or composition of the invention.
  • the agent as defined herein may be used alone, i.e. as the only active agent in a composition and/or medicament.
  • the additional active agent is an immunosuppressive compound.
  • immunosuppressive compounds include but are not limited to any one or more of glucocorticoids, cyclophosphamide, methotrexate, azathioprine, mercaptopurine, dactinomycin, anthracyclines, mitomycin C, bleomycin and mithramycin.
  • the additional active agent is an anti-inflammatory compound.
  • Suitable anti-inflammatory compounds include but are not limited to any one or more of diclofenac, ibuprofen, naproxen, celecoxib, mefenamic acid, etoricoxib, indometacin and aspirin.
  • the additional active agent is an antibiotic
  • antibiotic compounds include but are not limited to any one or more of Aminocoumarins (such as Novobiocin, Albamycin, Coumermycin and Clorobiocin), Aminoglycosides (such as Amikacin, Apramycin, Gentamicin,
  • Cephalosporins such as Cefadroxil, Cefazolin, Cefalothin
  • Ceftibuten Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil and
  • Ceftobiprole Glycopeptides (such as Teicoplanin, Vancomycin and Telavancin), Lincosamides (such as Clindamycin and Lincomycin), Lipopeptides (such as Daptomycin), Macrolides (such as Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin and Spiramycin), Monobactams (such as Aztreonam), Nitrofurans (such as Furazolidone and
  • Nitrofurantoin Oxazolidonones (such as Linezolid, Posizolid, Radezolid and Torezolid), Penicillins (such as Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin and Ticarcillin), Penicillin combinations (such as Amoxicillin/clavulanate, Ampicillin/sulbactam,
  • Piperacillin/tazobactam and Ticarcillin/clavulanate Polyethers (such as Monensin), Polypeptides (such as Bacitracin, Colistin and Polymyxin B), Quinolones (such as Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin and Temafloxacin); Sulfonamides (such as Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole,
  • Sulfanilimide Sulfasalazine, Sulfisoxazole, Sulfamethoxazole (Co-trimoxazole, TMP-SMX, 'Trimethoprim') and Sulfonamidochrysoidine
  • Tetracyclines such as Demeclocycline, Doxycycline, Minocycline, Oxytetracycline and Tetracycline
  • Others such as Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin ('Rifampin'), Rifabutin,
  • the additional active agent is an antifungal compound.
  • Suitable antifungal compounds include but are not limited to any one or more of Polyene antifungals (such as Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin and Rimocidin), Imidazoles (such as
  • Triazoles such as Albaconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Ravuconazole, Terconazole and Voriconazole
  • Thiazoles such as Abafungin
  • Allylamines such as Amorolfin, Butenafine, Naftifine and Terbinafine
  • Echinocandins such as Anidulafungin, Caspofungin and Micafungin
  • Others such as Benzoic acid, Ciclopirox, Flucytosine or 5-fluorocytosine, Griseofulvin, Haloprogin, Polygodial, Tolnaftate, Undecylenic acid and Crystal violet.
  • the additional active agent is a kinase inhibitor.
  • Suitable kinase inhibitors include but are not limited to any one or more of p38 MAPK inhibitors (such as VX-702, SB203580, VX-745, LY2228820, BIRB 796 (Doramapimod), PH-797804 and TAK-715) and class I PI3K inhibitors (such as AS- 605240, BYL719, CAL-101 , GDC-0941 (Bismesylate salt), GDC-0941 (free base), GSK2636771 , IC-871 14, IPI-145, LY294002, NVP-BKM120 (Buparlisib), PIK-75, TG100-1 15 and TGX-221 ).
  • p38 MAPK inhibitors such as VX-702, SB203580, VX-745, LY2228820, BIRB 796 (Doramapimod), PH-797804 and TAK-71
  • class I PI3K inhibitors such as AS- 605240, BY
  • a suitable dose may be defined as a dose that does not induce apoptosis, i.e. an apoptosis non-inducing dose.
  • a suitable dose may be defined as a "low dose” or “low amount” of the agent (e.g. oligopeptidic compound), which may be seen as a dose or amount that is not sufficient to cause or induce apoptosis either directly or indirectly.
  • a "low dose” or “low amount” of the agent is a dose or amount that is an effective dose or amount for reducing or inhibiting cytokine release from non- proliferating immune cells in blood.
  • a high dose or amount may be viewed as an effective dose or amount that is sufficient to cause or induce apoptosis either directly or indirectly (i.e. cytotoxic doses or doses that result in an increased sensitivity to other cytotoxic or cytostatic agents).
  • the effective dose or amount of agent may depend on the characteristics of the peptide, e.g. the strength of the interaction between the PCNA interacting motif and the binding domain of the target protein(s). Furthermore, effective dose or amount of the agent may depend upon the nature of the compound used (i.e.
  • the mode of administration the course of treatment, the age and weight of the patient, the medical indication, the body or body area to be treated and may be varied or adjusted according to choice.
  • a low dose or amount may result in an active concentration range of about 0.01 , 0.05, 0.1 , 0.25, 0.5, 0.75, 1 .0, 1 .25, 1.50, 1.75, 2.0, 3.0, 4.0, 5.0 to 10 ⁇ , e.g. 0.01 to 10 ⁇ , e.g. 0.05 to 7.5 ⁇ , such as 0.1 to 7.5 ⁇ , e.g. 0.5 to 5 ⁇ .
  • a high dose or amount may result in an active concentration range of about 1 .0, 2.0, 3.0, 4.0, 5.0, 7.5, 10, 15, 20, 25, 30, 40 to 50 ⁇ , e.g. 1.0 ⁇ 50 ⁇ , e.g. 2.0 to 40 ⁇ , such as 3.0 to 30 ⁇ , e.g. 5.0 to 25 ⁇ .
  • Said concentrations are determined by reference to the amount of the compound itself and thus appropriate allowances should be made to take into account the purity of the composition.
  • the subject is an animal (i.e. any human or non-human animal), preferably a mammal, most preferably a human.
  • peptide-mediated methods of delivery can be used, notably uptake (import) peptides, such as cell penetrating peptides (CPPs), which as discussed above, are short, in some cases polycationic, sequences which can facilitate cellular uptake of peptides, proteins or nucleotide molecules which contain CPPs or to which CPPs are linked, for example by enhancing uptake into endosomes of mammalian cells.
  • the oligopeptidic compound of defined herein typically comprises an uptake peptide, e.g. a CPP.
  • Microencapsulation provides a simple and cost-effective way to enclose bioactive materials within a semi-permeable polymeric membrane for the purpose of protecting the bioactive materials and releasing the enclosed substances or their products in a controlled fashion.
  • photochemical photochemical
  • PCI internalisation
  • Chitosan and water-soluble chitosan derivatives, in particular glycol chitosan, are emerging as the drug carriers of choice because of their
  • a preferred example is glycol chitosan hydrophobically modified with 5 ⁇ -cholanic acid.
  • Figure 1 shows that ATX-101 (SEQ ID NO: 1289, which contains the API M motif, RWLVK (SEQ ID NO: 1290)) affects MAPK and PI3K/Akt pathways in multiple myeloma cell lines, wherein: (A) shows cell growth (MTT assay) of JJN-3 cells untreated ( ⁇ ) and after addition of 6 ⁇ ATX-101 5 ⁇ LY294002 (PI3K
  • Figure 2 shows that ATX-101 reduces cytokine secretion of monocytes after TLR stimulation
  • a and B show multiplex analysis of cytokine levels produced by peripheral blood monocytes after 4h treatment with 4 ⁇ ATX-101 in combination with 10 ng/ml LPS (A) and 40 g/ml polylC stimulation (B).
  • Measured cytokine levels were normalized to cytokine levels from monocytes stimulated with TLR ligand alone.
  • a two fold or higher increase in cytokine level after TLR stimulation was defined as cytokine induction.
  • Data is from three donors (mean ⁇ SD);
  • C) shows an ELISA of IFN- ⁇ levels produced by peripheral blood monocytes from five donors.
  • Isolated monocytes were stimulated with 10 ng/ml LPS and 40 ⁇ g/ml polylC alone and in combination with 4 ⁇ ATX-101 for 4 h.
  • Donor 1 was treated with 6 ⁇ ATX-101 .
  • DRAQ5 was used for nuclear staining (red). Bar, 5 ⁇ .
  • Figure 3 shows that ATX-101 reduces Akt phosphorylation in monocytes, wherein: (A) shows a Western blot analysis of Akt Ser473 phosphorylation in peripheral blood monocytes stimulated with 10 ng/ml LPS and 40 ⁇ g/ml polylC alone and in combination with 4 ⁇ ATX-101 for 4 h. The upper panel shows the quantification of phosphorylated Akt corrected for loading differences ( ⁇ -tubulin) and normalized to untreated control cells.
  • (B-E) show multiplex analysis of cytokine levels produced by peripheral blood monocytes stimulated with 10 ng/ml LPS (B and D, same donor) and 40 ⁇ g/ml polylC (C and E, same donor) after 4 h.
  • B and C Stimulated- monocytes were treated with 4 ⁇ ATX-101 , 10 ⁇ SB203580 (p38 MAPK inhibitor), and combination of ATX-101 and SB203580.
  • (D and E) Stimulated- monocytes were treated with 4 ⁇ ATX-101 , 5 and 10 ⁇ LY294002 (PI3K class I inhibitor), and combination of ATX-101 and LY294002.
  • cytokine levels were normalized to cytokine levels from monocytes stimulated with TLR ligand alone. A two fold or higher increase in cytokine level after TLR stimulation was defined as cytokine induction. # Value is too low to be visible in the column diagram; n cytokine measurement was out of range. Data is from representative experiments out of two.
  • Figure 4 shows that ATX-101 reduces Akt phosphorylation and PACT protein levels in HaCaT cells, wherein: (A) shows quantification of Akt Ser473 phosphorylation and PACT protein levels in HaCaT cells treated with 12 ⁇ ATX- 101 , 2 ⁇ g/ml polylC, and combination of ATX-101 and polylC for 4 h by Western blot analysis. Protein levels were corrected for loading differences ( ⁇ -tubulin) and normalized to untreated control cells. Data is from three independent experiments (mean ⁇ SD); and (B) shows a confocal fluorescence image of immunofluorescently stained PCNA in HaCaT cells. Bar, 5 ⁇ .
  • Figure 5 shows that ATX-101 reduces cytokine secretion of monocytes after TLR stimulation, wherein: (A) and (B) show multiplex analysis of cytokine levels produced by peripheral blood monocytes after 4h treatment with 4 ⁇ ATX-101 in combination with 10 ng/ml PAM3Cys (A) and 10 ⁇ / ⁇ R848 stimulation (B).
  • cytokine levels were normalized to cytokine levels from monocytes stimulated with TLR ligand alone. A two fold or higher increase in cytokine level after TLR stimulation was defined as cytokine induction, n Cytokine measurement was out of range. Data is from one experiment out of two.
  • Figure 6 shows that ATX-A (SEQ ID NO: 1206, which contains the motif RALVK (SEQ ID NO: 1207), which is not capable of interacting with PCNA) reduces less efficiently cytokine secretion of monocytes after TLR stimulation, wherein multiplex analysis of LPS- and polylC-induced cytokine levels produced by peripheral blood monocytes after 4 h treatment with 4 ⁇ ATX-101 or 4 ⁇ ATX-A in combination with 10 ng/ml LPS and 40 ⁇ g/ml polylC (left and right panel, respectively). Measured cytokine levels were normalized to cytokine levels from monocytes stimulated with TLR ligand alone. Data is from one donor. We obtained similar results for IL-6 secretion in two other donors.
  • ATX-A SEQ ID NO: 1206, which contains the motif RALVK (SEQ ID NO: 1207), which is not capable of interacting with PCNA
  • Figure 7 shows that the Akt inhibitor SC66 reduces cytokine secretion of monocytes to or below the basal levels after TLR stimulation, wherein multiplex analysis of cytokine levels produced by peripheral blood monocytes after 4 h treatment with 4 ⁇ ATX-101 and 1 ⁇ g/ml SC66 in combination with 10 ng/ml LPS (left panel) and 40 ⁇ g/ml polylC stimulation (right panel).
  • Measured cytokine levels were normalized to cytokine levels from monocytes stimulated with TLR ligand alone. A two fold or higher increase in cytokine level after TLR stimulation was defined as cytokine induction. # Value is too low to be visible in the column diagram; n Cytokine measurement was out of range. Data is from one experiment out of two.
  • Figure 8 shows a graph showing the results of FRET analysis. Normalised FRET (NFRET) measurements are shown between EYFP (yellow fluorescent protein)/ ECFP (cyan fluorescent protein) (Lane 1 , background due to dimerisation of the tags). EYFP-APIM motif / ECFP-PCN A for various motifs are shown in the other lanes.
  • Figure 9 shows a graph showing the results of FRET analysis. Normalised FRET (NFRET) measurements are shown between EYFP (yellow fluorescent protein)/ ECFP (cyan fluorescent protein) (Lane 1 (EY-EC), background due to dimerisation of the tags).
  • EYFP-APIM motif / ECFP-PCNA for various motifs are shown in the other lanes, wherein the annotations on the X-axis refer to the following APIM sequences: WT (RWLVK, SEQ ID NO: 1290); LA (RWLAK, SEQ ID NO: 1291 ); AV (RWAVK, SEQ ID NO: 1292); LG (RWLGK, SEQ ID NO:1293); LS (RWLSK, SEQ ID NO: 1294); SV (RWSVK, SEQ ID NO: 1295); WF (RWFLVK, SEQ ID NO: 1258); FF (RFFLVK, SEQ ID NO: 1265); FL (RFLLVK, SEQ ID NO:
  • Figure 10 shows confocal microscope images which show that
  • RWYLVK SEQ ID NO: 1259
  • FF RWYLVK, SEQ ID NO: 1265
  • FY RFYLVK, SEQ ID NO: 1266
  • FV RVLVK, SEQ ID NO: 1269
  • Fl RFILVK, SEQ ID NO:
  • Figure 1 1 shows graphs showing the results of cytotoxicity assays with various APIM peptides, with (B) or without cisplatin (A) as described in Example 12.
  • the annotations in the legend refer to the following APIM sequences: WT-101 , MDRWLVKWKKKRKIRRRRRRRRRRRRR (SEQ ID NO: 1289); ATX-A,
  • MDRALVKWKKKRKIRRRRRRRRRRRRRRRRR (SEQ ID NO: 1206) (negative control); ATX-FF, MDRFFLVKWKKKRKIRRRRRRRRRRRRRRR (SEQ ID NO: 1 182); ATX-FL MDRFLLVKWKKKRKIRRRRRRRRRRRRRRR (SEQ ID NO: 1 187); ATX-WY,
  • Figure 12 shows bar charts of relative cytokine levels produced by LPS stimulated peripheral blood monocytes after addition of peptides. Cytokine levels from monocytes treated with LPS alone are set to 100 % (gray line). The bar charts, A-Q, show only cytokines with a two-fold or higher increase in cytokine level after LPS stimulation. Data are given as means +/- SD from two experiments. Four different blood donors were used. The abbreviations refer to the following peptide sequences: R1 1 , RRRRRRRRRRR (SEQ ID NO: 337); 101 ,
  • MDRWLVKWKKKRKIRRRRRRRRRRRRRRRRR (SEQ ID NO: 1289); 101 -Y, MDRYLVKWKKKRKIRRRRRRRRRRRRRRR (SEQ ID NO: 1300); 101 -FF,
  • MDRWLVKWKKKRKIRQIKIWFQNRRMKWKK (SEQ ID NO: 1304); 101 -FF-pen, MDRFFLVKWKKKRKIRQIKIWFQNRRMKWKK (SEQ ID NO: 1305); 101 -WW-pen, MDRWWLVKWKKKRKIRQIKIWFQNRRMKWKK (SEQ ID NO: 1306); 101 -trans MDRWLVKWKKKRKIAGYLLGKINLKALAALAKKIL (SEQ ID NO: 1307); 101 -prot, MDRWLVKWKKKRKIYGRKKRRQRRR (SEQ ID NO: 1308).
  • Figure 13 shows graphs showing the results of cytotoxicity assays with various "standard” and “extended” APIM peptides.
  • A)-(C) show the levels cell survival of HEK 293 cells treated with 6 ⁇ of peptides alone (A), with 0.5 ⁇ cisplatin (B) or with 1 ⁇ cisplatin (C) as described in Example 14.
  • D)-(l) show the levels of cell survival of HEK 293 cells treated with 12 ⁇ of peptides alone (D) and (G), with 0.5 ⁇ cisplatin (E) and (H) or with 1 ⁇ cisplatin (F) and (I) as described in Example 14.
  • MDRWLVKWKKKRKIRQIKIWFQNRRMKWKK (SEQ ID NO: 1304); 101 -FF-pen, MDRFFLVKWKKKRKIRQIKIWFQNRRMKWKK (SEQ ID NO: 1305); 101 -WW-pen, MDRWWLVKWKKKRKIRQIKIWFQNRRMKWKK (SEQ ID NO: 1306).
  • Figure 14 shows graphs showing the results of cytotoxicity assays with various "standard” and “extended” APIM peptides.
  • A)-(C) show the levels cell survival of U20S cells treated with 4 ⁇ of peptides alone (A), with 0.5 ⁇ cisplatin (B) or with 1 ⁇ cisplatin (C) as described in Example 14.
  • D)-(F) show the levels of cell survival of U20S cells treated with 6 ⁇ of peptides alone (D), with 0.5 ⁇ cisplatin (E) or with 1 ⁇ cisplatin (F) as described in Example 14.
  • MDRWLVKWKKKRKIRRRRRRRRRRRRRRRRR (SEQ ID NO: 1289); 101 -FF, MDRFFLVKWKKKRKIRRRRRRRRRRRRRRR (SEQ ID NO: 1 182); 101 -WW,
  • Figure 15 shows graphs showing the results of cytotoxicity assays with various "standard” and “extended” APIM peptides.
  • (A)-(C) show the levels cell survival of U20S cells treated with 12 ⁇ of peptides alone (A), with 0.5 ⁇ cisplatin (B) or with 1 ⁇ cisplatin (C) as described in Example 14.
  • the annotations in the legend refer to the following APIM sequences: 101 ,
  • MDRWLVKWKKKRKIRQIKIWFQNRRMKWKK (SEQ ID NO: 1304); 101 -FF-pen, MDRFFLVKWKKKRKIRQIKIWFQNRRMKWKK (SEQ ID NO: 1305); 101 -WW-pen, MDRWWLVKWKKKRKIRQIKIWFQNRRMKWKK (SEQ ID NO: 1306).
  • the inventors have determined that more than 20 different signalling kinases, mainly cytoplasmic, contain a "conventional" APIM sequence, suggesting that they interact with PCNA. These include three direct members of the PI3K/Akt pathway (p1 10-a, p1 10- ⁇ , and PI3K-C23) and several kinases directly or indirectly affecting the MAPK pathways (ERK8, MK2, MK5, MST4, and TA02). PCNA has heretofore not been linked to signal transduction. The inventors have determined that PCNA plays a role in signal transduction pathways downstream of TLRs.
  • the inventors have established that the signal transduction pathways can be disrupted by targeting PCNA oligopeptidic compounds comprising an APIM motif, including an extended or longer APIM motif as defined herein. These compounds are thought to compete with PCNA for PCNA interacting proteins.
  • the effects of oligopeptidic compounds comprising an APIM motif on signal transduction pathways have been established using an exemplary cell-penetrating APIM-containing peptide ATX-101 (SEQ ID NO: 1289, which contains the APIM motif, RWLVK (SEQ ID NO: 1290)).
  • the inventors have surprisingly found that ATX-101 treatment reduced cytokine secretion of peripheral blood monocytes after stimulation with different TLR ligands and reduced Akt phosphorylation.
  • This data indicates that targeting PCNA affects signal transduction pathways, including the PI3K/Akt and MAPK pathways, likely by inhibition of PCNA-binding of signaling proteins containing the APIM-sequence.
  • the "conventional" APIM sequence may be substantially modified as described herein to produce extended or longer APIM peptides that are capable of interacting with PCNA.
  • the extended or longer APIM peptides interact with PCNA with a similar or improved affinity relative to the conventional APIM peptides, but are not cytotoxic to normal, healthy cells.
  • peptides containing the extended or longer APIM sequence as defined herein would also find utility in the treatment or prevention of a disorder or condition associated with cytokine release from non-proliferating immune cells in blood, such as a disorder or condition resulting in or from, or associated with, hypercytokinemia.
  • the multiple myeloma cell lines JJN-3 (gift from J. Ball, University of Birmingham, United Kingdom) were grown in RPMI 1640 (Sigma-Aldrich,
  • HaCaT cells spontaneous transformed keratinocytes were cultured in DMEM (Sigma-Aldrich) containing FCS, glutamine, amphotericin B, and gentamicin. All cells were cultured at 37°C in a humidified atmosphere of 5% C0 2 .
  • Peripheral blood mononuclear cells were isolated from A+ buffy coats (Blood Bank, St. Olav's University Hospital, Trondheim, Norway) by density gradient centrifugation (Lymphoprep; Axis-Shield PoC, Oslo, Norway).
  • Mononucleated cells were seeded at 4 million cells/ml in serum-free RPMI 1640 supplemented with glutamine and gentamicin. After 60-90 min the adherent cell population was washed before cultured in medium with 10% heat-inactivated human serum (Blood Bank, St. Olav's University Hospital). ATX-101 (4 ⁇
  • ATX-A (4 ⁇ , [Innovagen])
  • ATX-A is represented in SEQ ID NO: 1206 and contains the motif RALVK (SEQ ID NO: 1207), which is not capable of interacting with PCNA), p38 inhibitor (SB203580, 10 ⁇ , [Sigma- Aldrich]), PI3K inhibitor (LY294002, 5 and 10 ⁇ , [Sigma-Aldrich]), and Akt inhibitor (Akt inhibitor XVIII SC66, 1 ⁇ g ml, [Sigma-Aldrich]) were added in serum-free medium and incubated for 5 min before LPS (10 ng/ml, [Sigma-Aldrich]), polylC (40 g/ml, [GE Healthcare, Little Chalfont, UK]), PAM3Cys (10 ng/ml, [Invivogen, San Diego, CA, USA]) or R848 (10 ⁇ g/ml, [Invivogen]) were added in serum
  • Mononucleated cells were seeded and stimulated as described above. JJN- 3 cells were treated with ATX-101 (6 ⁇ ) for 4 h. HaCaT cells were treated with ATX-101 (12 ⁇ ), polylC (2 Mg/ml), and LPS (100 ng/ml) for 4 h.
  • the cells were harvested, the cell pellet was resuspended in 1x packed cell volume of buffer 1 (10 mM Tris-HCI pH 8.0, 200 mM KCI) and diluted in the same volume (packed cell volume + buffer 1 ) of buffer 2 (10 mM Tris-HCI pH 8.0, 200 mM KCI, 10 mM EGTA, 10 mM MgCI 2 , 40% glycerol, 0.5% NP40, 1 mM DTT, 1 % phosphatase inhibitor cocktails 1 and 3 [Sigma-Aldrich], 2% Complete EDTA-free protease inhibitor
  • the proteins were visualized with the Odyssey infrared imaging system (LI-COR) and quantified using Odyssey Image Studio V2. Protein levels were compared to the protein level in untreated cells, which was set to 100%. ⁇ -tubulin was used as reference for data normalization. Immunofluorescence and confocal imaging
  • HaCaT cells and peripheral blood monocytes were grown on glass bottom dishes and stained as described using an antibody a-PCNA (PC10, Santa Cruz biotechnology Inc.; 1 :1000) and Alexa fluor 532 goat ormouse (Invitrogen).
  • the nuclei were stained with DRAQ5 prior to imaging according to the manufacturer's manual (eBioscience, San Diego, CA, USA).
  • the fluorescent images were acquired using a Zeiss LSM 510 Meta laser scanning microscope equipped with a Plan- Apochromate 63x/1.4 oil immersion objective in 2% FCS in PBS at RT using the Zeiss LSM 510 software.
  • the thickness of the slice was 1 ⁇ . All images were acquired with consecutive scans. No image processing was performed, except contrast and intensity
  • IFN- ⁇ was measured in supernatants with the VeriKine-HSTMHuman IFN- ⁇ Serum ELISA kit (Pestka Biomedical Laboratories, Piscataway, NJ, USA) according to the manufacturer's instructions with the following adaptions: the amount of supernatant was doubled from 50 to 100 ⁇ ; sample buffer was reduced from 50 to 25 ⁇ ; and antibody solution was reduced from 50 to 25 ⁇ (reducing assay diluent, keeping same volume of antibody concentrate and diluent additive). The OD was measured at 450 nm.
  • Cells were seeded into 96-well plates and ATX-101 , kinase inhibitors (SB203580 and BIRB0796), and polylC were added alone or in combination at the indicated concentrations. Cells were exposed continuously and harvested every day for the next four days using the MTT (3-(4.5-Dimethylthiazol-2-yl)-2.5 diphenyl- tetrazolium bromide) assay (Gilljam et al., 2009, supra). The average from at least 4 wells was used to calculate cell survival.
  • MTT 3-(4.5-Dimethylthiazol-2-yl)-2.5 diphenyl- tetrazolium bromide
  • Example 1 Targeting PCNA with APIM-containing peptides affects signal transduction in multiple myeloma cells
  • the MAPK and PI3K/Akt pathways have been suggested as potential therapeutic targets in multiple myeloma, alone and in combination because these pathways are frequently dysregulated and extensive cross-talk between these pathways exists. Furthermore, these pathways are important for cytokine production.
  • ATX-101 increased the growth-inhibitory effect of p38 MAPK and class I PI3K inhibitors in the multiple myeloma cell line JJN-3 ( Figure 1A).
  • ATX-101 increased the growth-inhibitory effects of ERK5 and MAPK kinase 1 (MEK1 ) inhibitors, and enhanced the cytotoxic effects of melphalan in combination with these inhibitors as well as the p38 inhibitor in another multiple myeloma cell line (RPMI-8226).
  • RPMI-8226 multiple myeloma cell line
  • ATX-101 affects important signal transduction pathways in multiple myeloma cells including the PI3K/Akt/mTOR and MAPK pathways.
  • Example 2 Targeting PCNA with APIM-containing peptides reduces cytokine secretion of monocytes after TLR stimulation
  • ATX-101 could affect cytokine secretion from primary monocytes, which is partly regulated by the PI3K Akt and MAPK pathways.
  • the secretion of LPS-induced cytokines was efficiently reduced by ATX-101 addition ( Figure 2A).
  • ATX-101 treatment reduced the secretion of CXCL10, CCL2, CCL3, and CCL4 after TLR3 stimulation with polylC ( Figure 2B).
  • ATX-101 and a p38 MAPK inhibitor both reduced cytokine secretion from stimulated monocytes, and the combination of ATX-101 and inhibitor resulted in an increased reduction of most cytokines (Figure 3B and C).
  • the PI3K inhibitor LY294002 on the other hand had less effect on induced cytokine secretion alone and in combination with ATX-101 , but still the combination showed a reduction in the secretion of some cytokines (e.g. IFNy, IL-1 RA, IL-2, 4, and 9 after LPS, and IL- 1 RA, CCL2, 3, and 4 after polylC stimulation) (Figure 3D and E).
  • PCNA has a regulatory role in signal transduction, affecting both cell growth and cytokine production.
  • Example 4 APIM-containing peptides reduce Akt phosphorylation and PACT protein levels in HaCaT keratinocytes
  • PACT may interact with PCNA and this interaction may have a regulatory role in the response to polylC in HaCaT cells.
  • ATX-101 an APIM-containing peptide, e.g. ATX- 101 , affects the cellular response to TLR agonists, supporting a role for PCNA in cellular stress responses.
  • ATX-101 treatment reduced the cytokine secretion from monocytes and decreased Akt phosphorylation in monocytes and HaCaT cells stimulated with different TLR ligands.
  • the APIM-sequence is found in over 200 proteins including several signaling kinases that have been described to act in TLR signaling. Therefore, ATX-101 likely affects several signal transduction pathways.
  • ATX-101 may also affect cytokine secretion by interfering with PI signaling.
  • ATX-101 did not reduce cytokine secretion by its cell- penetrating peptide but rather by targeting PCNA via the APIM-sequence because the mutant ATX-A with a single amino acid change in APIM reduced cytokine secretion far less than ATX-101 .
  • the small observed effects of ATX-A are likely because ATX-A still has some weak PCNA-binding ability.
  • Figure 12 shows that the cell-penetrating peptide that forms part of ATX-101 (R1 1 , SEQ ID NO: 337) has no effect on cytokine secretion when used alone.
  • ATX-101 interfered with signal transduction pathways including the PI3K/Akt pathway in the multiple myeloma cell line JJN-3 independent of TLR stimulation.
  • the ATX-101 -mediated effect on signal transduction pathways is therefore not restricted to TLR signaling.
  • the cellular target of ATX-101 is PCNA and thus our data suggest a new regulatory function of PCNA in signaling pathways likely by acting as cytoplasmic binding platform.
  • APIM-mediated PCNA interactions play a role in the cellular stress response to pathogens and damage- associated molecular patterns recognized by TLRs.
  • ATX-101 reduces cytokine secretion from monocytes at doses that do not induce apoptosis.
  • the inventors have performed sequence analyses to determine how much variation within the APIM motif occurs naturally, i.e. in native sequences across a number of species.
  • sequence variation of the APIM motif in orthologues of polypeptides that are thought to interact with PCNA is representative of the variation that may be used in the oligopeptidic compounds of the invention, i.e. variation of amino acids within the APIM motif at some positions may be permitted without losing affinity to PCNA.
  • the inventors used identified 657 human polypeptide sequences that comprise the motif [K/R]-[F/W/Y]-[A/LA /I]-[A/LA /I]-[K/R] (SEQ ID NO:1297) from a possible 21 ,673 polypeptide sequences. Of the 657 sequences identified, 291 were excluded because insignificant information about the function of the polypeptides was available.
  • LL LA, LV, AL, VL, VI, LI, IL, W, VA, IV, II, AV, IA, Al, AM, LM, LS, LT, IS, MV, TV, AA, IM, LN, LQ, VM, TL, SL, IT, VT, LG, MA, ML, NL, QL, Ql, Tl, SI, AS, VS, SV, CA, IG, LR, VR, TK and IR.
  • APIM motif In vivo characterisation of APIM consensus motif
  • PCNA tagged with green fluorescent protein forms distinct foci representing sites of replication and thus can be used as a S- phase marker.
  • PCNA tagged with cyan fluorescent protein was co-expressed with various APIM peptide constructs fused with yellow fluorescent protein (EYFP).
  • EYFP yellow fluorescent protein
  • FRET fluorescence resonance energy transfer
  • the thickness of the slice was 1 ⁇ .
  • Fluorescent resonance energy transfer occurs if the tags (EYFP and ECFP) are less than 100 A (10 nm) apart.
  • FRET Fluorescent resonance energy transfer
  • FRET/ (l-i x l 3 ) 1/2 FRET/ (l-i x l 3 ) 1/2 .
  • NFRET was calculated from mean intensities (I) within a region of interest (ROI) containing more than 25 pixels where all pixels had intensities below 250 and the average intensities were between 100 and 200 for both the donor and the acceptor constructs.
  • ROI region of interest
  • I D1 , D2 , D3 and I A1, A2, A3 were determined for cells transfected with ECFP and EYFP constructs only, with same settings and same fluorescence intensities as co-transfected cells (l 1 and l 3 ).
  • ECFP-PCNA and EYFP- PCNA were included as positive controls, and due to dimerisation of co-expressed tags, ECFP
  • Figure 8 shows that a significant FRET signal could be detected for all of the variants tested, which verifies that a variety of peptides within the API M motif definition described herein (and that occur in polypeptides that are expected to interact with PCNA) are capable of interacting with PCNA and would therefore be expected to find utility in the method and uses described herein, i.e. as
  • immunosuppressive peptides e.g. for inhibiting or preventing cytokine release for non-proliferating immune cells in blood and treating or preventing disorders or conditions associated therewith.
  • Example 7 Identification of cytosolic proteins involved in cellular signalling that interact with PCNA
  • API M containing proteins detected include MST4 and PLK3, which are involved in regulating ERK and Akt respectively.
  • PACT protein was also detected. These proteins are involved in cytokine and chemokine expression pathways.
  • PCNA also pulled down the API M containing protein BRE, a protein part of the
  • a mock antibody should ideally target an unrelated protein.
  • PCNA has many interaction partners and likely acts as a signalling hub in the cytoplasm
  • an ideal mock antibody is difficult to obtain.
  • the mock protein chosen here (TOM20) was detected in the PCNA pull down by MS (Mascot-score 32.53, not detected by Sequest; data not shown).
  • Table 4 includes several interesting proteins that are not exclusively found, but enriched, in the PCNA pull down compared to mock pull down, based on the number of detected peptides and Mascot/Sequest score. These include several proteins in the MAPK pathways, including TA03, MAPKK3, ERK1/2 and the APIM-containing proteins MK2 and TA01 .
  • Akt1 and p38 alpha were also enriched in PCNA pull down, however, these proteins were detected in gel pieces corresponding to different molecular weights from the a-PCNA and ormock pull down suggesting that regulation by PTMs is important for their localization to PCNA complexes.
  • the APIM-protein PACT was detected from the gel-piece with the expected molecular weight (MW) of PACT in the PCNA pull down only (Table 3). PACT was found in both PCNA and mock pull down (less in mock), and in a gel piece with 5-10 kDa higher molecular weight than predicted (Table 4). The increase in molecule weight suggests that PACT may be regulated by PTMs, e.g. polyubiquitination which leads to degradation.
  • Example 8 Determination of the level of apoptosis in non-proliferating immune cells (monocytes) from blood when treated with APIM-containing peptides
  • Annexin staining was used to determine cell viability and cells were counted using flow cytometry.
  • Peripheral blood lymphocytes were isolated from A+ buffy coats (Blood Bank, St. Olav's University Hospital) by density gradient centrifugation
  • monocytes The adherent cell fraction after 90 minute incubation, termed monocytes, were washed twice with HANKS and maintained in RPMI 1640 supplemented with 2mM glutamine, 100 mg/ml gentamicin and 25% heat-inactivated HS over-night. Cells were cultured at 37°C in a humidified atmosphere of 5% C0 2 over-night. The next day cells were treated with different concentrations of API M-containing peptides ATX-101 (SEQ I D NO: 1289) and ATX-P (MDRWLVPWKKKRKI RRRRRRRRRRR, SEQ I D NO: 1299).
  • Table 6 shows that there was no significant increase in annexin positive cells (apoptotic cells) when treated with API M peptides.
  • a 4 ⁇ dose of ATX-101 reduces the cytokine production from TLS-ligand stimulated monocytes in absence of apoptosis. It is noted that the same dose showed low toxicity on normal cells in BMCS assays (Muller et a/,. PloS One, 2013, v. 8: e70430).
  • Microscale thermophoresis was used to determine the dissociation constant for various APIM containing peptides.
  • PCNA was labeled with a fluorescent molecule.
  • concentration of PCNA was kept constant, whereas dilutions of each APIM containing peptide were prepared (1 :1 ).
  • the signal was recorded in all capillaries with varying concentrations of the unlabeled peptide, and any change of thermophoretic properties was observed as a change in fluorescence intensity.
  • Table 7 shows that ATX-101 and ATX-101 -P (natural variant of APIM in ABH2) both show stronger interactions with PCNA than ATX-A, a negative control (a low Kd value indicates a strong interaction, whereas a high Kd value indicates a weak interaction). Furthermore, no data on specific interaction with R1 1 could be obtained by MST, indicating that this peptide does not interact with PCNA.
  • the inventors have determined unexpectedly that the "conventional" APIM sequence may be substantially modified without reducing the affinity interaction of the peptide with PCNA.
  • an aromatic or hydrophobic amino acid may be inserted into the APIM sequence to generate a new consensus sequence, as defined by SEQ ID NO: 1.
  • the inventors have also determined that the insertion of an aromatic or hydrophobic amino acid, particularly an aromatic amino acid, allows an increase in flexibility at the C-terminal end of the APIM sequence.
  • an aromatic or hydrophobic amino acid, particularly an aromatic amino acid, within the APIM sequence improves the affinity of the peptide, such that it is not essential to include a basic amino acid at the C-terminal end of the APIM sequence to maintain the capacity of the peptide to bind to PCNA.
  • FRET assays were used to determine the capacity of the extended APIM peptides (peptides containing an extended or longer APIM sequence) to bind to PCNA, as described in Example 6.
  • Figure 9 shows the FRET signal for a variety of APIM peptides containing a pentamer motif and three peptides containing an "extended" hexamer APIM sequence. A significant FRET signal could be detected for all of the "extended"
  • APIM variants tested Furthermore, all of the "extended" APIM peptides generate a signal that is equivalent to, or higher than, the APIM peptides containing a pentamer motif. These results verify that a variety of peptides within the APIM motif definition described herein are capable of interacting with PCNA. Accordingly, peptides containing the "extended” APIM sequence would therefore be expected to find utility in the method and uses described herein akin to peptides containing the pentamer APIM sequence, i.e. as immunosuppressive peptides, e.g. for inhibiting or preventing cytokine release for non-proliferating immune cells in blood and treating or preventing disorders or conditions associated therewith as evidenced by the data in the Examples above.
  • immunosuppressive peptides e.g. for inhibiting or preventing cytokine release for non-proliferating immune cells in blood and treating or preventing disorders or conditions associated therewith as evidenced by the data in the Examples above.
  • the "extended” APIM sequence is capable of facilitating the interaction between peptides containing the sequence and PCNA because the extended sequence does not typically occur in proteins that are known to interact with PCNA. Moreover, it was completely unexpected that this "extended” sequence would facilitate the interaction with PCNA with a similar or improved affinity relative to various pentamer sequences.
  • Fluorescently-labelled (FAM-tagged) extended APIM peptide constructs were incubated with HeLa cells.
  • HEK293 cells Human embryonic kidney cells
  • 96 well plates 6000 cells/well
  • peptides were added to the cells in serum free media and incubated for 1 h.
  • Fresh media was added and the cells were harvested after additional 24, 72 and 96 hours.
  • MTT was added to the cells (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and OD was measured at 565 nm, and the average from at least 6 wells was used to calculate cell survival.
  • Data is presented in Figure 1 1 as growth from one representative experiment and has been reproduced at least 2 times.
  • Example 13 Effects of "extended” APIM-containing peptides on cytokine secretion of monocytes after TLR stimulation
  • the levels of 27 cytokines were measured in LPS-stimulated peripheral blood monocytes treated with 4 ⁇ or 6 ⁇ of peptides akin to the experiment in Example 2.
  • APIM sequences were coupled to a variety of cell penetrating peptides and linker sequences and the results demonstrate that the peptides are effective at reducing the secretion of cytokines irrespective of the linker and cell- penetrating peptide sequence.
  • Example 12 (without a FAM-tag) was investigated by an MTT assay as described in Example 12, using both HEK 293 and U20S cells.
  • the ATX-101 peptide ("101 "), which contains a "standard” pentamer APIM sequence was used as a control.
  • peptides were tested in combination with various concentrations of a cytostatic agent, cisplatin.
  • Figures 13-15 show that the peptides containing an "extended" APIM sequence do not effect considerably the growth of normal healthy cells, i.e. the peptides are not cytotoxic to healthy cells, and do not significantly potentiate the effects of cytostatic agents.

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Abstract

La présente invention concerne un agent, ou une composition contenant un agent, pour son utilisation dans le traitement ou la prévention de l'hypercytokinémie chez un sujet résultant de la libération de cytokine par des cellules immunitaires non-proliférantes dans le sang, ledit agent comprenant : (i) un composé oligopeptidique qui comprend un motif d'interaction avec PCNA et un domaine qui facilite l'absorption cellulaire de ce composé, dans lequel le motif d'interaction avec PCNA est X1X2X3X4X5X6 (SEQ ID NO :1 ) et dans lequel : X1 est un acide aminé basique; X2 est un acide aminé aromatique; X3 est un acide aminé aromatique ou un acide aminé hydrophobe qui a un groupe R comprenant au moins trois atomes de carbone; X4 est un acide aminé non chargé autre qu'un acide aminé aromatique, la glycine (G) et la proline (P) ; X5 est un acide aminé quelconque autre qu'un acide aminé acide ou un acide aminé aromatique ; et X6 est un acide aminé quelconque autre qu'un acide aminé acide ou un acide aminé aromatique, de préférence un acide aminé basique ou la proline (P), dans lequel lorsque X3 n'est pas un acide aminé aromatique, X5 n'est pas la lysine (K) et X6 est un acide aminé basique ou la proline (P) ; ou (ii) une molécule d'acide nucléique comprenant une séquence codant pour le composé oligopeptidique de (i). Dans certains aspects, l'agent et la composition selon l'invention peuvent être utilisés comme des agents uniques. Dans d'autres aspects de l'invention, l'agent et la composition selon l'invention peuvent être utilisés conjointement à un ou plusieurs autres principes actifs, tels que des inhibiteurs de kinase.
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US11793884B2 (en) 2018-01-29 2023-10-24 Ohio State Innovation Foundation Cyclic peptidyl inhibitors of CAL-PDZ binding domain
WO2021224068A1 (fr) 2020-05-06 2021-11-11 Therapim Pty Ltd Peptides contenant un motif d'interaction pcna destinés à être utilisé dans le traitement du cancer solide
US11741686B2 (en) 2020-12-01 2023-08-29 Raytheon Company System and method for processing facility image data

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