WO2023007182A1 - Inhibiteurs d'interactions de protéines - Google Patents

Inhibiteurs d'interactions de protéines Download PDF

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Publication number
WO2023007182A1
WO2023007182A1 PCT/GB2022/052007 GB2022052007W WO2023007182A1 WO 2023007182 A1 WO2023007182 A1 WO 2023007182A1 GB 2022052007 W GB2022052007 W GB 2022052007W WO 2023007182 A1 WO2023007182 A1 WO 2023007182A1
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WIPO (PCT)
Prior art keywords
agent
antibody
subject
gpiba
platelets
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PCT/GB2022/052007
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English (en)
Inventor
Julie RAYES
Martina COLICCHIA
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The University Of Birmingham
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Publication date
Priority claimed from GBGB2110970.7A external-priority patent/GB202110970D0/en
Priority claimed from GBGB2111043.2A external-priority patent/GB202111043D0/en
Application filed by The University Of Birmingham filed Critical The University Of Birmingham
Publication of WO2023007182A1 publication Critical patent/WO2023007182A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4727—Calcium binding proteins, e.g. calmodulin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7095—Inflammation

Definitions

  • the present invention relates to agents for use in treating or preventing inflammatory and/or thrombotic diseases.
  • Platelets are crucial to haemostasis and arterial thrombosis and support venous thrombosis.
  • platelet glycoprotein lb (GPIb) UniProt P07359) interaction with VWF mediates the rolling of platelets on the exposed endothelium.
  • the interaction between GPVI-collagen leads to platelet activation and firm adhesion, the release of secondary mediators such as ADP and thromboxane A2, the recruitment of circulating platelets, GPIIbllla activation and binding to fibrinogen and thrombus formation.
  • Platelets play a critical role in atherogenesis and thrombosis- mediated myocardial ischemia, processes that are accelerated in diabetes.
  • Activated platelets promote a proinflammatory phenotype in innate immune cells, through receptor-ligand interactions (including P-selectin-PSGLl and GPIb-MAC-1 interactions), secretion of proinflammatory cytokines and chemokines and/or release of prothrombotic and proinflammatory vesicles.
  • Activated platelets also shed GPIb- positive microvesicles which bind preferentially to monocytes via P-selectin and stabilized through PS, which has been demonstrated to increase monocyte adhesion to TGF i -activated human umbilical vein endothelial cells (HUVECs).
  • Activated and procoagulant platelet levels are increased in patients with cardiovascular diseases and diabetes, and their activation status correlates with an increased risk of thrombosis.
  • Hyperglycemia is associated with the release of large platelets harbouring higher levels of GPIb, GPIIbllla and P2Y12, which have lower levels of cAMP and have higher intracellular calcium mobilisation and TXA2 release following activation.
  • Anti platelet drugs are classically used to reduce vascular events in patients with cardiovascular diseases.
  • anti-platelet therapies were shown to be less effective in reducing ischemic events in patients living with diabetes. This suggests alternative pathways of platelet activation and thrombo- inflammation, which are not targeted by classical anti -platelet drugs.
  • the present invention fulfils these needs and further provides other related advantages.
  • the invention provides an agent for use in preventing or reducing the interaction between SI 00 A8/A9 and GPIba.
  • the invention is based on the finding that S100A8/A9 induces thrombosis independently from inflammation, in a signalling axis that works via GPIba to induce platelet activation and the formation of procoagulant platelets.
  • the inventors have identified this previously-unknown signalling axis, for which known inhibitors targeting classical SI 00 A8/A9 receptor signalling pathways have little or no effect.
  • the inventors have demonstrated that this signalling axis can be blocked or reduced in a number of ways, and that in doing so, procoagulant platelet formation and platelet activation, both of which are strongly linked to inflammatory and thrombotic diseases, is significantly decreased. This paves the way for new treatments for such diseases, where known inhibitors and treatments are ineffective.
  • S100A8 (UniProt) P05109 and S100A9 (UniProt P06702), also known as MRP-8/MRP- 14, calprotectin, and calgranulin A/calgranulin B, are two proteins of the 24-member S100 family of cytoplasmic EF-hand helix-loop-helix Ca2+-binding proteins.
  • S100A8 and S100A9 can form noncovalently associated homo- and heterodimers, with the heterodimeric complex being the predominantly occurring form.
  • S100A8/A9 is abundant in myeloid cells, comprising up to 45% of cytosolic proteins in neutrophils and ⁇ 5% of monocyte/ macrophage cytosolic proteins.
  • S100A8/A9 is significantly upregulated under inflammatory conditions, both in the plasma and in immune cells, in particular in cardiovascular diseases such as myocardial ischemia- reperfusion injury (Li Yet al., Circulation. 2019;140(9):751-764) and atherosclerosis (Ionita MG et al., Arterioscler Thromb Vase Biol. 2009;29(8): 1220-1227.).
  • S100A8/A9 is also present in platelets and megakaryocytes (Lood C, Tyden H, Gullstrand B, et al., Arthritis Rheumatol.
  • S100A8/A9 functions as an extracellular DAMP with bacteriostatic activity by chelating transition metal ions (iron, zinc and manganese) (Yang et al., Front Immunol. 2018;9: 1298). Extracellular S100A8/A9 also promotes the expression of inflammatory mediators and migration of neutrophils and monocytes and increases the expression of adhesion receptors on endothelial cells, chemokines and endothelial permeability. S100A8/A9 has three known receptors: RAGE, TLR4 and CD36.
  • S100A8/A9 is not only a biomarker of immune activation in infection and chronic inflammatory diseases but also promotes a thrombo-inflammatory state making S100A8/A9 an important target to reduce ischemic events. Elevated plasma levels of S100A8/A9 predict increased risk of first and recurrent cardiovascular events independently of traditional cardiovascular risk factors and C-reactive protein (Kraakman et ah, J Clin Invest. 2017;127(6):2133- 2147).
  • Hyperglycemia stimulates neutrophil release of S100A8/A9 (Nagareddy et al., Cell Metab. 2013;17(5):695-708) and macrophage and monocyte from humans with T1DM and T2DM express higher levels of S100A8/A9. Moreover, higher expression of S100A8/A9 and RAGE is observed in atherosclerotic lesions, in particular in diabetic patients 12 and these levels correlates with the extent of coronary and carotid atherosclerosis and with a vulnerable plaque phenotype. S100A8/A9-deficient mice are protected against vascular injury and have smaller atherosclerotic lesions, less plaque inflammation and decreased re-stenotic response (Croce et al., Circulation. 2009; 120(5):427-436). Previously, how S100A8/A9 contributes to thrombosis via platelet interaction was unknown.
  • the invention provides an agent which prevents or reduces the interaction between SI 00 A8/A9 and GPIba, for use in treating or preventing one or more inflammatory and/or thrombotic disease.
  • the invention provides a method of treating or preventing one or more inflammatory and/or thrombotic disease, comprising administering to a subject in need thereof: a therapeutically effective amount of one or more agents of the invention.
  • the invention provides a pharmaceutical composition comprising an agent of the invention, for use in treating or preventing one or more inflammatory and/or thrombotic disease.
  • the invention provides the use of an agent of the invention for the manufacture of a medicament for treating or preventing one or more inflammatory and/or thrombotic disease.
  • the one or more inflammatory and/or thrombotic disease of any aspect may be known to have as one of its causative effects increased platelet activation and/or increased procoagulant platelet formation.
  • the one or more inflammatory and/or thrombotic disease of any aspect of the invention may be selected from the group comprising or consisting cardiovascular diseases, chronic inflammatory diseases, infectious diseases and autoimmune diseases.
  • the cardiovascular disease may be selected from the group comprising or consisting myocardial ischemia-reperfusion injury, peripheral artery disease, deep vein thrombosis, myocardial infarction, stroke and atherosclerosis.
  • the chronic inflammatory diseases disease, infectious diseases or autoimmune disease may be selected from the group comprising or consisting rheumatoid arthritis, asthma, inflammatory bowel disease, Crohn’s disease, COVID-19, long-COVID, chronic and/or acute inflammation associated with SARS-COV-2 infection, and sepsis or diseases associated with platelet and/or innate immune cell activation.
  • the agent which prevents or reduces the interaction between SI 00 A8/A9 and GPIba may be an antibody, a small molecule inhibitor, a peptide, a polypeptide, a recombinant protein, a nucleic acid, a T-cell receptor (TCR), an affibody, an aptamer, or a glycosaminoglycan (e.g. heparin).
  • the polypeptide or recombinant protein may be snake venom protein, or plasma derived-protein such as Glycocalicin.
  • the agent which can present or reduce this interaction between SI 00 A8/A9 and GPIba may be a drug inhibiting and/or limiting the release of S100A8/A9 from innate immune cells or from platelets.
  • a peptide may be under 50 amino acids in length.
  • a polypeptide may be 50 amino acids or more in length.
  • a small molecule inhibitor may be a chemical compound which has a molecular weight of 900Da or less. The molecular weight may be accurately determined using a number of techniques available in the art, such as mass spectrometry.
  • the agent may be an anti-GPlba antibody, an anti-SlOO A8 antibody, an anti-SlOO A9 antibody, or an anti-SlOO A8/A9 antibody.
  • the agent may be a bispecific antibody which binds to both GPlba and S100 A8/A9.
  • the antibody may be an antibody listed in Table 1, all of which are commercially available.
  • the antibody may be an antibody listed in Table 2.
  • the antibody may be 42C01, AK2 or SZ2.
  • the antibody may be an antibody that competes for binding with a monoclonal antibody listed in Table 1, such as anti- GPlba antibody 42C01, AK2 or SZ2.
  • the antibody may be an antibody that competes for binding with a monoclonal antibody listed in Table 2, such as anti- GPlba antibody 42C01, AK2 or SZ2.
  • the antibody may be an antibody which has the same variable heavy and light regions as a monoclonal antibody listed in Table 1, such as anti- GPlba antibody 42C01, AK2 or SZ2.
  • the antibody may be an antibody which has the same variable heavy and light regions as a monoclonal antibody listed in Table 2, such as anti-GPlba antibody 42C01, AK2 or SZ2.
  • the antibody may be an antibody which has the same CDRs as a monoclonal antibody listed in Table 1, such as anti- GPlba antibody 42C01, AK2 or SZ2, as defined by the Kabat numbering system.
  • the antibody may be an antibody which has the same CDRs as a monoclonal antibody listed in Table 2, such as anti-GPlba antibody 42C01, AK2 or SZ2, as defined by the Kabat numbering system.
  • the antibody may be an antibody which binds to the same epitope as a monoclonal antibody listed in Table 1, such as anti- GPlba antibody 42C01.
  • the antibody may be an antibody which binds to the same epitope as a monoclonal antibody listed in Table 2, such as anti-GPlba antibody 42C01.
  • the antibody may refer to a combination of antibodies listed in Table 1 or Table 2, such as AK2 and SZ2.
  • AK2 and SZ2 bind to 2 distinct binding sites on GPIba and can be used in combination to reduce S 100A8/A9-induced platelet activation.
  • the agent may be recombinant GPIba or a non-active variant thereof, recombinant SI 00 A8 or a non-active variant thereof, or recombinant SI 00 A9 or a non-active variant thereof.
  • the recombinant GPIba may be glycocalicin, which is the soluble proteolytic fragment of GPIba.
  • the agent may be a glycosaminoglycan such as heparin which binds to S100A8/A9 and blocks the formation of procoagulant platelets. Heparin is used as anti-coagulant and can limit S 100A8/A9-induced platelet activation.
  • the agent may be an inhibitor for S100A8/A9 release from platelets or from immune cells such as small molecule inhibitor, NSAIDs or a steroid.
  • a non-active variant of a protein may refer to a fragment of the full- length protein which has no or reduced levels of its known biological activity.
  • a non active variant of a protein may also refer to a variant of the full-length protein, for example which has one or more, two or more, three of more, four or more, five or more amino acid modifications, and which has no or reduced levels of its known biological activity.
  • a non-active variant of GPIba may bind to S100 A8/A9, or may not bind to SI 00 A8/A9, but would exhibit reduced or none of the biological effects demonstrated by the inventors, such as platelet activation and/or pro-coagulant platelet formation.
  • An active or non-active variant of the proteins named above may be confirmed using one of a number or techniques available to the skilled person, including those described in the accompanying drawings.
  • a non-active protein above may be confirmed by assessing its effect on the level of Annexin V or Lactadherin binding to Phosphatidylserine on the surface of platelets, as a readout of whether platelets are of the procoagulant phenotype.
  • a non active protein above may promote a reduced level Annexin V or Lactadherin binding to phosphatidylserine on the surface of platelets compared to an active variant of the same protein.
  • the effect on procoagulant platelets can be confirmed using thrombin generation assay, perfusion of whole blood over coated surfaces at arterial or venous shear rate, fibrin generation in whole blood or plasma and binding of coagulation factors on the surface of platelet using immunofluorescence staining.
  • a non-active protein above may be confirmed by assessing its effect on the level of P-selectin in platelets, as a readout of platelet activation.
  • a non-active protein above may promote a reduced level P-selectin in platelets compared to an active variant of the same protein.
  • An agent which is a recombinant protein or a non-active variant thereof may comprise or consist of GPIba, as defined above, or an amino sequence with about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more identity to GPIba as defined above.
  • An agent which is a recombinant protein or a non-active variant thereof may comprise or consist of SI 00 A8, as defined above, or a sequence with about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more identity to SI 00 A8 as defined above.
  • An agent which is a recombinant protein or a non-active variant thereof may comprise or consist of SI 00 A9, as defined above, or a sequence with about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more identity to SI 00 A9 as defined above.
  • An agent which is a plasma-derived protein, such as Glycocalicin, or a non-active variant thereof may comprise or consist of soluble plasma derived-GPIba, the soluble proteolytic fragment of GPIba generated by cleavage by enzymes such as calpain, plasmin, trypsin, elastase.
  • the agent may sterically inhibit the interaction between SI 00 A8/A9 and GPIba.
  • the agent may allosterically inhibit the interaction between SI 00 A8/A9 and GPIba.
  • the agent may indirectly inhibit the interaction between SI 00 A8/A9 and GPIba, for example by inhibiting a protein which interacts with SI 00 A8/A9 and GPIba, thereby preventing or reducing signalling via this pathway.
  • the agent may block the binding of S100A8 to SI 00 A9, thereby preventing or reducing the formation of the SI 00 A8/A9 heterodimer.
  • the term ‘interaction’ may encompass the direct binding of proteins of interest to each other to induce a biological effect.
  • the term may also encompass indirect relationships between proteins of interest, for example where the proteins of interest are part of the same signalling pathway but where there are one or more other proteins which act between the proteins of interest, either by binding in the same complex but not permitting direct interaction between the proteins of interest, or by inducing signalling events where the proteins of interest are spatially separated and not in the same complex.
  • the interaction between SI 00 A8/A9 and GPIba may not be direct, and may be part of a signal cascade.
  • the invention provides a method of reducing the number of or preventing the formation of procoagulant platelets in a subject, comprising administering to the subject an effective amount of an agent of the invention.
  • the invention provides a method of diagnosing an inflammatory and/or thrombotic disease in a subject, comprising determining the level of SI 00 A8/A9 in a sample obtained from a subject for example by ELISA, comparing the level of SI 00 A8/A9 in the sample obtained from a subject with the level of S 100 A8/A9 in a reference sample, and concluding that the subject has an inflammatory and/or thrombotic disease when the level of SI 00 A8/A9 is above a threshold level.
  • the method may further comprise administering a suitable treatment to the subject.
  • the skilled person will be able to identify a suitable treatment for a given disease, which may comprise or consist of an agent of the invention.
  • the treatment may comprise or consist of anti-coagulants and/or anti -platelet drugs.
  • the invention provides a method of prognosing a subject who has been diagnosed with an inflammatory and/or thrombotic disease, comprising determining the level of S100 A8/A9 in a sample obtained from a subject, comparing the level of S100 A8/A9 in the sample obtained from a subject with the level of S 100 A8/A9 in a reference sample, and concluding that the subject has a poor prognosis when the level of SI 00 A8/A9 is above a threshold level.
  • a poor prognosis may be a poor likelihood of survival.
  • a poor prognosis may be a high level of clinical severity of the inflammatory and/or thrombotic disease.
  • the method may further comprise administering a suitable treatment.
  • a suitable treatment for a given disease, which may comprise or consist of an agent of the invention.
  • the threshold level may be about 5 pg/mL of SI 00 A8/A9 in the sample obtained from the subject.
  • the threshold level may be about 10 pg/mL of S100 A8/A9 in the sample obtained from the subject.
  • the threshold level may be adjusted for gender and age.
  • a reference sample may be an equivalent sample from a healthy subject.
  • a healthy subject may be defined as a subject who is not suffering from an inflammatory and/or thrombotic disease.
  • the reference sample may be an average value taken from a population of healthy subjects, for example from five or more subjects of the same gender and age or age group.
  • the reference sample or sample obtained from the subject refers to a sample of biological material obtained from a subject, e.g., a human subject, including tissue, a tissue sample, a biopsy, a cell sample, a tumour sample, a stool sample and a sample of biological fluid, e.g., plasma, serum, blood, urine, lymphatic fluid, ascites, saliva.
  • a subject e.g., a human subject
  • tissue e.g., a human subject
  • biological fluid e.g., plasma, serum, blood, urine, lymphatic fluid, ascites, saliva.
  • the reference sample or sample obtained from the subject is a blood sample.
  • Any method referred to herein may be performed in vitro, ex vivo, or in vivo.
  • the method is performed in vitro.
  • the level of Annexin V or Lactadherin binding to Phosphatidylserine on the surface of platelets may be measured, as a readout of whether platelets are of the procoagulant phenotype.
  • a reduced or prevented interaction between SI 00 A8/A9 and GPIba may promote a reduced level Annexin V or Lactadherin binding to Phosphatidylserine on the surface of platelets compared to the level when the interaction has not been reduced or prevented.
  • the level of P-selectin in platelets may be measured, as a readout of platelet activation.
  • a combination of Annexin-V binding and P-selectin can be used for selective populations of procoagulant platelets.
  • a reduced or prevented interaction between SI 00 A8/A9 and GPIba may promote a reduced level P-selectin in platelets compared to the level when the interaction has not been reduced or prevented.
  • thrombin generation as a readout of procoagulant platelets may be measured.
  • a reduced or prevented interaction between SI 00 A8/A9 and GPIba may promote a reduced level of thrombin formation, compared to the level when the interaction has not been reduced or prevented.
  • Fibrin generation in plasma or whole blood can also be used as readout.
  • a reduced or prevented interaction between SI 00 A8/A9 and GPIba may promote a reduced level of fibrin in plasma or whole blood, compared to compared to the level when the interaction has not been reduced or prevented.
  • biochemical and biological techniques may also be employed, such as immunoprecipitation, to determine whether to determine whether a direct interaction between SI 00 A8/A9 and GPIba has been reduced or prevented.
  • An amino acid modification as recited herein may refer to a substitution, deletion, addition or combination thereof. It may be determined that the interaction between SI 00 A8/A9 and GPIba has been reduced or prevented by an agent of the invention, when the level of interaction as measured using any of the techniques available in the art, is reduced by 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more relative to the level of interaction as measured under the same conditions but in the absence of an agent of the invention.
  • An antibody or antigen binding fragment thereof may refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies, single-chain Fvs (scFv), single-chain antibodies, nanobodies, immunologically active antibody fragments (e.g., antibody fragments capable of binding to an epitope, e.g., Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fv fragments, fragments containing a VL and/or VH Domain, or that contain 1, 2, or 3 of the complementary determining regions (CDRs) of such VL Domain (i.e., CDRL1, CDRL2, and/or CDRL3) or VH Domain (i.e., CDRH1, C
  • antibody is intended to encompass immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.
  • the term “antibody” may encompass immunoglobulin-like or mimetic molecules, such as affibodies.
  • a bispecific antibody comprises two different variable regions, each of which specifically bind different epitopes, either on the same or on different antigens.
  • An agent which is a nucleic acid molecule may be a DNA or RNA molecule.
  • the nucleic acid molecule may bind SI 00 A8/A9 or GPIba, or DNA or RNA which encodes S100 A8, S100 A9, S10 0A8/A9 or GPIba.
  • the nucleic acid molecule may be a DNA or RNA aptamer which binds S100 A8, S100 A9, S10 0A8/A9 or GPIba.
  • the nucleic acid molecule may prevent or reduce the expression of SI 00 A8, SI 00 A9, S10 0A8/A9 or GPIba, such as an oligonucleotide, an siRNA or an shRNA.
  • An agent which is a small molecule inhibitor may refer to a molecule which can bind to S100 A8, S100 A9, S100 A8/A9 or GPIba.
  • An agent referred to herein may additionally or alternatively reduce the level of procoagulant platelets.
  • An agent referred to herein may additionally or alternatively reduce the release of S100A8/A9 from immune cells, thereby reducing the levels of SI 00 A8/A9 in the plasma or sequestrating SI 00 A8/A9 in the plasma.
  • a pharmaceutical composition may comprise or consist of one or more different agents disclosed herein, such as one, two or more, three or more, four or more of the agents disclosed herein and a pharmaceutically acceptable excipient or carrier.
  • the agent or pharmaceutical may be administered to the subject indefinitely or for a specified period of time.
  • the agent or pharmaceutical may be administered at regular intervals.
  • the pharmaceutical composition may further comprise one or more carriers or excipients.
  • an excipient can give form or consistency, or act as a diluent.
  • Suitable excipients include, but are not limited to, stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers.
  • the compositions can be in any suitable form, for example tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
  • Such compositions may be prepared by any known method, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate for oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances that increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol, and /or dextran.
  • the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.
  • the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulation may be used simultaneously to achieve systemic administration.
  • Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
  • these agents are formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.), although other forms of administration (e.g., oral, mucosal, etc.) can be also used.
  • agents of the invention are preferably combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like.
  • compositions can also be formulated so as to provide quick, sustained or delayed release of their active ingredients after administration to the patient by employing procedures known in the art.
  • the physical and chemical characteristics of the compositions of the invention may be modified or optimized according to the skill in the art, depending on the mode of administration and the particular disease or disorder to be treated.
  • the compositions may be provided in unit dosage form, a sealed container, or as part of a kit, which may include instructions for use and/or a plurality of unit dosage forms.
  • agents may be formulated together in the same formulation or may be formulated into separate pharmaceutical compositions.
  • the separate compositions may be administered concurrently, sequentially or separately.
  • a variety of administration routes for the agents or pharmaceutical compositions of the invention are available. The particular mode selected will depend upon the particular agent or composition selected, whether the administration is for prevention, or treatment of disease, the severity of the medical disorder being treated and dosage required for therapeutic efficacy.
  • the methods of this invention may be practiced using any mode of administration that is medically acceptable, and produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
  • modes of administration include, but are not limited to, oral, buccal, sublingual, inhalation, mucosal, rectal, intranasal, topical, ocular, periocular, intraocular, transdermal, subcutaneous, intra-arterial, intravenous, intramuscular, parenteral, or infusion methodologies.
  • compositions of the invention may be desirable to administer locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • the term “therapeutically effective amount” refers to the total amount of the agent or each active component of the pharmaceutical composition or method that is sufficient to provide patient benefit, i.e., prevention or amelioration of the condition to be treated, a reduction in symptoms, an increase in rate of healing, or a detectable change in the levels of a substance in the treated or surrounding tissue.
  • patient benefit i.e., prevention or amelioration of the condition to be treated, a reduction in symptoms, an increase in rate of healing, or a detectable change in the levels of a substance in the treated or surrounding tissue.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in concurrently, sequentially or separately.
  • the precise dose to be employed in the formulations of the present invention may depend on the route of administration, and the seriousness of the condition, and should be decided according to the judgment of the practitioner and each patient’s circumstances and can be determined by standard clinical techniques. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the particular dosage regimen, i.e., dose, timing and repetition, will thus depend on the particular individual and that individual's medical history, as well as the route of administration.
  • the agents or compositions may be delivered at intervals ranging from about 24hrs to about 2 days, to about 1 week, to about 2 weeks, to about 3 weeks to about 1 month to about 2 months, to about 3 months, to about 4 months, to about 5 months, to about 6 months.
  • the scheduling of such dosage regimens can be optimized by the clinician.
  • the agents or compositions may be administered using a treatment regimen comprising one or more doses, wherein the treatment regimen is administered over 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, 14 days, 30 days.
  • agent, pharmaceutical composition or method of treatment of the invention may be combined with other known therapies for the treatment or prevention of one or more inflammatory and/or thrombotic disease.
  • the subject in need thereof of any aspect of the invention may have been diagnosed with of one or more inflammatory and/or thrombotic disease as defined above.
  • Figure 1 shows that S100 A8/A9-positive cells are present the lung of severe COVID-19 patients.
  • A Immunofluorescence imaging of SI 00 A8/A9 and platelets (CD42b) in formalin-fixed and paraffin-embedded lung autopsies from severe COVID- 19 patients and non-COVID-19 patients.
  • B Platelets colocalise with S100A8/A9 on the vessel wall in COVID-19 patients
  • C Immunofluorescence imaging of SI 00 A8/A9 and myeloperoxidase (MPO) in lung autopsies showing S100A8/A9 lining the vessel wall. Images was captured using Epi fluorescence microscope and slide scanner Axio Scan. Zl.
  • Figure 2 shows that S100 A8/A9 plasma levels are increased in patients with COVID-19.
  • A, B SI 00 A8/A9 level was measured by ELISA in the plasma of uncomplicated and complicated (ICU and death) COVID-19 patients during the first 7 days of admission to hospital.
  • A SI 00 A8/A9 levels one day 1 of admission.
  • FIG. 3 shows that S100 A8/A9 induces human platelet activation in vitro.
  • Human washed platelets (lxlO 6 ) were incubated with different concentrations of recombinant S100 A8/A9 (10, 20 and 40 pg/ml) for 30 min at 37°C in the presence of Ca2+ (2mM).
  • CRP (10 pg/ml) and TRAP-6 (100 pM) were used as positive control.
  • B, E, H Data is shown as the percentage of positive population within the CD41+ gate and
  • C, F, I shows the fold change in the median fluorescence intensity (MFI) of treated platelets over unstimulated platelets (control). The statistical significance was analysed using one-way ANOVA (Kruskal-Wallis Test). * p ⁇ 0.05, ** p ⁇ 0.005, ***p ⁇ 0.001, **** p ⁇ 0.0001.
  • Figure 4 shows that S100 A8/A9 does not induce platelet aggregation.
  • Figure 5 shows that S100 A8/A9 induces the formation of phosphatidylserine positive pro-coagulant platelets.
  • Human washed platelets (lxlO 6 ) were incubated with different concentrations of S100 A8/A9 (10, 20 and 40 pg/ml) for 30 min at 37°C in the presence of Ca2+ (2mM).
  • A Representative histogram for Annexin V staining.
  • Figure 6 demonstrates a partial decrease in S100 A8/A9-dependent GPIIbllla activation and P-selectin expression by CD36 blockade and ITAM receptor inhibitors, respectively.
  • Human washed platelets (lxlO 6 ) were incubated with S100 A8/A9 20 pg/ml with or without different inhibitors for 30 min at 37°C. Platelet activation was determined by flow cytometry using (A, D) CD41/CD61 PAC-1 antibody (activated GPIIb/IIIa), (B, E) anti-P-selectin antibody, antibody and (C, F) PE-Cy7- labelled Annexin V.
  • Leaf SI 00 A8/A9 antibody blocking binding to RAGE and TLR4 (S100 A8/A9 blocking antibody, 20 pg/ml), CD36 antibody blocking binding to OxLDL (CD36 blocking antibody), Sulfosuccinimidyl oleate (SSO, 25 mM), Paquinimod blocking S100A9 binding to RAGE and TLR4 (10 mM), RAGE inhibitor Azeliragon (1 mM), P2Y12 inhibitor Cangrelor (10 mM), COX inhibitor Indomethacin (10 mM), Syk inhibitor RT- 060318 (10 mM), Src inhibitor PP2 (20 mM), BTK inhibitor Ibrutinib (500 nM), were used.
  • FIG. 7 shows that murine S100 A8/A9 activates mouse platelets independent of GPVI and/or CLEC-2.
  • Mouse washed platelets (lxlO 6 ) were incubated with different doses of recombinant mouse S100 A8/A9 for 30 min at 37°C in the presence of Ca2+ (2mM). Platelet activation was determined by flow cytometry using (A, B) anti-P- selectin antibody.
  • mice (C, D, ) Mouse washed platelets (lxlO 6 ) were isolated from wild type (WT), GPVI (GPVI -/-), CLEC-2 (CLEC-2-/-) and GPVI/CLEC-2 knock-out mice
  • Figure 8 shows that extracellular GPIba deficiency inhibits platelet activation by S100 A8/A9.
  • Mouse washed platelets (lxlO 6 ) from WT (Control) and IL4R/GPIba-Tg mice lacking extracellular domain of GPIba (GPIba KO mice) were incubated in the absence (control) or presence of mouse recombinant SI 00 A8/A9 (10 pg/ml) for 30 min at 37°C. Platelet activation was determined by flow cytometry using (A, B, C) anti-P- selectin antibody, (D, F, F) Annexin-V and (G, H, I) anti-CD41/61 JON/A antibody.
  • Figure 9 demonstrates that recombinant human GPIba inhibits platelet response to S100 A8/A9.
  • Platelet activation was assessed by flow cytometry using (A, B, C) anti- CD41/61 PAC-1 and (D, F, F) anti-P-selectin antibodies.
  • G, H, I Phosphatidylserine exposure was assessed using Annexin V.
  • Figure 10 shows that platelets from Bernard-Soulier patient (GPIb-IX-V deficiency) failed to respond to S100 A8/A9.
  • Human washed platelets (lxlO 6 ) from a healthy donor or Bernard-Soulier Syndrome (BSS) patient were incubated with S100 A8/A9 (20 and 40 pg/ml) for 30min at 30°C.
  • CRP (10 pg/ml) is used as positive control.
  • B P-selectin and
  • C Phosphatidylserine exposure were assessed using anti-CD41/61 PAC-1 antibody, anti-P-selectin antibody and Annexin V, respectively.
  • FIG. 11 Shows that anti-GPlb antibodies can block the effects of S100 A8/A9 on platelet activation and formation of procoagulant platelets human washed platelets (lxlO 6 ) were incubated with S100 A8/A9 40 pg/ml with or without different GPIb antibodies 80 pg/ml for 30 min at 37°C. Platelet activation was assessed by flow cytometry using anti- CD41/61 PAC-1 (A,D) anti-P-selectin antibodies (B,E) and phosphatidylserine exposure was assessed using Annexin V (C,F). Data shown as total (%) and MFI.
  • Figure 12 shows that anti-GPlb antibodies AK2 and SZ2, alone or combined, can block the effects of S100 A8/A9 on platelet activation and formation of procoagulant platelets.
  • Washed human platelets (10 6 /condition) were pre-incubated with or without anti human CD42b/GPIb alpha antibody (clone AK2) Ab (Novusbio) (40 pg/ml), anti-human CD42b/GPIb alpha (clone SZ2) (Beckman Coulter) (40 pg/ml) or combination of both antibodies AK2+SZ2. for 10 min at 37 °C and then stimulated with S100A8/A9 (20 pg/ml) for 30 minutes at 37 °C.
  • Platelet activation was determined by flow cytometry using (A, B) Anti-CD41/CD61 PAC-1 antibody, (C, D) anti-P- selectin antibody and (E, F) Annexin-V binding. Data is shown as percentage platelets (CD41+) positive for different markers (A, C, E) or MFI (fold change over control) (B, D, F). Data is shown as mean + SD. The statistical significance was analyzed using ordinary one-way ANOVA. * p ⁇ 0.05, ** p ⁇ 0.01 *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • Figure 13 Shows that heparin can block the effects of S100 A8/A9 on platelet activation and formation of procoagulant platelets.
  • Washed human platelets (106/condition) were pre-incubated with or without Heparin (0.5 U/ml) for 10 min at 37 °C and then stimulated with S100A8/A9 (20 pg/ml) for 30 minutes at 37 °C.
  • Platelet activation was determined by flow cytometry using (A,B) Anti-CD41/CD61 PAC-1 antibody, (C, D) anti-P-selectin antibody and (E, F) Annexin-V binding.
  • Data is shown as percentage platelets (CD41+) positive for different markers (A, C, E) or MFI (fold change over control) (B, D, F). Data is shown as mean + SD. The statistical significance was analyzed using ordinary one-way ANOVA. * p ⁇ 0.05, *** p ⁇ 0.001.
  • Figure 14 shows that S100A8/A8 induces intracellular calcium release in platelets and increases phosphatidylserine exposure.
  • A, B S100A8/A9 was immobilized at different concentrations (20 and 40 pg/ml) and platelet spreading was assessed by immunofluorescence imaging and compared to immobilized collagen (10 pg/ml). Immobilized S100A8/A9 induces P-selectin and Annexin-V expression on platelets.
  • C, D Measurement of calcium release in platelets. Immobilized S100A8/A9 increases calcium release in platelets as measured using O regon green-488 BAPTA-l-AM calcium sensitive dye. Peak fluorescence at time point zero (FO/Fmax). Data is shown as mean + SD. The statistical significance was analyzed using nonparametric test (Kruskal-
  • MATERIALS AND METHODS Platelet preparation Blood was taken from consenting patients or healthy volunteers, into 3.2% vacutainer sodium citrate tubes.
  • Mouse blood was drawn from mice on 10% ACD-A Anticoagulant Citrate Dextrose solution.
  • Platelet-rich plasma was obtained by centrifugation for 20min at room temperature at 200g. Washed human and mouse platelets were obtained by further centrifugation of the platelet rich plasma in the presence of prostacyclin PGI2 (lpg/ml) for 6min at room temperature at lOOOg. The pellet was resuspended in modified Tyrode buffer containing PGI2 to wash platelets and platelets collected by centrifugation was described above. Platelets were used at a cell density of 2xl0 8 /mL for aggregometry and lxl0 6 /ml for flow cytometry.
  • Aggregation was assessed by light transmission using a Model 700 aggregometer (Chronolog, Havertown, PA, USA) as previously described (Nicolson PLR et al. 2018). Aggregation was performed using washed platelets (2xl0 8 /mL) for 20 minutes in the presence of recombinant SI 00 A8/A9 (40pg/ml) (Biolegend) and 6 minutes for classical platelet agonist (collagen related peptide CRP 10 pg/ml).
  • Plasma was prepared as previously described (Mussbacher et al., 2017) by centrifugation for lOmin at lOOOg (4°C) followed by lOmin at lO.OOOg (RT). Plasma was stored at -80°C until use. Plasma levels of Calprotectin were measured by LEGEND MAX Human MRP8/14 (Calprotectin) ELISA Kit (Biolegend) according to manufacturer’s instructions.
  • Washed platelets (1 million) were incubated with conjugated antibodies for 30 minutes at 37 degrees in the dark in the presence or absence of agonists (S100A8/A9, CRP, TRAP-6).
  • platelets were pre-incubated with inhibitors (recombinant soluble GPIba (rGPIba) (R&D), small molecule inhibitors (TAK-242, S100 A8/A9 blocking Ab, CD36 blocking Ab, SSO, Paquinimod (BioVision), Azeliragon (MCE), Cangrelor (Medicines company), Indomethacin (Sigma), PRT- 060318, PP2, Ibrutinib, CD36 Ab (stemcell), TAK-242 (Tocris), SSO (Cayman Chemicals), Ultra-LEAFTM Purified anti -human S100A8/A9 Heterodimer Antibody (Biolegend)), anti-GPIb monoclonal antibodies (see table below) for 10 minutes and then treated with agonists for 30min
  • EM staining and immunohistochemistry Whole blood aggregation was performed in the presence or absence of recombinant S100 A8/A9 (40pg/ml) for 20min at 37C followed by fixation using PFA 4%. Sample was processed to electron microscopy. For human lung sections, paraffin sections, following rehydration and antigen retrieval, lung sections were treated with H202 3% for 15 minutes and blocked with PBS containing 5% bovine serum albumin and 10% goat serum for lh. Antibodies against platelet CD42b (Abeam) and S100A8/A9 were incubated overnight at 4°C. Secondary antibodies were added for lh at room temperature. Nuclei was staining using DAPI.
  • Lung autofluorescence was quenched using commercial kit (Vector laboratories) and slides mounted using ProLong Gold Antifade Mountant (Life Technologies). Sections were imaged using Epi fluorescent microscopy or Zeiss Axio Scan.Zl microscope and analysed using ZEN software and image J.
  • PE-conjugated anti-mouse P-selectin PE-conjugated anti-mouse activated aIII b3 antibodies (JON/A) and PEcy7 Annexin V were from Biolegend.
  • FITC conjugated anti human PAC-1 was from biolegend and Invitrogen
  • PE-conjugated P-selectin, pecy7 conjugated Annexin V and APC conjugated CD41 were from Biolegend.
  • Example 1 - S100 A8/A9 is increased in the lungs and plasma of COVID-19 patients.
  • SI 00 A8/A8 is elevated in severe COVID- 19-patients and these levels are higher in patients with thrombosis (Zuo et al., Journal of thrombosis and thrombolysis, 2020).
  • SI 00 A8/A9 positive cells were more present in the lungs of COVID-19 patients, both with and without confirmed thrombosis (Figure 1).
  • a fraction of SI 00 A8/A9 positive cells colocalizes with CD42b, a marker of platelets, suggesting platelet-bound cells and or S100 A8/A9-positive platelets.
  • S100 A8/A9 is highly present in neutrophils and plasma levels suggesting neutrophil activation.
  • anti-myeloperoxidase antibody it was observed that a large proportion of S100A8/A9 positive cells are positive for MPO.
  • the levels of S100 A8/A9 were measured in the plasma of 87 COVID-19 patients by ELISA.
  • Example 2 - SI 00 A8/A9 induces human platelets activation but not aggregation in vitro.
  • SI 00 A8/A9 affects platelet function.
  • the effect recombinant SI 00 A8/A9 on human platelet activation in vitro was assessed. Human washed platelets were incubated with different concentration of SI 00 A8/A9 and platelet activation markers were measured by flow cytometry.
  • SI 00 A8/A9 in the range found in the plasma of COVID-19 patients, induces alpha-granule secretion and GPIIb/IIIa activation as measured using anti-P-selectin ( Figure 3 A, B, C) and anti CD61/Cd41 PAC-1 antibody binding ( Figure 3D, E, F).
  • Example 3 - S100A8/A9 induces phosphatidylserine exposure and the formation of procoagulant platelets. Segregation of platelet aggregatory and procoagulant properties has been previously described (Rendu et al., 1995; Munnix et al., 2007) with procoagulant platelets showing high levels of phosphatidylserine (PS) membrane exposure. The surface expression of PS on S100A8/A9-activated platelets was therefore measured. S100A8/A9 induces PS exposure on the surface of platelets as measured by platelet-positive PS and by increase in the MFI compared to control ( Figure 5 A, B, C).
  • S100 A8/A9 also increases the levels of PS on CD41+ microvesicles (MV), suggesting the formation of procoagulant platelets and release of procoagulant MV ( Figure 5D, E).
  • the procoagulant phenotype and fibrin generation was confirmed using scanning electron microscopy after addition of S100A8A9 in whole blood ( Figure 5F).
  • SI 00 A8/A9 induces the formation of procoagulant platelets and microvesicles, supporting fibrin generation and thrombosis.
  • Example 4 - CD36 blockade and ITAM-receptor inhibitors only partially decreased platelets activation induced by S100 A8/A9
  • S100A8/A9 has three known receptors: CD36, RAGE and TLR4, which are all expressed on platelets (Hally et al., 2020).
  • platelet inhibitors targeting different platelet receptors, were used. It was first assessed whether platelet activation and procoagulant platelets are induced through SI 00 A8/A9 known receptors. Inhibition of TLR-4, using the small molecule inhibitor TAK-242 or an antibody anti SI 00 A8/A9 blocking binding to TLR4 and RAGE or blocking RAGE using Paquinomod or Azeliragon did not alter platelet activation (Figure 6A, B, C).
  • Example 5 Murine recombinant SI 00 A8/A9 activates mouse platelets in a dose- dependent manner through GPIba.
  • CLEC-2, GPVI, GPIb and CD36 signal through ITAM receptor pathways
  • the effect of S100A8/A9 was assessed on platelets deficient in CLEC-2, GPVI, CLEC-2/GPVI or platelets deficient in the extracellular domain of GPIba (IL4R/GPIba-Tg mice, GPIba KO) and compared to wild type (WT) platelets.
  • SI 00 A8/A9 induces platelet activation in a dose dependent manner with similar ranges to human platelets (Figure 7a).
  • GPIba is the main receptor for S100A8/A9 on mouse platelets inducing platelet activation and procoagulant platelets.
  • Example 6 Recombinant human GPIba completely inhibits platelet response to SI 00 A8/A9
  • GPIba is the receptor for SI 00 A8/A9 on human platelets.
  • platelets were activated in the presence of recombinant GPIb (rGPIba).
  • the inhibitory effect was observed for GPIIbllla activation ( Figure 9A-C), P- selectin upregulation ( Figure 9D-F) and PS exposure (Figure 9 G-I).
  • rGPIba inhibits the formation of PS+-microvesicles ( Figure 9 L, M).
  • Example 7 - S100A8/A9 did not induce procoagulant platelets isolated from a patient with Bernard Soulier Syndrome (BSS).
  • GPIba is the main receptor for S100A8/A9 on platelets
  • S100A8/A9 were added on platelets isolated from healthy donor or BSS patient.
  • platelets from BSS patient failed to respond to S100A8/A9, whereas the response to CRP was normal.
  • S100A8/A9 does not induce P-selectin expression, GPIIbllla activation or PS exposure on platelets from BSS patient compared to healthy donor ( Figure 10).
  • Example 8 - anti GPIba antibodies are able to reduce the effects of extracellular SI 00 A8/A9 on platelet activation and procoagulant platelet formation.
  • SI 00 A8/A9 induced platelet activation and procoagulant platelet formation and supporting their roles in treating or preventing thrombotic and/or inflammatory diseases associated with such increased platelet activation and procoagulant platelet formation
  • a panel of commercially available anti-GPlba antibodies was tested (Figure 11). The results indeed demonstrate that anti-GPlba antibodies are able to reduce SI 00 A8/A9 induced platelet activation and procoagulant platelet formation.
  • Example 9 Combination of two anti GPIba recognising the sequences Leu36-Gln59 and Tyr276-Glu282 on GPIba are able to significantly reduce the formation of procoagulant platelets.
  • Example 10 Heparin reduces platelet activation and formation of procoagulant platelets induced by S100 A8/A9
  • heparin a glycosaminoglycan used as an anticoagulant was able to block platelet activation and formation of procoagulant platelets (Figure 13).
  • Example 11 Immobilised S100A8/A9 induce PS exposure and calcium release in platelets.
  • S100A8/A9 induces the expression of P-selectin and PS on platelets along increase in intracellular calcium release ( Figure 14). These parameters can be used as a readout to assess the efficacy of drugs targeting platelet activation induced by S 100A8/A9.

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Abstract

L'invention concerne des agents destinés à être utilisés dans la prévention ou la réduction de l'interaction entre S100 A8/A9 et GPIbα. De tels agents sont particulièrement destinés à être utilisés dans le traitement ou la prévention d'une ou de plusieurs maladies inflammatoires et/ou thrombotiques, telles que des maladies cardiovasculaires, des maladies inflammatoires chroniques, des maladies infectieuses et des maladies auto-immunes. Un agent selon l'invention peut être un anticorps, un inhibiteur de petite molécule, un peptide, un polypeptide, une protéine recombinante, un acide nucléique, un récepteur de lymphocytes T (TCR), un afficorps, un aptamère ou un glycosaminoglycane.
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