WO2022123062A1 - Blocage de la caspase et/ou de la fasl pour prévenir une issue fatale chez des patients atteints de la covid-19 - Google Patents

Blocage de la caspase et/ou de la fasl pour prévenir une issue fatale chez des patients atteints de la covid-19 Download PDF

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WO2022123062A1
WO2022123062A1 PCT/EP2021/085315 EP2021085315W WO2022123062A1 WO 2022123062 A1 WO2022123062 A1 WO 2022123062A1 EP 2021085315 W EP2021085315 W EP 2021085315W WO 2022123062 A1 WO2022123062 A1 WO 2022123062A1
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fasl
fasr
covid
cells
caspase
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PCT/EP2021/085315
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English (en)
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Jérôme ESTAQUIER
Pierre Corbeau
Fabrizio Mammano
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université de Paris
UNIVERSITé LAVAL
Université De Montpellier
C.H.U. De Nîmes
Centre National De La Recherche Scientifique (Cnrs)
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Publication of WO2022123062A1 publication Critical patent/WO2022123062A1/fr

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    • 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
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22036—Caspase-1 (3.4.22.36), i.e. interleukin-1-beta-convertase
    • 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
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses

Definitions

  • the present invention is in the field of medicine, in particular virology and immunology.
  • the lung injury caused by SARS-CoV-2 has been related to local inflammation in which the infiltration in the lung is linked to the respiratory syndrome severity, as well as an increase in vascular permeability.
  • Several inflammatory biomarkers such as IL-6 has been linked to a worst prognosis 5 ’ 6 , and impaired interferon response 7 .
  • Expansion of neutrophils associated with a lymphopenia is observed in COVID-19 patients that is predictive for serious outcomes 8 ' 11 ; however, the mechanisms behind lymphopenia remains elusive.
  • coronavirus including MERS-CoV and HCoVs induced a cell death program namely apoptosis 12 13 .
  • apoptosis was observed in SARS-CoV-infected lung and thyroid tissues 14 .
  • SARS-CoV-2 infected hamsters demonstrated extensive apoptosis 15 .
  • coronavirus have been reported to induce apoptosis of T cells 16 17 that has been associated with lymphopenia in some HCoV diseases.
  • CD4 T cells are require to coordinate immune response against microbes in particular the genesis of humoral response, such T cell defect could impact on immunoglobulin response, leading to a T-cell independent humoral immunity 18 19 .
  • lymphoid cells from individuals are more sensitive to die during SARS-CoV-2 infection.
  • Fas and FasL are upregulated in alveolar epithelial cells 21,22 .
  • Fas/FasL interaction was implicated in cystic fibrosis airway pathway 23 , in pneumonitis 24 , and in the acute respiratory distress syndrome (ARDS) 25 .
  • FasL can be released as a biologically active death-inducing mediator capable of inducing apoptosis during acute lung injury 26 . Therefore, whether COVID-19 patients displayed higher levels of FasL is unknown and could be one of the main actors contributing in COVID-19 pathology.
  • the present invention is defined by the claims.
  • the present invention relates to methods of determining the risk of worsening in CO VID-19 patients.
  • the present invention also relates to methods of treating CO VID-19.
  • CD4 and CD8 T cells from COVID-19 patients are more prone to undergo death and that blocking caspase activation prevents T cell death.
  • higher levels of soluble FasL in the plasma of COVID-19 individuals is associated with higher levels of T cell death.
  • the presence of higher levels of activated caspase is associated with lower levels of Thl cytokines and of IL-18. It also indicated an inflammasome activation pathway, which can be link with several endothelial biomarkers of cell damage.
  • the term "subject” or “subject in need thereof', is intended for a human or non-human mammal. Typically the patient is affected or likely to be infected with SARS- Cov-2.
  • SARS-Cov-2 severe Acute Respiratory Syndrome coronavirus 2
  • SARS-Cov-2 has its general meaning in the art and refers to the strain of coronavirus that causes coronavirus disease 2019 (COVID-19), a respiratory syndrome that manifests a clinical pathology resembling mild upper respiratory tract disease (common cold-like symptoms) and occasionally severe lower respiratory tract illness and extra-pulmonary manifestations leading to multi-organ failure and death.
  • the term refers to the severe acute respiratory WO 2022/123062 PCT/EP2021/085315 syndrome coronavirus 2 isolate 2019-nCoV_HKU-SZ-005b_2020 for which the complete genome is accessible under the NCBI access number MN975262.
  • COVID-19 refers to the respiratory disease induced by the Severe Acute Respiratory Syndrome coronavirus 2.
  • the term "worsening” means that the COVID-19 evolves at a later stage with respect to the first measured time point phase.
  • CT chest computed tomography
  • CRP C-reactive protein
  • risk of worsening is to be understood as referring to the probability of worsening subject.
  • risk in the context of the present invention, relates to the probability that an event will occur over a specific time period and can mean a subject's "absolute risk” or “relative risk”.
  • Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.
  • Odds ratios the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion.
  • "Risk evaluation,” or “evaluation of risk” in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of relapse, either in absolute or relative terms in reference to a previously measured population.
  • the methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk of conversion.
  • the invention can be used to discriminate between normal and other subject cohorts at higher risk.
  • FasR or “CD95”has its general meaning in the art and refers to the the receptor present on the surface of mammalian cells, which has been originally shown to have the capacity to induce apoptosis upon binding of the trimeric form of its cognate ligand, FasL (CD95L) (Krammer, P.H. (2000). CD95's deadly mission in the immune system. Nature 407, 789-795).
  • CD95 is also known as FasR or Apo-1.
  • SEQ ID NO:1 UniProtKB/Swiss-Prot accession number : P25445).
  • FasL or “CD95L” has its general meaning in the art ant refers to the cognate ligand of FasR/CD95.
  • soluble FasL has its general meaning in the art and refers to the soluble ligand produced by the cleavage of the transmembrane FasL (Matsuno et al., 2001; Vargo-Gogola et al., 2002; Kiaei et al., 2007; Kirkin et al., 2007; or Schulte et al., 2007).
  • An exemplary amino acid sequence of FasL is shown as SEQ ID NO:2 (UniProtKB/Swiss-Prot accession number : P48023).
  • FasR antagonist or “CD95 antagonist” means any molecule that attenuates signal transduction mediated by the binding of FasR (CD95) to the soluble FasL (CD95L). Such inhibition may result where: (i) the FasR antagonist of the invention binds to FasR without triggering signal transduction, to reduce or block signal transduction mediated by soluble FasL; (ii) the FasR antagonist binds to the soluble FasL, preventing its binding to FasR; (iii) the FasR antagonist binds to, or otherwise inhibits the activity of, a molecule that is part of a regulatory chain that, when not inhibited, has the result of stimulating or otherwise facilitating FasR signal transduction mediated by soluble FasL; or (iv) the FasR antagonist inhibits FasR WO 2022/123062 PCT/EP2021/085315 expression or FasL expression, especially by reducing or abolishing expression of one or more genes encoding Fa
  • a “functional equivalent of FasR” is a compound which is capable of binding to soluble FasL, thereby preventing its interaction with FasR.
  • the term “functional equivalent” includes fragments, mutants, and muteins of FasR.
  • the term “functionally equivalent” thus includes any equivalent of FasR obtained by altering the amino acid sequence, for example by one or more amino acid deletions, substitutions or additions such that the protein analogue retains the ability to bind to soluble FasL. Amino acid substitutions may be made, for example, by point mutation of the DNA encoding the amino acid sequence.
  • Functional equivalents include molecules that bind soluble FasL and comprise all or a portion of the extracellular domains of FasR.
  • a functionally equivalent fragment as used herein also may mean any fragment or assembly of fragments of FasR that binds to soluble FasL.
  • the present invention provides a polypeptide capable of inhibiting binding of FasR to soluble FasL, which polypeptide comprises consecutive amino acids having a sequence which corresponds to the sequence of at least a portion of an extracellular domain of FasR, which portion binds to soluble FasL.
  • the polypeptide corresponds to an extracellular domain of FasR.
  • immunoadhesin designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains.
  • the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is “heterologous"), and an immunoglobulin constant domain sequence.
  • the adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand.
  • the immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
  • immunoglobulin such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
  • an “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. Therefore, an “inhibitor of FasR or FasL expression” denotes a natural or synthetic compound that has a biological effect to inhibit the expression of FasR or FasL gene.
  • caspase refers to a family of cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases that are a family of cysteine proteases that play essential roles in apoptosis (programmed cell death), necrosis, and WO 2022/123062 PCT/EP2021/085315 inflammation. Effector caspases (e.g., CASP3, CASP6, and CASP7) cleave protein substrates within the cell to trigger the apoptotic process. The initiation of this cascade reaction is regulated by caspase inhibitors.
  • Caspases are first synthesized as inactive pro-caspases that consist of a prodomain, a small subunit and a large subunit.
  • Granzyme B released by cytotoxic T lymphocytes and NK cells is known to activate caspase-3 and -7.
  • caspase inhibitor refers to any compound, molecule, or protein that is capable of inhibiting, or reducing the activity of a caspase enzyme.
  • Q-VD-OPH has its general meaning in the art and refers to 5 -(2, 6-Difluorophenoxy)-3 - [ [3 -methyl- 1 -oxo-2- [(2-quinolinylcarbonyl)amino]butyl]amino] - 4-oxo-pentanoic acid having the formula of:
  • Q-VD(Ome)-OPH has its general meaning in the art and refers to (S)-methyl 5-(2,6-difluorophenoxy)-3-((S)-3-methyl-2-(quinoline-2- carboxamido)butanamido)-4-oxopentanoate having the formula of:
  • blood sample means any blood sample derived from the subject. Collections of blood samples can be performed by methods well known to those skilled in the art. In some embodiments, the blood sample is a serum sample or a plasma sample. WO 2022/123062 PCT/EP2021/085315
  • high refers to a measure that is greater than normal, greater than a standard such as a predetermined reference value or a subgroup measure or that is relatively greater than another subgroup measure.
  • high levels of FasL refers to a level of FasL that is greater than a normal FasL level.
  • a normal FasL level may be determined according to any method available to one skilled in the art.
  • High level of FasL may also refer to a level that is equal to or greater than a predetermined reference value, such as a predetermined cutoff.
  • High level of FasL may also refer to a level of FasL wherein a high FasL subgroup has relatively greater levels of FasL than another subgroup.
  • two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a subgroup whose measure is high (i.e., higher than the median) and another subgroup whose measure is low.
  • a “high” level may comprise a range of level that is very high and a range of level that is “moderately high” where moderately high is a level that is greater than normal, but less than “very high”.
  • low refers to a level that is less than normal, less than a standard such as a predetermined reference value or a subgroup measure that is relatively less than another subgroup level.
  • low level of FasL means a level of FasL that is less than a normal level of in a particular set of samples of patients.
  • a normal level of FasL measure may be determined according to any method available to one skilled in the art.
  • Low level of FasL may also mean a level that is less than a predetermined reference value, such as a predetermined cutoff.
  • Low level of FasL may also mean a level wherein a low level FasL subgroup is relatively lower than another subgroup.
  • two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a group whose measure is low (i.e., less than the median) with respect to another group whose measure is high (i.e., greater than the median).
  • a mathematically determined point such as, without limitation, a median
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen.
  • two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond.
  • light chains There are two types of light chains, lambda (1) and kappa (k).
  • k kappa
  • the light chain includes WO 2022/123062 PCT/EP2021/085315 two domains, a variable domain (VL) and a constant domain (CL).
  • the heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH).
  • the variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen.
  • the constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).
  • the Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain.
  • the specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant.
  • Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) can participate in the antibody binding site, or influence the overall domain structure and hence the combining site.
  • Complementarity Determining Regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.
  • the light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively.
  • An antigen-binding site therefore, typically includes six CDRs, comprising the CDRs set from each of a heavy and a light chain V region.
  • Framework Regions refer to amino acid sequences interposed between CDRs.
  • the variable regions of the light and heavy chains typically comprise 4 framework regions and 3 CDRs of the following sequence: FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al.
  • Kabat et al. 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NTH, USA (Kabat et al., 1992, hereafter “Kabat et al ”).
  • the Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences.
  • the actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure.
  • CDR complementarity determining region
  • the correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence.
  • the CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system.
  • the CDRs of the light WO 2022/123062 PCT/EP2021/085315 chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system.
  • the CDRs have been determined using CDR finding algorithms from www.bioinf.org.uk - see the section entitled « How to identify the CDRs by looking at a sequence » within the Antibodies pages.
  • the predicted CDRs of some agonist antibodies, such as 11B6, 12E2, 12B4, CP (CP-870,893 from Pfizer) or 24A3 are described in the Examples below.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • the term "therapeutically effective amount" of the agent of the present invention is meant a sufficient amount of the compound to treat a coronavirus infection at a WO 2022/123062 PCT/EP2021/085315 reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the specific agonist employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • An object of the present invention relates to a method of determining the risk of worsening in a patient suffering from COVID-19 comprising determining the level of soluble FasL in a sample obtained from the patient wherein said level indicates the risk of worsening.
  • the method of the present invention is particularly suitable for determining the outcome in patient diagnosed with COVID-19.
  • the method of the present invention is particularly suitable for predicting whether the patient has to be admitted in intensive care unit.
  • the method of the present invention is particularly suitable for predicting the survival of a patient suffering from COVID-19.
  • the sample is any sample liable to contain an amount of FasL. In some embodiments, the sample is a blood sample.
  • the level of the FasL is determined by an immunoassay.
  • immunoassays include, for example, competition assays, direct reaction assays sandwich- type assays and WO 2022/123062 PCT/EP2021/085315 immunoassays (e.g. ELISA).
  • the assays may be quantitative or qualitative.
  • the detecting step can comprise performing an ELISA assay, performing a lateral flow immunoassay, performing an agglutination assay, analyzing the sample in an analytical rotor, or analyzing the sample with an electrochemical, optical, or opto-electronic sensor.
  • the devices are useful for performing an immunoassay according to the present invention.
  • the device is a lateral flow immunoassay device.
  • the device is an analytical rotor.
  • the device is a dot blot.
  • the device is a tube or a well, e.g., in a plate suitable for an ELISA assay.
  • the device is an electrochemical sensor, an optical sensor, or an opto-electronic sensor. The presence and amount of the immunocomplex may be detected by methods known in the art, including label- based and label-free detection.
  • label-based detection methods include addition of a secondary antibody that is coupled to an indicator reagent comprising a signal generating compound.
  • the secondary antibody may be an anti-human IgG antibody.
  • Indicator reagents include chromogenic agents, catalysts such as enzyme conjugates, fluorescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums, ruthenium, and luminol, radioactive elements, direct visual labels, as well as cofactors, inhibitors and magnetic particles.
  • enzyme conjugates include alkaline phosphatase, horseradish peroxidase and beta-galactosidase.
  • Methods of label-free detection include surface plasmon resonance, carbon nanotubes and nanowires, and interferometry.
  • Label- based and label-free detection methods are known in the art and disclosed, for example, by Hall et al. (2007) and by Ray et al. (2010) Proteomics 10:731- 748. Detection may be accomplished by scanning methods known in the art and appropriate for the label used, and associated analytical software.
  • fluorescence labeling and detection methods are used to detect the immunocomplexes.
  • a particularly useful assay format is a lateral flow immunoassay format.
  • Antibodies to human or animal (e.g., dog, mouse, deer, etc.) immunoglobulins, or staph A or G protein antibodies can be labeled with a signal generator or reporter (e.g., colloidal gold) that is dried and placed on a glass fiber pad (sample application pad or conjugate pad).
  • a signal generator or reporter e.g., colloidal gold
  • Another assay is an enzyme linked immunosorbent assay, i.e., an ELISA.
  • the FasLs are adsorbed to the surface of a microtiter well directly or through a capture matrix (e.g., an antibody).
  • Residual, non-specific proteinbinding sites on the surface are then blocked with an appropriate agent, such as bovine serum albumin (BSA), heat-inactivated normal goat serum (NGS), or BLOTTO (a buffered solution WO 2022/123062 PCT/EP2021/085315 of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent).
  • BSA bovine serum albumin
  • NGS heat-inactivated normal goat serum
  • BLOTTO a buffered solution WO 2022/123062 PCT/EP2021/085315 of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent.
  • the sample can be applied neat, or more often it can be diluted, usually in a buffered solution which contains a small amount (0.1-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO.
  • an appropriate anti-immunoglobulin antibody e.g., for human subjects, an anti-human immunoglobulin (aHulg) from another animal, such as dog, mouse, cow, etc. that is conjugated to an enzyme or other label by standard procedures and is dissolved in blocking buffer.
  • the label can be chosen from a variety of enzymes, including horseradish peroxidase (HRP), beta-galactosidase, alkaline phosphatase, glucose oxidase, etc.
  • the method of the present invention further comprises comparing the level of FasL with a predetermined reference value wherein detecting a difference between the level of FasL and the predetermined reference value indicates the risk of worsening.
  • the predetermined reference value is a threshold value or a cut-off value.
  • a “threshold value” or “cut-off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).
  • the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • the full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests.
  • ROC curve is a comprehensive indicator that WO 2022/123062 PCT/EP2021/085315 reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1- specificity). It reveals the relationship between sensitivity and specificity with the image composition method.
  • a series of different cut-off values are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis.
  • AUC area under the curve
  • the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values.
  • the AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate.
  • This algorithmic method is preferably done with a computer.
  • Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE- ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.
  • the method of the invention thus comprises the use of an algorithm.
  • the algorithm is a classification algorithm typically selected from Multivariate Regression Analysis, Linear Discriminant Analysis (LDA), Topological Data Analysis (TDA), Neural Networks, Support Vector Machine (SVM) algorithm and Random Forests algorithm (RF).
  • the method of the present invention thus comprises a) determining the level of FasL in the sample obtained from the subject; b) implementing an algorithm on data comprising the level of FasL so as to obtain an algorithm output; c) determining the risk of worsening from the output obtained at step c).
  • a further object of the invention relates to a kit for performing the above described method, said kit comprising means for measuring the level of soluble FasL in the sample obtained from the patient.
  • said means for measuring the level of soluble FasL is an antibody that interacts specifically with soluble FasL as described above.
  • Said binding partner can be tagged for an easier detection. It may or may not be immobilized on a substrate surface (e.g., beads, array, and the like).
  • a substrate surface e.g., beads, array, and the like.
  • an inventive kit may include an array for predicting the risk of having a cardiovascular event as provided herein.
  • a substrate surface e.g. membrane
  • kits of the invention generally also comprises at least one reagent for the detection of a complex between binding partner included in the kit and biomarker of the invention.
  • the kit may further comprise one or more of: extraction buffer and/or reagents, western blotting buffer and/or reagents, and detection means. Protocols for using these buffers and reagents for performing different steps of the procedure may be included in the kit.
  • the different reagents included in a kit of the invention may be supplied in a solid (e.g. lyophilized) or liquid form.
  • kits of the present invention may optionally comprise different containers (e.g., vial, ampoule, test tube, flask or bottle) for each individual buffer and/or reagent.
  • Each component will generally be suitable as aliquoted in its respective container or provided in a concentrated form.
  • Other containers suitable for conducting certain steps of the disclosed methods may also be provided.
  • the individual containers of the kit are preferably maintained in close confinement for commercial sale.
  • a kit comprises instructions for using its components for the prediction of the risk of worsening in the patient according to a method of the invention.
  • Instructions for using the kit according to methods of the invention may comprise instructions for processing the biological sample obtained from the patient and/or for performing the test, or instructions for interpreting the results.
  • a kit may also contain a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products.
  • a further object of the present invention relates to a method of treating CO VID-19 in patient in need thereof comprising administering to the subject a therapeutically effective amount of a FasR antagonist and/or a caspase inhibitor.
  • the method of the present invention is suitable for preventing worsening of a patient suffering from COVID-19. In some embodiments, the method of the present invention is suitable for treating a patient suffering from COVID-19 who worsens.
  • the method is carried out for a patient that has been determined as being at risk of worsening by the diagnostic method of the present invention.
  • the method comprises the steps of i) determining the risk of worsening in the patient suffering from COVID- 19 comprising determining the level of soluble FasL in a sample obtained from the patient wherein said level indicates the likelihood of worsening and ii) administering to the subject a therapeutically effective amount of a FasR WO 2022/123062 PCT/EP2021/085315 antagonist and/or a caspase inhibitor when it is determined that the patient is at risk of worsening.
  • the FasR antagonist includes but is not limited to an antibody, a small organic molecule, a polypeptide and an aptamer.
  • the antibodies of the invention have the ability to block the interaction between soluble FasL and FasR or have the ability to block the induction of the signaling pathway mediated by soluble FasL (e.g. by inhibiting the oligomerisation of FasR).
  • the antibodies may have specificity to soluble FasL or FasR.
  • the antibodies or fragment of antibodies are directed to all or a portion of the extracellular domain of FasR.
  • the antibodies or fragment of antibodies are directed to an extracellular domain of FasR.
  • this invention provides an antibody or portion thereof capable of inhibiting binding of FasR to soluble FasL, which antibody binds to an epitope located within a region of FasR, which region of FasR binds to soluble FasL. Even more particularly, the invention provides an antibody or portion thereof capable of binding to an epitope located within a region of FasR, which region of FasR is involved the oligomerisation of the receptor. Typically, the antibody binds to the cysteine-rich domain 1 of FasR which is called the pre-ligand assembly domain (PLAD) (Edmond V, Ghali B, Penna A, Taupin JL, Daburon S, Moreau JF, Legieri P.
  • PAD pre-ligand assembly domain
  • the antibody of the invention binds to the regions delimitated between the amino acid at position 43 and the amino acid at position 66 in SEQ ID NO: 1.
  • the antibody is a monoclonal antibody. In some embodiments of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In some embodiments of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In some embodiments of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In some embodiments of the antibodies or portions thereof described herein, the antibody is a full-human antibody. In some embodiments of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion. In some embodiments of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab')2 portion.
  • the FasR antagonist is a polypeptide.
  • the polypeptide is a functional equivalent of FasR.
  • the functional equivalents include soluble forms of the FasR.
  • a suitable soluble form of these proteins, or functional equivalents thereof, might comprise, for example, a truncated form of the protein from which the transmembrane domain WO 2022/123062 PCT/EP2021/085315 has been removed by chemical, proteolytic or recombinant methods.
  • the functional equivalent is at least 80% homologous to the corresponding protein.
  • the functional equivalent is at least 90% homologous as assessed by any conventional analysis algorithm such as for example, the Pileup sequence analysis software (Program Manual for the Wisconsin Package, 1996).
  • polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art.
  • expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention.
  • the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.
  • polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy.
  • modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution.
  • the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.
  • adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.
  • a strategy for improving drug viability is the utilization of water- soluble polymers.
  • water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body.
  • water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.
  • Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity.
  • PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule.
  • copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.
  • the polypeptides of the invention may be fused to a heterologous polypeptide (i.e. polypeptide derived from an unrelated protein, for example, from an immunoglobulin protein).
  • the fusion protein is an immunoadhesin.
  • Immunoadhesins can possess many of the valuable chemical and biological properties of human antibodies. Since immunoadhesins can be constructed from a human protein sequence with a desired specificity linked to an appropriate human immunoglobulin hinge and constant domain (Fc) sequence, the binding specificity of interest can be achieved using entirely human components. Such immunoadhesins are minimally immunogenic to the patient, and are safe for chronic or repeated use.
  • the Fc region is a native sequence Fc region. In some embodiments, the Fc region is a variant Fc region. In some embodiments, the Fc region is a functional Fc region.
  • the FasR portion and the immunoglobulin sequence portion of the FasR immunoadhesin may be linked by a minimal linker.
  • the immunoglobulin sequence preferably, but not necessarily, is an immunoglobulin constant domain.
  • the immunoglobulin moiety in the chimeras of the present invention may be obtained from IgGl, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD or IgM, but preferably IgGl or IgG3.
  • the FasR antagonist is a FasR fusion protein is a fusion of the FasR polypeptide with human serum albumin-binding domain antibodies (AlbudAbs) according to the AlbudAbTM Technology Platform as described in Konterman et al. 2012 AlbudAbTM Technology Platform-Versatile Albumin Binding Domains for the Development of Therapeutics with Tunable Half-Lives
  • FasR fusion protein is a fusion of the FasR polypeptide with human serum albumin-binding domain antibodies (AlbudAbs) according to the AlbudAbTM Technology Platform as described in Konterman et al. 2012 AlbudAbTM Technology Platform-Versatile Albumin Binding Domains for the Development of Therapeutics with Tunable Half-Lives
  • the FasR is APG101 which is developed by Apogenix TM.
  • APG101 is a fully human fusion protein consisting of the extracellular domain of the FasR receptor and the Fc domain of an IgG antibody.
  • the FasR antagonist is an inhibitor of FasR expression (or FasL expression).
  • said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme.
  • anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of FasR or FasL mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of FasR or FasL, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding FasR or FasL can be synthesized, e.g., by conventional phosphodiester techniques.
  • Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; WO 2022/123062 PCT/EP2021/085315
  • Small inhibitory RNAs can also function as inhibitors of expression for use in the present invention.
  • FasR or FasL gene expression can be reduced by contacting a patient or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that FasR or FasL gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector” is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing FasR or FasL.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus
  • adenovirus adeno-associated virus
  • SV40-type viruses polyoma viruses
  • Epstein-Barr viruses Epstein-Barr viruses
  • papilloma viruses herpes virus
  • vaccinia virus
  • caspase inhibitors that can be used in the method provided herein have been reported in the literature. Certain exemplary caspase inhibitors for use in the methods are described by Linton in Current Topics in Medicinal Chemistry, (2005) 5: 1-20; and Linton et al. in J. Med. Chem., 2005, 11, 295-322 295, U.S. Pat. Nos.
  • the caspase inhibitor is selected from the group consisting of VX- 166, VX-799, LB 84318, LB 84451, MX-1013, IDN6556, Emricassan (IDN7314), IDN7568, VX-166 VX-765, belnacasan, Pralnacasan (VX-740), IDN-6556 and VX-765.
  • Typical examples of caspase inhibitors that are suitable for use with the invention also include WO 2022/123062 PCT/EP2021/085315
  • the caspase inhibitor is Q-VD-OPH or Q-VD(Ome)-OPH.
  • the agent of the present invention i.e. caspase inhibitor or FasR antagonist
  • pharmaceutically acceptable excipients such as biodegradable polymers
  • sustained- release matrices such as biodegradable polymers
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the active principle in the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • Galenic adaptations may be done for specific delivery in the small intestine or colon.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol ; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising the compound of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, WO 2022/123062 PCT/EP2021/085315 such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the compound of the invention can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifusoluble agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are WO 2022/123062 PCT/EP2021/085315 especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the compound of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.
  • other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations ; time release capsules ; and any other form currently used.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 Caspase activity of CD4 and CD8 T cells.
  • Figure 2 Apoptosis of CD4 and CD8 T cells.
  • D) Histograms show the means ⁇ SEM of CD4 and CD8 T cell subsets from 4 HD and 4 ICU patients. Statistical analyses were performed using a Mann Whitney test. *p ⁇ 0.05, and ****p ⁇ 0.0001.
  • Figure 3 Correlation between CXCL10 and caspase activity in COVID 19 patients.
  • AUCs of pROC were computed using Meval R package by adding genes, ranked by information gain using Biocomb R package, to a naive Bayes model computed with the R caret Package.
  • D and E Correlation between levels of CXCL10 and plasma sFasL and between caspase activity in CD4 T cells. Values of Spearman correlation are shown in the panels.
  • FIG. 4 Caspase-3 activation in T cells correlates with plasma sFasL and CD95 in COVID 19 patients.
  • a and B Caspase activity of CD4 and CD8 T cells was quantified by flow cytometry using fluorescent caspase-3 substrate. Percentages of CD4 (A) and CD8 T cells (B) expressing fluorescent caspase substrates are shown. Each dot represents one individual. Statistical analysis was performed using a Mann- Whitney U test. ***p ⁇ 0.001 and ****p ⁇ 0.0001).
  • D Correlation between the percentages of CD45RA- T cells expressing CD95 and caspase-3 activation in CD4 and CD8 T cells. Values of Spearman correlation are shown in the panels.
  • Figure 5 Inhibition of caspase prevents cell death.
  • A) Flow cytometry of CD4 and CD8 T cells expressing caspase activity in the absence (Med) or presence of Q-VD-OPH.
  • C Flow cytometry of CD4 and CD8 T cells expressing annexin V in the absence (Med) or presence of Q-VD-OPH.
  • D) Inhibition of apoptosis in the presence of IDN6556, VX-765, Q-VD and MCC950. The percentages were calculated as followed: (Med- Inh/Med)*100. Each dot represents one individual.
  • Statistical analyses were performed using a Mann- Whitney test. *p ⁇ 0.05, ***p ⁇ 0.001, and ****p ⁇ 0.000
  • FIG. 6 Higher levels of FasL and cell death.
  • Statistical analyses were performed using a Mann- Whitney test. ***p ⁇ 0.001.
  • FIG. 7 Caspase-3 inhibition enhances Thl mRNA expression.
  • A) Flow cytometry of CD4 and CD8 T cells expressing active caspase-3 (Caspase-3 mAb) in the absence (Med) or WO 2022/123062 PCT/EP2021/085315 presence of Q-VD.
  • Statistical analysis was performed using a paired Mann- Whitney U test (
  • Figure 8 Inhibition of caspase prevents FasL-mediated T cell death.
  • C and D Q-VD reduced CD4 and CD8 T cell death in the presence of rhFasL Histograms show the percentages of CD4 (C) and CD8 T cells (D) expressing annexin-V. Each dot represents one individual.
  • Statistical analysis was performed using a paired Mann- Whitney U test (**p ⁇ 0.01).
  • the trial was registered as Eudract/IDRCB 2020-A00875-34 and ClinicalTrials NCT04351711. Blood samples were collected at a single time point upon hospital entry and plasma supernatant obtained after blood centrifugation was frozen at -80°C. Cells were used for phenotyping and cell death quantification.
  • Blood cells (5x105 cells per ml) were cultured at 37°C for 12hrs in RPMI 1640 supplemented with 10% FCS (PAA Laboratories, Inc), penicillin/streptomycin (50 U/mL, Life technologies), glutamine (2 mM, Life technologies), sodium pyruvate (1 mM, Life technologies) and HEPES buffer (10 mM, Life technologies) at 37°C and 5% CO2.
  • FCS PAA Laboratories, Inc
  • penicillin/streptomycin 50 U/mL, Life technologies
  • glutamine (2 mM, Life technologies)
  • sodium pyruvate (1 mM, Life technologies
  • HEPES buffer 10 mM, Life technologies
  • T cells were activated with either CD3 mAb (0.5 pg/ml, Becton Dickinson) or Staphylococcal enterotoxin B (SEB) (1 pg/ml, Sigma) in the absence or presence of either Q-VD (10 pM, MedChemExpres) or recombinant human Fas-Fc (5 pg/ml, Enzolifesciences).
  • CD3 mAb 0.5 pg/ml, Becton Dickinson
  • SEB Staphylococcal enterotoxin B
  • Cell death was assessed by measuring caspase- 1 activity with FAM-FLIC A caspase- 1, caspase-3 activity with FAM-FLICA caspase-3 (Bio-Rad), active caspase-3 conjugated antibodies (R&D systems), and measuring phosphatidylserine (PS) exposure using labeled annexin V (Beckman Coulter Coultronics) according to the manufacturer’s instructions. Cells were stained with specific antibodies. Samples were analyzed by flow cytometry (Attune NxT, ThermoFisher) and using FlowJo software (Tree Star, Inc.).
  • cells were incubated in the absence or presence of several inhibitors, including IDN-6556 (10 pM, MedChemExpress), Q-VD-OPH (10 pM, MedChemExpress), VX-765 (10 pM, MedChemExpress), a NLPR3 inhibitor, MCC950 (1 pM, Merck) as well as necroptosis inhibitors GSK-872 and Dafrafenib (2 pM, MedChemExpress). The efficacy of these compounds was tested either in primary human cells or THP1 cell lines.
  • inhibitors including IDN-6556 (10 pM, MedChemExpress), Q-VD-OPH (10 pM, MedChemExpress), VX-765 (10 pM, MedChemExpress), a NLPR3 inhibitor, MCC950 (1 pM, Merck) as well as necroptosis inhibitors GSK-872 and Dafrafenib (2 pM, MedChemExpress).
  • peripheral blood mononuclear cells PBMC, 106 cells per ml
  • PBMC peripheral blood mononuclear cells
  • ELISA was used to quantify IL-ip (R&D Systems) in the supernatants of stimulated PBMC.
  • THP1 monocytic cells were primed with LPS (1 pg/lml) for 6 hrs and then treated with ATP for 2 hrs (40 mM, Sigma).
  • THP1 cells were also cultured with actinomycin D (ActD, Sigma) a conventional pro-apoptotic stimulus.
  • Jurkat cells were incubated in the presence of recombinant human FasL (Enzolifesciences). PS exposure was measured by flow cytometry.
  • ELISA enzyme-linked immunosorbent assay
  • CD4 and CD8 T cells were purified using anti-CD4 and anti-CD8 microbeads (Milenyibiotec) and lysed with TRIzol reagent (Thermo Fischer scientific).
  • the mRNAs were extracted using a kit from Qiagen.
  • RT-PCR was performed with 150 ng of RNA by a SensiFAST cDNA synthesis kit. Gene expression was assessed by qPCR using SensiFAST SYBR Hi-ROX kit (Bioline) in 10 pL reactions with 2.5 ng of cDNA.
  • Thermocycling settings consisted of one hold for 15 min at 95°C followed by a two-step temperature (95°C for 15s and 60°C for 30s) over 40 cycles in CFX384 Touch Real-Time PCR Detection System (Bio-Rad).
  • CD4 and CD8 T cells from COVID-19 patients were more prone to express caspase activity than CD4 and CD8 T cells of healthy donors.
  • the percentages of CD4 and CD8 T cells expressing caspase activity were higher in ICU than in non-ICU patients, in which half of them displayed higher percentages caspase activity compared to T cells of healthy donors ( Figures IB and 1C).
  • T cells can be subdivided into four populations on the basis of their relative surface expression of CD27 and CD45RA molecules 12 . Undifferentiated populations have been shown to express CD27 + CD45RA + (naive), those that are at an early stage of differentiation are CD27 + CD45RA‘(CM), whereas more differentiated T cells are CD27"CD45RA” (EM) ( Figure 2B).
  • COVID-19 is characterized by inflammation, in which the expression of CXCL10 correlates with disease severity 37 ’ 40 .
  • Plasma levels of CXCL10, IL-IRa, hepatocyte growth factor (HGF), and soluble CD 14 (sCD14,) are biomarkers that discriminate ICU from HDs ( Figures 3A-3C).
  • the AUC (Area Under the ROC Curve) was 0.98 based on these 4 biomarkers and associated with disease severity ( Figure 3B).
  • sCD14 considered as a biomarker of disease severity and comorbidities in HIV-infected individuals 42 when it increases, actually decreased in COVID-19 patients (ICU, 122 ⁇ 33 pg/ml versus HDs, 723 ⁇ 175 pg/ml, p ⁇ 0.0001; Figure 3A).
  • HGF concentration was also higher in ICU than in HDs (1516 ⁇ 626 pg/ml versus 381 ⁇ 67 pg/ml, pO.OOOl, Figure 3A). This could be of importance as it has been previously reported that stimulation of the hypoxia inducible factor- 1 by HGF was associated with pulmonary arterial hypertension 43 .
  • PCA Principal Component Analysis
  • Figure 3C the heat map
  • caspase inhibitor may have a preventive effect on T cells from HIV-infected individuals 13 ' 15 .
  • inhibitor of caspases may also prevent T cell death in the context of SARS-CoV-2 infection.
  • VX-765 Belnacasan, specific of caspase-1 and -4
  • IDN-6556 Emricasan, a caspase inhibitor
  • Q-VD-OPH a pan-caspase inhibitor that we recently demonstrated to prevents Aids
  • MCC950 an inhibitor of NLRP3 inflammasome complex WO 2022/123062 PCT/EP2021/085315 in which caspase- 1 is activated.
  • FasL may contribute in the death of immune cells during viral infection 13 18 ' 20 .
  • FasL is a confounding biomarker associated with T cell death in COVID-19 individuals.
  • T cells from COVID-19 patients are more prone to die and that SARS-CoV-2 infection is associated with increased levels of FasL.
  • Q-VD-OPH prevents the death of CD4 and CD8 T cells.
  • Q-VD represents an attractive molecule for COVID-19 patients.
  • Q-VD blocks active caspase-3 in CD4 (60 ⁇ 4% of decrease) and CD8 T cells (47.9 ⁇ 3.5% of decrease) ( Figures 7A and 7B).
  • Q-VD prevented the death of EM CD4 T cells, which are essential for cell-mediated immunity, and of memory CD8 T cells subsets, which are strongest in expressing cytotoxic effector molecules 28, 29 (Data not shown . Having observed that COVID-19 individuals displayed lower levels of IFN-y, we assessed whether Q-VD restores functional T cells.
  • RIPK3 inhibitors including GSK-872 and dafrafenib.
  • RIPK3 inhibitors did not prevent spontaneous T cell death in the absence of Q-VD, they had no synergetic effect with Q-VD suggesting that necroptosis had a minor role in the occurrence of T cell death (Data not shown ⁇ . This observation may be consistent with the absence of increased expression of necroptotic genes in T cells (Data not shown ⁇ .
  • Q-VD may represent an attractive molecule for COVID-19 patients by preventing the apoptosis of both CD4 and CD8 T cells and increasing Thl profiles.
  • CD4 and CD8 T cells from COVID-19 patients are more prone to undergo death and that blocking caspase activation prevents T cell death.
  • higher levels of soluble FasL in the plasma of COVID-19 individuals is associated with higher levels of T cell death.
  • the presence of higher levels of soluble caspase- 1 in the plasma and of IL- 18 also indicated an inflammasome activation pathway, which can be link with several endothelial biomarkers of cell damage. Therefore, strategy aims to block caspase and/or FasL could be beneficial for restoring full immune competent immune cells and preventing fatal outcome in COVID-19 patients.
  • FasL plays a major role in a model of acute immune complex alveolitis, and of bleomycin-induced pulmonary fibrosis. Fas activation causes lung injury by inducing alveolar epithelial cell apoptosis and inducing local inflammatory responses, associated with disruption of the paracellular tight junction proteins 41 .
  • Fas and FasL are upregulated and associated WO 2022/123062 PCT/EP2021/085315 with apoptosis of bronchiolar and alveolar epithelial cells. Fas/FasL interaction was also proposed to be implicated in the human cystic fibrosis (CF) airway apoptotic pathway, hypersensitivity pneumonitis (HP), in acute lung injury (ALI) and in the acute respiratory distress syndrome (ARDS). Furthermore, there is also evidence that soluble FasL can be capable of inducing apoptosis of cells of the distal pulmonary epithelium during acute lung injury.
  • CF cystic fibrosis
  • HP hypersensitivity pneumonitis
  • ALI acute lung injury
  • ARDS acute respiratory distress syndrome
  • FasL in addition to the lungs, epithelial cells from kidney, as renal tubule epithelial cell injury or kidney allograft injury have been also described to be sensitive to FasL-mediated cell death. Furthermore, in the context of retinal degenerative diseases, such as age-related macular degeneration, FasL has been proposed. Our observation of circulating FasL in patients may provide a support of several symptoms reported to occur during COVID-19. In addition to its role as a death ligand, engagement of Fas has been reported to activate NF-kB and may contribute in the release of inflammatory interleukins such as IL-6 and IL-8 enhancing neutrophilic infiltrates.
  • Caspases which are the main effectors of programmed cell death, have been also demonstrated to be essential for the processing of IL-18.
  • higher levels of IL-18 are generally associated with tissue injury, and previously reported to be elevated at early days post fever onset, and higher in the non-survival SARS patients.
  • a multiprotein complex termed inflammasome, is formed after activation leading to caspase- 1 activation and release.
  • caspase activation may contribute in a rapid lytic cell death known as pyroptosis that contributes in the release of inflammatory cytokines in mice. This could be consistent with increased levels of active caspase- 1 observed in COVID-19 individuals.
  • Q-VD-OPH may offer additional interest for COVID-19 patients.
  • MERS coronavirus induces apoptosis in kidney and lung by upregulating Smad7 and FGF2.
  • TGF-beta in intestinal lymphoid organs contributes to the death of armed effector CD8 T cells and is associated with the absence of virus containment in rhesus macaques infected with the simian immunodeficiency virus.
  • T helper type 1/T helper type 2 cytokines and T cell death preventive effect of interleukin 12 on activation- induced and CD95 (FAS/APO-l)-mediated apoptosis of CD4+ T cells from human immunodeficiency virus-infected persons. J Exp Med, 182 (6), 1759-1767 (1995).

Abstract

Les inventeurs ont mis en évidence que les lymphocytes T CD4 et CD8 de patients atteints de COVID-19 étaient plus susceptibles de mourir et que le blocage de l'activation de la caspase empêchait la mort des lymphocytes T. En outre, il a été découvert que des niveaux supérieurs de FasL soluble dans le plasma d'individus atteints de la COVID-19 étaient associés à des niveaux supérieurs de mort des lymphocytes T. La présence de niveaux supérieurs de CXCL10 et d'IL-18 indique également une voie d'activation de l'inflammasome, qui peut être liée à plusieurs biomarqueurs endothéliaux de lésion cellulaire et associée à une immunité Th1 plus faible. Le blocage de FasL ou l'activation de la caspase augmente les ARNm de la cytokine Th1. Par conséquent, la stratégie qui vise à bloquer la caspase et/ou la FasL pourrait être bénéfique pour restaurer des cellules immunitaires immunocompétentes complètes et prévenir une issue fatale chez des patients atteints de la COVID-19.
PCT/EP2021/085315 2020-12-11 2021-12-10 Blocage de la caspase et/ou de la fasl pour prévenir une issue fatale chez des patients atteints de la covid-19 WO2022123062A1 (fr)

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