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WO2025202222A1 - Utilisation thérapeutique d'agonistes de sting et de tlrs pour induire l'expression de p16 dans des cellules immunitaires - Google Patents

Utilisation thérapeutique d'agonistes de sting et de tlrs pour induire l'expression de p16 dans des cellules immunitaires

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Publication number
WO2025202222A1
WO2025202222A1 PCT/EP2025/058170 EP2025058170W WO2025202222A1 WO 2025202222 A1 WO2025202222 A1 WO 2025202222A1 EP 2025058170 W EP2025058170 W EP 2025058170W WO 2025202222 A1 WO2025202222 A1 WO 2025202222A1
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Prior art keywords
cells
immune cells
agonist
mice
bnt162b2
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Inventor
Barbara Polski
Dmitry Bulavin
Francisco TRIANA-MARTINEZ
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Nice Sophia Antipolis UNSA
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Nice Sophia Antipolis UNSA
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Publication of WO2025202222A1 publication Critical patent/WO2025202222A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • 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

Definitions

  • p16 high immune cells surprisingly play a key role in establishing disease tolerance, and can be useful for counteracting different lethal conditions, including LPS-induced sepsis, acute lethal SARS-CoV-2 infection, cancer and ionizing irradiation.
  • activation of the TLR7 and/or TLR5 and/or STING pathways induces an increase in p16 high immune cells subsets that, in turn, establishes a low adenosine environment and disease tolerance. Maintaining a beneficial level of p16 high immune cells by activating these pathways is shown here for the first time to advantageously delay organ deterioration upon aging and extend healthspan.
  • tolerance mechanisms are largely unknown, they are expected to prevent, to reduce, or counter the pathological alterations caused by infections or other inducers of tissue damages including autoimmune diseases, stress or aging (Medzhitov, R., Schneider, D.S., and Soares, M.P. (2012). Science 335, 936-941).
  • senescence cells accumulate in adipose tissue of patients with diabetes and age-related metabolic dysfunction, in osteoarthritic joints, in the aorta in vascular hyporeactivity and atherosclerosis and in the lungs in idiopathic pulmonary fibrosis.
  • SASPs senescence-associated secretory phenotypes
  • cytokines pro-inflammatory cytokines
  • proteases proteases
  • growth factors are upregulated and secreted by senescent cells and are part of the extrinsic arm of cellular senescence.
  • selective elimination of p16 + senescent cells was proposed to confer benefits in aging tissues and to extend lifespan (Baker DJ.
  • the present invention is based on the surprising realization that a number of different Pattern-Recognition Receptors (PRR) agonists can activate p16 expression in immune cells, and that p16 high immune cells play a key role in establishing an early and broad disease tolerance in response to multiple lethal conditions following severe inflammation and tissue damage, such as sepsis, SARS-CoV-2 infection and ionizing radiations.
  • PRR Pattern-Recognition Receptors
  • PRR agonists leading to p16 increased expression in immune cells are able to play a key role in broad and early disease tolerance in response to lethal conditions following severe inflammation and tissue damage due to sepsis (example 5, example 15, example 18, example 19), radiations (example 7), viral infections (examples 7 and 12), cancer (examples 14 and 15), thereby promoting disease tolerance and extending health span (example 11), even after cell transplantation (example 16).
  • Pattern-recognition receptors agonists of the invention are provided.
  • Pattern recognition receptors are a class of receptors that can directly recognize the specific molecular structures on the surface of pathogens, apoptotic host cells, and damaged senescent cells. PRRs bridge nonspecific immunity and specific immunity. Through the recognition and binding of ligands, PRRs can produce nonspecific anti-infection, antitumor, and other immunoprotective effects.
  • PRRs in the innate immune system of vertebrates can be classified into the following five types based on protein domain homology: Toll-like receptors (TLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene-l (RIG-l)-like receptors (RLRs), C-type lectin receptors (CLRs), and absent in melanoma-2 (AIM2)-like receptors (ALRs).
  • TLRs Toll-like receptors
  • NOD nucleotide oligomerization domain
  • RLRs retinoic acid-inducible gene-l
  • CLRs C-type lectin receptors
  • AIM2 melanoma-2
  • Nucleic acid sensors which can detect extracellular or intracellular DNA or RNA as damage-associated molecular pattern signals, are the essential part of the PRRs as they can induce type I interferons (IFNs).
  • IFNs type I interferons
  • the secreted Type I IFNs will act on producing and neighboring cells via IFNa/preceptor 1 or IFNAR1-IFNAR2 heterodimer and support cytotoxic T lymphocytes (CTLs) via several mechanisms.
  • CTLs cytotoxic T lymphocytes
  • TLR5 is a transmembrane receptor expressed by keratinocytes and immune cells such as T cells, macrophages and monocytes. It is an extracellular sensor activated mostly by bacterial flagellin whose interaction activates NF-KB signaling and triggers an innate immune response to the invading pathogen.
  • the present invention relates to a new use of the toll-like receptor 5 (TLR5) agonist.
  • TLR5 agonist designates a compound that binds to the human TLR5 receptor and activates the signaling pathway through this receptor specifically.
  • the TLR5 signaling cascade is commonly triggered by the binding of bacterial flagellum to TLR5 on the cell surface. Binding of flagellum induces the dimerization of TLR5, which in turn recruits MyD88 and Mal/TIRAP. The recruitment of MyD88 leads to subsequent activation of IRAK4, IRAKI , TRAF6, and eventually IKB kinases. Activation of IKB kinases contributes to the nuclear localization of NF-KB. NF-KB induces many downstream gene expressions, which initiates the canonical proinflammatory pathway. This TLR5/flagellum interaction results in different responses in difference cell types. In epithelial cells, binding of flagellum to TLR5 induces IL8 production. In human monocytes and dendritic cells, this interaction results in the secretion of proinflammatory cytokines such as TNF.
  • the agonist effect of a compound toward TLR5 can be measured by any conventional means, e.g., by detecting the activation of NF-KB and IRAKs and IKB kinases and/or the expression of pro-inflammatory cytokines IL8 or TNF, depending on the cells which are used for the test.
  • the TLR5 agonist used in the invention is capable of increasing the number of p16 high macrophages, p16 high T cells, p16 high B cells, p16 high neutrophils, p16 high monocytes and/or p16 high dendritic cells, more preferably of p16 high T cells and/or p16 high macrophages, in patients to which it is administered.
  • TLR5 agonists have been already described, all of them being herewith encompassed. As mentioned previously, the most well-known TLR5 agonist is flagellin.
  • Alternative TLR5 agonist small molecules can however be used in the context of the invention, such as : Caveolin-1 (Lim JS., et al, Molecules and Cells. 38(12): 1 111 -7), CBLB502 (entolimod) and MAP1 S (the references of the studies describing these molecules are given in Table 1 of Chi H et al, 2017), or Heat Killed Salmonella typhimurium (HKST).
  • the TLR5 agonist used in the invention is flagellin, because it has been shown to trigger the expression of p16 in immune cells (figure 2G).
  • flagellin refers to a protein that makes up the filament of bacterial flagella, which are whip-like appendages that protrude from the surface of certain bacteria. These flagella are used by bacteria for locomotion, allowing them to move toward or away from stimuli in their environment. Flagellin is a crucial component of the bacterial flagellum structure.
  • the TLR5 agonist can also be a flagellin derivative like the CBLB502 peptide (Bai H. et al, 2019, Biol. Reprod. 100(1):281-291).
  • Stimulator of interferon genes also known as “transmembrane protein 173” (TMEM173) and “MPYS”, “MITA”, “ERIS”, “NET23”, “SAVI”, “STING1”, “hMITA”, “hSTING”, “Stimulator of interferon genes”, “STING-beta”, or “stimulator of interferon response cGAMP interactor 1” is a transmembrane protein that in humans is encoded by the STING1 gene, which plays an important role in innate immunity. STING works as both a direct cytosolic DNA sensor (CDS) and an adaptor protein in Type I interferon signaling through different molecular mechanisms.
  • CDS direct cytosolic DNA sensor
  • STING-activated TBK1 is able to phosphorylate IRF3, promoting IRF3 dimerization and translocation to the nucleus where it induces the transcription of many inflammation genes especially IFN
  • STING-activated IKK could phosphorylate IKBO, which leads to the translocation of NF-KB to the nucleus, and then activates the transcription of pro- inflammatory cytokines (Chen et al., 2020).
  • the present invention relates to a new use of a STING agonist.
  • the present inventors found for the first time that inhibition of the STING pathway impairs the increase of p16 high immune cells in animals treated with TLR7 agonist (example 8). They also show that direct activation of STING with the specific agonist 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is sufficient to increase the number of p16 Hi0h immune subsets including within Tregs as well as PD1- and PD-L1-positive T cells in different tissues ( Figure 4E) and to protect mice from LPS-induced sepsis (figure 4G), when administered at low doses in animals.
  • DMXAA 5,6-dimethylxanthenone-4-acetic acid
  • STING agonist designates a compound that binds to the human STING receptor and activates the signaling pathway through this receptor.
  • the interaction of the agonist of the invention with other receptors is weaker as compared to its interaction with human STING.
  • Amino acid sequences of human STING are known (see, for example. GenBank Accession Nos. NP_001288667; NP_938023; NP_001354187).
  • the agonist effect of a compound toward STING can be measured by any conventional means, e.g., by detecting the phosphorylation of IRF3, and/or its translocation to the nucleus, or the expression of I NFp, or the translocation of NF-KB to the nucleus or the expression of pro-inflammatory ctytokines in immune cells contacted with the compound. It is also possible to detect with adequate antibodies if the Ser366 of the STING protein is phosphorylated.
  • the STING agonist used in the invention is capable of increasing the number of p16 high macrophages, p16 high T cells, p16 high B cells, p16 high neutrophils, p16 high monocytes and/or p16 high dendritic cells, more preferably of p16 high T cells and/or p16 high macrophages, in patients to which it is administered.
  • STING agonists have been already described, all of them being herewith encompassed.
  • the second messenger 2’3’-cGAMP, and a number of synthetic or natural double-stranded DNAs (dsDNAs) can be used in this respect.
  • the STING agonist used in the invention is 5,6-dimethylxanthenone-4-acetic acid (DMXAA), because it has been shown to trigger the expression of p16 in immune cells (figure 4).
  • the STING agonist is preferably administered subcutaneously, intramuscularly, intranasally or intravenously.
  • DMXAA 5,6-dimethylxanthenone-4-acetic acid
  • p16 has its general meaning in the art and refers to the “p16INK4a” or “p16INK4” or “multiple tumor suppressor-1 (MTS-1)’’ or “cyclin-dependent kinase inhibitor 2a (CDKN2A)” protein, which is a 16 kDa protein encoded by the CDKN2A gene, within the INK4/ARF tumor suppressor locus on Chromosome 9 (9p21.3).
  • This protein plays a crucial role in the regulation of the cell cycle by inhibiting cyclin-dependent kinases (CDK4 and CDK6 and CDK2) that are required to phosphorylate the retinoblastoma protein (Rb).
  • CDK4 and CDK6 and CDK2 cyclin-dependent kinases
  • Rb retinoblastoma protein
  • the term “p16 high ” or “p16 + ” has its general meaning in the art and refers to “high” or elevated level or activity of the p16 protein as defined above, within the cells of interest for the present invention (namely, immune cells).
  • the term “p16 low ” or “p16'“ has its general meaning in the art and refers to low or reduced level or activity of the p16 protein as defined above, within the cells of interest for the present invention (namely, immune cells).
  • the designation “p16 high cells” or “p16 + cells” is used to describe immune cells exhibiting a high level or abundance or activity of the p16 protein as defined above.
  • the designation “p16 low cells” or “p16 _ cells” is used to describe immune cells exhibiting a low level or abundance or activity of the p16 protein as defined above.
  • level refers to the “expression level” of the p16 protein in the target cells of the invention. Specifically, it refers to the quantity, amount or concentration of the p16 protein expressed in the studied cells.
  • the measurement of the level of p16 in cells can be carried out using standard protocols known in the art. For example, it can be performed by contacting the cells with a compound capable of selectively interacting with the p16 protein, for example with antibodies, such as, for example, monoclonal antibodies or even aptamers.
  • the interaction may be detected by using a competitive immunoassay, a non-competitive assay system using techniques such as western blots, a radioimmunoassay, an ELISA (enzyme linked immunosorbent assay), a “sandwich” immunoassay, an immunoprecipitation assay, a precipitin reaction, a gel diffusion precipitin reaction, an immunodiffusion assay, an agglutination assay, a complement fixation assay, an immunoradiometric assay, a fluorescent immunoassay, a protein A immunoassay, an immunoprecipitation assay, an immunohistochemical assay, a competition or sandwich ELISA, a radioimmunoassay, a Western blot assay, an immunohistological assay, an immunocytochemical assay, a dot blot assay, a fluorescence polarization assay, a scintillation proximity assay, a homogeneous time resolved fluorescence assay
  • the aforementioned assays generally involve the binding of the interacting compound (e.g., antibody or aptamer) to a solid support.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • Measuring the level of p16 may also include centrifugation based on the p16 molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on p16’s affinity for the particular solid-phase that is used.
  • p16 may be detected and measured by, for example, a mass spectrometer.
  • “High” or “low” level of p16 is typically identified by comparing the p16-associated signal obtained in a defined amount of the studied cells with a pre-determined reference value.
  • This pre-determined reference value is preferably obtained by measuring the p16-associated signal generated by the same amount of cells that are known to be expressing high levels of p16.
  • a “reference value” is obtained from several subjects known to be free of the disease or, alternatively, from the general population. Yet, it can also be adjusted to specific subject populations in the context of the invention, e.g., to inflammatory patients or to young / aged subjects, in which p16 expression is known to be either high or low.
  • the reference value or reference level can be an absolute value; a relative value; a value that has an upper or a lower limit; a range of values; an average value; a median value, a mean value, or a value as compared to a particular control or baseline value.
  • a reference value can be based on an individual sample value such as, for example, a value obtained from a sample from the subject being tested, but at an earlier point in time.
  • the reference value can be based on a large number of samples, such as from population of subjects of the chronological age matched group, or based on a pool of samples including or excluding the sample to be tested.
  • p16 hi0h immune cells are induced with age and in patients suffering from an inflammatory condition (see Safwan-Zaiter et al., 2022, Life, 12, 1332, and also example 1).
  • “reference samples” containing p16 hi0h cells are biological samples containing immune cells which have been obtained in patients older than 65, more preferably older than 68 or 70.
  • Other “reference samples” can be for example collected from healthy persons at least 7 days after their vaccination with the BNT162b2 COVID-19 vaccine ( Figure 7D).
  • the reference value used in the context of the invention can be the mean value of p16 expression measured in the CD45 + immune cells contained in these reference samples.
  • the immune cells of the patient would be qualified as “pi6 high ”. If the p16 expression level in the immune cells of the patient is lower than this reference value, then the immune cells of the patient would be qualified as “pi6 low ”. It is possible to determine the p16 expression level in target cells in reference to the expression of housekeeping genes (e.g., beta-actin) these expression levels being for example measured by western blot and immunofluorescence.
  • housekeeping genes e.g., beta-actin
  • Assessing p16 activity in a tested sample can be performed by any conventional means, for example by measuring the activity of the cyclin-dependent kinases (CDK4 and CDK6 and CDK2) and/or the phosphorylation state of the retinoblastoma protein (Rb).
  • CDK4 and CDK6 and CDK2 the cyclin-dependent kinases
  • Rb the retinoblastoma protein
  • the activity of p16 in a tested sample containing a define amount of immune cells can be compared to the activity of p16 in a reference sample containing the same amount of immune cells.
  • This reference sample is, for example, a biological sample containing immune cells which have been collected in patients older than 65, more preferably older than 68 or 70, or a sample of immune cells collected from healthy persons at least 7 days after they have been vaccinated with the BNT162b2 COVID-19 vaccine (Figure 7D). If the activity of p16 in the immune cells of the patient is the same or higher than in the reference sample, then the immune cells of the patient would be qualified as “pi6 high ”. If the activity of p16 in the immune cells of the patient is lower than in the reference sample, then the immune cells of the patient would be qualified as “p16 low ”.
  • p16 high patients or “p16 + patients” is herein used to describe patients whose biological samples contain a high percentage of p16 high cells as defined above.
  • high percentage it is herein meant that the immune cells contained in the biological sample of the patient are predominantly p16 high . Specifically, this means that more than 50%, 60%, 70%, 80%, or 90% of the immune cells contained in the biological sample of p16 high patients are p16 high as defined above. Conversely, this means that less than 50%, 40%, 30%, 20%, or 10% of the immune cells contained in the biological sample of p16 high patients are p16 low as defined above.
  • p16 low patients or “p16 _ patients” is used to describe patients whose biological samples contain a low percentage of these p16 high cells.
  • low percentage it is herein meant that few of the immune cells contained in the biological sample of the patient are p16 high . Specifically, this means that less than 50%, 40%, 30%, 20%, or 10% of the immune cells contained in the biological sample of p16 low patients are p16 high as defined above. Conversely, this means that more than 50%, 60%, 70%, 80%, or 90% of the immune cells contained in the biological sample of p16 low patients are p16 low as defined above.
  • An exemplary biochemical test for identifying the number of p16 high immune cells contained in a sample employs a standardized test format, such as ELISA test wherein the wells of a microtiter plate are coated with a set of antibodies which recognize immune cells. A sample containing p16 high immune cells (or not) is then added to the coated wells. After a period of incubation sufficient to allow the binding of the immune cells on the plate, the plate is washed to remove unbound cells. The lysis or permeabilization of the cells can be performed so as to permit the contact between a detectably labelled p16 binding molecule and intracellular p16. The plate is washed and the presence of the p16 binding molecule is detected using methods well known in the art.
  • immune cells refers to natural killer (NK) cells, lymphocytes (e g., natural killer T (NKT) lymphocytes, T lymphocytes (also called T cells), and B lymphocytes (also called B cells)); phagocytes (e.g., macrophages, monocytes, and dendritic cells); granulocytes (e.g. basophils, eosinophils and neutrophils); mast cells, plasma cells, and memory cells.
  • NK natural killer T
  • T lymphocytes also called T cells
  • B lymphocytes also called B cells
  • phagocytes e.g., macrophages, monocytes, and dendritic cells
  • granulocytes e.g. basophils, eosinophils and neutrophils
  • mast cells plasma cells, and memory cells.
  • the p16 high immune cells of the invention are p16 high NK cells, p16 high macrophages, p16 high T cells, p16 high B cells, p16 high neutrophils, p16 high monocytes or p16 high dendritic cells.
  • Immune cells can be detected with conventional surface markers such as CD3, CD14, CD33, CD19, CD20, CD66b, B220, CD11c, Ly6C, Ly6G, F4/80, CD45, CD4, CD8, CD25, PD1 or PD-L1.
  • they are detected, isolated and/or sorted by flow cytometry.
  • the expression level and activity of p16 in immune cells can be increased by treating same with any of the pattern-recognition receptor agonists described above.
  • NNMT activation is a critical mechanism of p16 induction either directly or indirectly.
  • the present inventors show here for the first time that the PRR agonists of the invention require a functional NNMT enzyme to favor the expression of p16 in immune cells, and its further beneficial effects. Therefore, to perform the various methods of the invention, it is important to ensure that the expression and/or activity of the NNMT (nicotinamide N-methyltransferase) enzyme is normal in the immune cells in which p16 should be overexpressed.
  • NNMT neurotinamide N-methyltransferase
  • Nicotinamide N-methyltransferase (NNMT, NCBI Ref Seq is NP_006160) is an enzyme that in humans is encoded by the NNMT gene (gene ID: 4837).
  • NNMT catalyzes the methylation of nicotinamide and similar compounds using the methyl donor S-adenosyl methionine (SAM-e) to produce S-adenosyl-L-homocysteine (SAH) and 1 -methylnicotinamide.
  • SAM-e methyl donor S-adenosyl methionine
  • SAH S-adenosyl-L-homocysteine
  • the present invention relates to an in vitro method for selecting patients that will benefit from a treatment involving a TLR7 agonist, a TLR5 agonist, ora STING agonist, said method comprising the steps of : a) Detecting the expression and/or activity of the nicotinamide N- methyltransferase (NNMT) enzyme in immune cells present in a biological sample of a patient, b) Concluding that said patient will benefit from a treatment involving a TLR7 agonist, a TLR5 agonist, or a STING agonist if the NNMT enzyme is expressed and functional in said immune cells, c) Optionally, administering to said patient a treatment involving a TLR7 agonist, a TLR5 agonist, or a STING agonist, if the NNMT enzyme is expressed and functional in the immune cells present in the tested sample.
  • NNMT nicotinamide N- methyltransferase
  • composition of the invention contains, as active principle, the PRR agonist described herein, and optionally a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipient means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • Compositions according to the invention are usually be administered by parenteral, topical, intravenous, intratumoral, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means. A typical route of administration is intravenous or intratumoral, although other routes can be equally effective.
  • 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.
  • Sterile injectable solutions are prepared by incorporating the active ingredient at the required amount in the appropriate solvent with several 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.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the composition of the invention will be under liquid form. It will thus contain, apart from the PRR agonist, a pharmaceutically- acceptable diluent that does not affect the biological activity of the cells of the invention.
  • a pharmaceutically- acceptable diluent that does not affect the biological activity of the cells of the invention.
  • diluents are physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • the composition of the invention is under a liquid form.
  • PRRs pattern-recognition receptors
  • the pharmaceutical composition of the invention contains, apart from the optional pharmaceutically acceptable excipient, a combination of two of the pattern-recognition receptors (PRRs) agonists disclosed above, for example:
  • the present invention concerns a composition
  • a composition comprising, apart from the optional pharmaceutically acceptable excipient, a STING agonist as disclosed above and a TLR7 agonist as disclosed above.
  • this composition can for example contain a low dose of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and an effective amount of the BNT162b2 vaccine, which have been successfully tested in the examples below.
  • the composition of the invention may comprise a low dose of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and either the R837 or the R848 compound, as also tested in the examples below.
  • composition may also advantageously comprise an anti-CD3 antibody, which is shown in the examples 17-18 below to maintain the viability of T cells.
  • Inhibitory immune checkpoints include PD-1 , SIRPa, CD47, PD-L2, LAG3, Tim3, BTLA, and CTLA4, and immune checkpoint inhibitors according to the invention are therefore preferably selected from antagonist antibodies :
  • PD-1 e.g. those described in WO2004/004771; W02004/056875; W02006/121168; WO2008/156712; W02009/014708; W02009/114335; WO2013/043569; and W02014/047350, in particular nivolumab, pembrolizumab and cemiplimab),
  • SIRPa e.g. WO2019/023347
  • CD47 e.g. W02020/019135
  • Tim3 e.g. W02020/093023
  • BTLA e.g. W02010/106051
  • CTLA4 e.g. those described in US 8,491 ,895, W02000/037504, W02007/113648, WO2012/122444 and WO2016/196237 among others, and in particular ipilimumab marketed by Bristol Myer Squibb as Yervoy® (see e.g. US 6,984,720; US 8,017,114), MK-1308 (Merck), AGEN-1884 (Agenus Inc.; WO20 16/196237) and tremelimumab (AstraZeneca; US 7,109,003 and US 8,143,379) and single chain anti-CTLA4 antibodies (see e.g. WO97/20574 and WG2007/123737).
  • Immune checkpoint inhibitors are however not always requested. See e.g., examples 13 and 14 showing that for treating KRAS driven lung tumors or lung implanted melanoma cells, a TLR7 agonist alone is sufficient to reduce tumor size and improve mice survival.
  • a “low dose” of 5,6-dimethylxanthenone-4-acetic acid is for example a dose of DMXAA that can enhance the expression of p16 in immune cells without inducing any inflammatory side effects on its own, when injected in the patient.
  • DMXAA 5,6-dimethylxanthenone-4-acetic acid
  • Exemplary appropriate doses to start with are comprised between 0.1 and 10 mg/kg. Specifically, this dose would induce the secretion of IFNy but not of TNFa in the contacted immune cells.
  • low doses of DMXAA are for example comprised between 6 and 400 mg/m 2 .
  • an “effective amount” of the BNT162b2 vaccine is for example a standard vaccination dose of 30pg.
  • the pharmaceutical composition of the invention contains, apart from the optional pharmaceutically acceptable excipient, a combination of the three pattern-recognition receptors (PRRs) agonists disclosed above. Accordingly, it may contain an agonist of STING as disclosed above, an agonist of TLR7 as disclosed above, and an agonist of TLR5 as disclosed above.
  • the composition of the invention can for example contain a low dose of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and an effective amount of the BNT162b2 vaccine, or of the R837 / R848 compound, or of an effective amount of flagellin, which have been successfully tested in the examples below. Specifically, these doses would induce the secretion of IFNy but not of TNFa in the contacted immune cells.
  • DMXAA 5,6-dimethylxanthenone-4-acetic acid
  • R848 in human can be of 250 ng/mL as proposed in the art in human studies. In an injectable solution, it can be at 1.0 mg/mL as proposed in the AGADIR study combining resiquimod sulfate with atezolizumab (an immune checkpoint inhibitor) or in other clinical trials in combination with pembrolizumab. When used topically, R848 can be used between 0.01% and 0.05% in a cream (see NCT01583816).
  • the “patient in need thereof” is not necessarily suffering from a disease and might display no symptom of an illness. He / she may be an elderly person in non-pathological state or in good health. He/she may however suffer from (or likely to develop) symptoms of frailty due to aging.
  • the pharmaceutical composition of the invention is for use in preventing and/or treating tissue inflammation and/or tissue damage.
  • the term “pathogen infection” designates any infection from any pathogen, in particular from any bacteria, virus, parasite or fungi that is likely to induce inflammatory adverse effects.
  • it can be a viral infection due to an inflammatory virus chosen in the group consisting of: the influenza virus, the rabies virus, the Ebola virus, the smallpox virus, the Marburg virus, the Nipah virus, the hantavirus, the anthrax virus, the Human Immunodeficiency Virus (HIV) virus, the Epstein-Barr virus, the Zika virus, the Hepatitis A, B and C virus, and the SARS-COV-2 virus, that can trigger rapid and strong inflammation in the infected patient.
  • HIV Human Immunodeficiency Virus
  • it can be a bacterial infection due to an inflammatory bacteria chosen in the group consisting of: Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bacteroides fragilis, Bordetella pertussis, Borrelia sp. (burgdorferi, garinii, afzelii, recurrentis, crocidurae, duttonii, hermsii etc), Brucella sp. (abortus, canis, melitensis, suis), Campylobacter jejuni, Chlamydia sp.
  • an inflammatory bacteria chosen in the group consisting of: Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bacteroides fragilis, Bordetella pertussis, Borrelia sp. (burgdorferi, garinii, afzelii, recurrentis, crocidurae, duttonii, hermsi
  • the term “cancer” designates any kind of cancer, preferably those that can induce inflammation.
  • the patient of the invention can suffer from a cancer selected from the group consisting of oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, lung cancer (in particular KRAS lung cancer), bone cancer, pancreatic cancer, skin cancer, head cancer, cancer of the neck, skin cancer, melanoma, adenocarcinoma, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, perianal cancer, endometrial carcinoma, carcinoma of the vagina, Hodgkin's disease, esophageal cancer, bladder cancer, gall bladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, renal cancer, ureters
  • the inventors show that p16 upregulation in immune cells protect these cells from apoptosis during severe inflammation, thereby reducing the cytokine storm and deleterious effects of inflammation in patients treated with the agonists of the invention.
  • the pharmaceutical composition of the invention is for use in inhibiting the apoptosis of immune cells during inflammation and/or for preventing a cytokine storm linked to inflammation.
  • the inventors show that p16 upregulation in immune cells protect the treated animals against the deleterious effects of LPS-induced sepsis.
  • the pharmaceutical composition of the invention is for use in preventing and/or treating the deleterious inflammatory effects of sepsis.
  • the inventors show that the agonists of the invention have a rapid and noticeable radioprotective effect, so that p16 upregulation in immune cells protect the treated animals against the deleterious effects of radiation-induced tissue damages.
  • BNT162b2 vaccine-induced disease tolerance has a rapid and noticeable radioprotective effect that potentially could be improved by strategies that increase the presence of p16 high immune cells in the intestine.
  • the pharmaceutical composition of the invention is for use in preventing / treating the deleterious inflammatory effects of a radiation therapy.
  • the inventors show that patients suffering from severe COVID-19 disease display a low level of p16 hi0h immune cells.
  • the inventors show that p16 upregulation in immune cells after treatment with a TLR agonist provide a rapid and broad tissue protection from SARS-COV-2 induced severe inflammation, before the development of inhibitory antibodies against SARS-COV-2 infection.
  • the pharmaceutical composition of the invention is for use in preventing and / or treating a SARS-COV-2 infection, in particular for preventing the deleterious inflammatory effects induced by a SARS-COV-2 infection.
  • the inventors show that the TLR agonists of the invention induce the expression of p16 hi0h immune cells associated to melanoma tumors, and that KRAS lung cancer progression can be inhibited by administering in the animals the TLR agonists of the invention.
  • the pharmaceutical composition of the invention is for use in preventing and I or treating cancer, in particular for preventing the tissue inflammation and I or lesions associated with or induced by cancer, such as melanoma or lung cancer.
  • the present invention refers to the use of a STING agonist, of a TLR5 agonist or of a TLR7 agonist, or of a combination thereof, for the preparation of a medicament that is intended to be used for preventing and/or treating tissue inflammation and/or tissue damage, preferably induced by a pathogen infection, a cancer, a chemical or physical treatment, or by a physical damage. All the embodiments disclosed above apply to this use.
  • the present invention refers to the non-therapeutic use of a STING agonist, of a TLR5 agonist or of a TLR7 agonist, or of a combination thereof, to prevent or improve age-induced frailty and other side effects of aging, to maintain the good health of elderly persons, and therefore to extend their health expectancy and their healthspan. All the embodiments disclosed above apply to this use. Ex vivo cell therapy
  • Example 16 and example 18 below the inventors have shown that treating animals with a p16 high CD45 + immune cells collected from animals that have been treated with a TLR agonist or from human PBMCs that have been treated with the composition of the invention become also tolerant against septic shock.
  • Example 16 shows that adoptive transfer of p16 High immune cells works effectively in mice that have already started to develop a severe inflammatory response, suggesting that this strategy could potentially be used to treat patients with already developed severe inflammation, such as sepsis.
  • Example 18 demonstrates that the adoptive transfer of human p16-positive PBMCs, achieved using the protocol described in Figure 16, can protect mice from severe inflammation, such as LPS- induced sepsis.
  • These patients are for example elderly or aged patients (typically aged 70 and over) and/or those who suffer from an inflammatory disease when the treatment of the invention is administered.
  • the p16 level in both situations increases in immune cells (see also in example 1).
  • the adoptive cell therapies of the invention are very useful for inducing disease tolerance, for protecting tissues against deleterious effect of aging and/or for extending health span.
  • the proposed protocol for adoptive cell transfer could be especially effective in patients who are unable to mount a p16-induction response in their immune cells (a condition that could be detected using the procedure described in Figure 16 A-D as a diagnostic test).
  • p16 High immune cells in these cases, could not only protect against tissue damage but also improve various treatments, including cancer and severe inflammation treatments (such as sepsis). These patients may also suffer from cancer, as adoptive immunotherapy with suitable recombinant immune cells has already been proposed for the treatment of a number of cancers in humans.
  • immune cells can be purified from the circulating blood of patients, cultured ex vivo and activated to induce their differentiation and increase their tumoricidal power, then reinjected into the same or other patients. It is also possible, using suitable drugs and vectors, to treat the collected immune cells and/or to transfer genes in these cells, thereby enabling them to be endowed with superior properties as proposed herein.
  • the proposed adoptive transfer protocol for p16 High immune cells represents a more physiological, less toxic yet equally efficient alternative for anti-cancer therapies when the anti-cancer drugs are limited by their systemic toxicity.
  • p16 overexpression or activation in immune cells can be performed in vitro by any means, e.g., by using one, two or three of the PRR agonists disclosed above, or by genetic means with a p16-encoding vector (see below).
  • the invention targets the use of a STING agonist, a TLR5 agonist and/or a TLR7 agonist or the use of the composition of the invention, which is as disclosed above (i.e., comprising a STING agonist, a TLR5 agonist and/or a TLR7 agonist or combinations thereof) for ex vivo or in vitro inducing the expression or enhancing the activity of p16 in immune cells.
  • the present invention thus relates to an in vitro method to induce the expression or activity of p16 in immune cells, said method comprising the step of in vitro contacting immune cells with an agonist of a PRR, said agonist being chosen from a STING agonist, a TLR5 agonist or a TLR7 agonist, as defined above, or with a combination thereof.
  • an agonist of a PRR said agonist being chosen from a STING agonist, a TLR5 agonist or a TLR7 agonist, as defined above, or with a combination thereof.
  • These agonists would be provided in appropriate doses, as discussed above. Specifically, these doses would induce the secretion of IFNy but not of TNFa in the contacted immune cells.
  • the immune cells are contacted in vitro with a combination of a STING agonist and a TLR7 agonist, more preferably with a combination of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and of an effective amount of the BNT162b2 vaccine.
  • a STING agonist and a TLR7 agonist more preferably with a combination of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and of an effective amount of the BNT162b2 vaccine.
  • DMXAA 5,6-dimethylxanthenone-4-acetic acid
  • the in vitro overexpression of p16 in immune cells can be achieved by using a replication-defective recombinant virus encoding the p16 protein, under a promoter that is functional in lymphocytes or in monocytes.
  • This virus can be in T1 particular an adenovirus or a lentivirus or an AAV that are viruses known to be able to efficiently transduce immune cells.
  • This replication-defective virus would contain the human CDKN2A gene (NCBI gene 1029; one mRNA thereof being NM_000077.5), under the control of a promoter that is functional in these cells, e g., the promoter E2F1 (E2 promoter binding factor 1) or the promoter of EFS (elongation factor 1a short), SFFV (silencing-prone spleen focus forming virus), CMV (cytomegalovirus), RSV (Rous sarcoma virus).
  • a promoter that is functional in these cells e g., the promoter E2F1 (E2 promoter binding factor 1) or the promoter of EFS (elongation factor 1a short), SFFV (silencing-prone spleen focus forming virus), CMV (cytomegalovirus), RSV (Rous sarcoma virus).
  • E2F1 E2 promoter binding factor 1
  • SFFV stress-prone spleen focus forming virus
  • CMV cytomegalo
  • Enhancing the expression or activity of p16 would be very advantageous in immune cells expressing a chimeric antigen receptor (CAR), because these p16 hi0h CAR immune cells would be less prone to apoptosis once administered in vivo. Also, these p16 high immune cells expressing CAR would induce the beneficial anti-cancer and anti-inflammatory effects observed when p16 is enhanced in immune cells in vivo (see examples 13, 14, 15), especially when the treatment is combined with radiotherapy (example 6).
  • CAR chimeric antigen receptor
  • the present invention thus concerns p16 high immune cells expressing a functional recombinant CAR molecule and their incorporation into pharmaceutical compositions that can be used in cancer therapy, more particularly, in cancer immunotherapy.
  • the immune cells can originate from the patient himself (the composition therefore contains autologous cells) or from a donor (the composition therefore contains allogeneic or heterologous cells).
  • the composition therefore contains allogeneic or heterologous cells.
  • HLA compatibility and matching between the donor and the patient receiving the cells is required.
  • the invention relates to the isolation, culture, activation and/or treatment of these cells of the immune system, and their use in cell therapy, for example in adoptive immunotherapy. It also relates to immunotherapeutic methods using these particular cells.
  • the present invention proposes to use a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of autologous or heterologous p16 high immune cells, said immune cells being preferably T cells or macrophages expressing CAR.
  • said pharmaceutical composition contains a pharmaceutically acceptable excipient and at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 95% of autologous or heterologous p16 high immune cells. This means that, in this pharmaceutical composition, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 95% of the cells are p16 high immune cells.
  • Example 17 describes a particular protocol for the in vitro induction of p16 High human immune cells from PBMCs, including within the CD3 and CD11 b populations, following simultaneous stimulation of the TLR7 and STING pathways. Briefly, TLR7 and TLR7/8 agonists (R837 and R848, respectively), and a STING agonist (DMXAA) were used, in combination with anti-CD3, anti-CD28, and anti-CD2 stimulatory antibodies.
  • Example 18 demonstrates that the adoptive transfer of human p16-positive PBMCs, achieved using the protocol described in Example 17, can protect mice from severe inflammation, such as LPS- induced sepsis.
  • inflammatory disease it is herein meant a disease resulting from or including an inflammation reaction.
  • An inflammation reaction is the response of body tissues to harmful stimuli. Inflammatory disease can be classified as either acute or chronic. In particular, chronic inflammation is associated with various inflammatory diseases, such as hay fever, periodontal disease, atherosclerosis, and osteoarthritis.
  • inflammatory disease examples include notably acute pancreatitis; amyotrophic lateral sclerosis; Alzheimer's disease; cachexia/anorexia; asthma; atherosclerosis; chronic fatigue syndrome, fever; diabetes (e.g., insulin diabetes); glomerulonephritis; graft versus host rejection; hemorrhagic shock; hyperalgesia, inflammatory bowel diseases; inflammatory conditions of a joint, including osteoarthritis, psoriatic arthritis and rheumatoid arthritis; ischemic injury, including cerebral ischemia (e.g., brain injury as a result of trauma, epilepsy, hemorrhage or stroke, each of which may lead to neurodegeneration); lung diseases; multiple myeloma; multiple sclerosis; leukemias; myopathies; osteoporosis; Liver diseases like Non-Alcoholic Fatty Liver Disease (NAFLD), Non-Alcoholic Steatohepatitis (NASH), and Acute-on-chronic liver failure
  • patients in need of this adoptive treatment are preferably experiencing an inflammatory state, or they are suffering from an inflammatory disease, or from cancer, or from a pathogenic infection. They can be of any age.
  • This adoptive treatment can be also administered in healthy human beings expressing high levels of p16, typically in healthy people older than 65, to whom it can prevent age-associated diseases and/or extend lifespan.
  • p16 expression or activity in immune cells can be used as a prognostic marker of disease tolerance in humans. This marker can be easily detected by analyzing a blood sample collected from a patient. As a matter of fact, when the percentage of p16 high immune cells is low in a blood sample of a patient, then it can be concluded that the patient has a poor disease tolerance and will be very sensitive to an infection or an aged-related disease.
  • the present invention relates to an in vitro method for prognosing the disease tolerance of a patient, said method comprising the steps of : a) Detecting the expression and/or activity of the p16 protein in the immune cells present in a biological sample of said patient, b) Determining the percentage of p16 high immune cells in said sample, c) Concluding that the patient has a poor disease tolerance if the percentage of p16 high immune cells in the tested sample is low.
  • biological sample designates any sample collected in a patient that might contain immune cells as defined herein. It can be, for example, a blood sample, a saliva sample, a tissue sample, a bone-marrow sample, etc.
  • a blood sample is however preferred, as it is easily collected and contains many immune cells.
  • blood sample it is herein meant a whole blood, serum, or plasma sample obtained from the patient.
  • the blood sample according to the invention is a plasma sample.
  • the peripheral bone-marrow cells (PBMCs) contained in the blood sample are isolated or purified by conventional means, so as to be studied for p16 expression / activity.
  • the percentage of p16 high immune cells is low herein means for example that less than 50%, 40%, 30%, 20%, or 10% of the immune cells contained in the tested biological sample are p16 high . Conversely, this means for example that more than 50%, 60%, 70%, 80%, or 90% of the immune cells contained in the tested biological sample are p16 low .
  • the “immune cells” on which p16 should be detected are preferably lymphocytes, granulocytes, natural killer (NK), and macrophages.
  • this method is completed with a treatment step consisting in administering to the p16 low patient the pharmaceutical compositions disclosed above, containing either the PRR agonist(s) of the invention, or heterologous / autologous p16 hi0h immune cells obtained by contacting immune cells with the PRR agonist(s) of the invention. All the embodiments exposed above apply, mutatis mutandis, without needing to be repeated.
  • the prognostic method of the invention preferably can be implemented to detect specifically if a patient infected with an inflammatory virus (e.g., the SARS-COV-2 virus) will suffer from severe symptoms, or not.
  • an inflammatory virus e.g., the SARS-COV-2 virus
  • the prognostic method of the invention thus comprises the steps of : a) Detecting the expression and/or activity of the p16 protein in the immune cells contained in a blood sample of a patient infected with a SARS-COV-2 virus, b) Determining the percentage of p16 high immune cells in said sample, c) Concluding that the patient will suffer from a severe COVID19 if the percentage of p16 high immune cells in the tested sample is low.
  • This method can be furthermore completed by an optional treatment step consisting of administering to the p16 low patient, along with the pharmaceutical compositions of the invention as defined above, a strong anti-COVID19 treatment.
  • This anti-COVID19 treatment can be chosen in the approved treatments such as nirmatrelvir plus ritonavir (paxlovid), sotrovimab (Xevudy), molnupiravir (Lagevrio), dexamethasone and/or remdesivir (Veklury).
  • supplemental oxygen may be needed, and/or immunomodulators (baricitinib, tocilizumab, abatacept, infliximab).
  • the present invention relates to an in vitro method for selecting patients that will benefit from a treatment involving a TLR7 agonist, a TLR5 agonist, or a STING agonist, based on all the embodiments disclosed above.
  • This method comprises the steps of : a) Detecting the expression and/or activity of the p16 protein in immune cells present in a biological sample of a patient, b) Determining the percentage of p16 high immune cells in said sample, c) Concluding that the patient will benefit from a treatment involving a TLR7 agonist, a TLR5 agonist, ora STING agonist if the percentage of p16 high immune cells in the tested sample is low.
  • this method is completed with a treatment step consisting in administering to the p16 low patient the pharmaceutical compositions disclosed above, containing either the PRR agonist(s) of the invention, or heterologous / autologous p16 high immune cells obtained by contacting immune cells with the PRR agonist(s) of the invention. All the embodiments exposed above apply, mutatis mutandis, without needing to be repeated.
  • this method for selecting patients can contain the further steps of: i) Detecting the expression and/or activity of the nicotinamide N- methyltransferase (NNMT) enzyme in the immune cells present in a biological sample of said patient, and ii) Concluding that the patient will benefit from a treatment involving a TLR7 agonist, a TLR5 agonist, or a STING agonist if the percentage of p16 high immune cells in the tested sample is low and if the NNMT enzyme is expressed and functional in the immune cells present in the tested sample.
  • NNMT nicotinamide N- methyltransferase
  • NNMT nicotinamide N-methyltransferase
  • the term “higher” or “increased”, as used herein, refers to a level of p16 or to an activity of p16 at least 1.5 folds greater (preferably 2, 3, 4, 5, 6, 7, 8, 9, 10 folds greater) than the level of p16 or the activity of p16 in the reference sample.
  • the term “lower” or “decreased”, as used herein, refers to a level of p16 or to an activity of p16 at least 1 .5 folds lower (preferably 2, 3, 4, 5, 6, 7, 8, 9, 10 folds lower) than the level of p16 or the activity of p16 in the reference sample.
  • cancer is not limited to any stage, grade, histomorphological feature, invasiveness, aggressiveness or malignancy of an affected tissue or cell aggregation.
  • stage 0 cancer stage I cancer, stage II cancer, stage III cancer, stage IV cancer, grade I cancer, grade II cancer, grade III cancer, malignant cancer, primary carcinomas, and all other types of cancers, malignancies etc. are included.
  • tissue damage refers to harm, injury, or impairment to biological tissues that is either linked to the presence and progression of a specific disease or condition, or to an external event (wound, bruising, dislocation, sprain, strains, limb injuries, bone fracture, pealing, scarification, shaving, etc.).
  • tissue damage impacts the affected tissues at the cellular or structural level where inflammation is induced.
  • the term “radiation therapy” refers to a treatment involving particles or electromagnetic waves with sufficient energy to ionize atoms or molecules by removing tightly bound electrons.
  • This type of radiation includes alpha particles, beta particles, gamma rays, X-rays, and certain types of neutrons. Ionizing radiation has enough energy to produce ions when it interacts with matter, and it can cause damage to biological tissues, often leading to inflammatory side effects.
  • SARS-CoV-2 designates not only the firstly identified SARS-CoV-2 but also any variant or mutant thereof.
  • the terms “treat”, “treating” and “treatment”, are meant to include alleviating, attenuating or abrogating a condition or a disease, in particular, an inflammation, a cancer or a tissue damage and/or the signs, symptoms and/or complications associated therewith.
  • the signs or symptoms associated with a condition or a disease may be biochemical, cellular, histological, functional or physical, subjective or objective ones. This includes the complications associated with a condition or a disease.
  • prevent are meant to include not only delaying or precluding the onset of a condition or disease, and/or the signs, symptoms and/or complications associated therewith but also barring a patient from acquiring a condition or disease, in particular, or reducing a patient’s risk of acquiring a condition or disease.
  • nucleic acid refers to DNA, RNA, single-stranded, doublestranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof. Modifications include, but are not limited to, those providing chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole.
  • in vitro and ex vivo are equivalent and refer to studies or experiments that are performed using biological components (e.g. cells or population of cells) that have been isolated from their usual host organisms (e.g. animals or humans).
  • in vivo or “in situ” refer to studies that are conducted on whole living organisms (e.g., humans), after administration of the composition of the invention in a living subject.
  • Figure 1 results of the analysis of the fraction of p16 high cells in different populations of the immune cells.
  • p16 high T lymphocytes express PD-1 , PD-L1 , PD-L2 and Foxp3.
  • the percentage of p16 high cells was determined in T lymphocytes obtained from peritoneal cavity, liver, bone marrow and abdominal fat SVF defined as CD3 + CD4 + PD1 + , CD3 + CD4 + PD-L1 + , CD3 + CD4 + PD-L2 and CD3 + CD4 + Foxp3 + (Bar graphs) in 16-month-old mice.
  • Pie charts show abundance and distribution of PD-1 , PD-L1 , PD-L2 and Foxp3 in the total CD3 + CD4 + p16 high population. Data are mean ⁇ S.D.
  • p16 High peritoneal macrophages are not proliferative.
  • Peritoneal macrophages from 12-month-old p16-Cre/R26-mTmG mice were incubated with 5-ethynyl-2’-deoxyuridine (EdU) for 24 h.
  • the percentage of EdU positive cells was analyzed in p16 High and p16 Low populations. Data are mean ⁇ SD. Significance was analyzed by t-test. **p ⁇ 0,01 .
  • RNA expression of both populations was analyzed by RNA sequencing. Kegg pathway analysis of downregulated genes from RNA-seq data set reveals a set of clusters of genes related with the negative control of cell cycle. Individually analyzed genes are shown. Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) were analyzed by t-test. Data are mean ⁇ SD. *p ⁇ 0,05; **p ⁇ 0,01 and
  • Figure 2 Results of the analysis of p16 high immune cells in young animals in response to inflammation and tissue damage.
  • p16 High T lymphocytes express higher level of Foxp3, PD-1 , and PD-L1 after DSS treatment.
  • the percentage of p16 High cells was determined in T lymphocytes obtained from peritoneal cavity and liver defined as CD3 + CD4 + PD1 + , CD3 + CD4 + PD-L1 + , and CD3 + CD4 + Foxp3 + in 12-month-old mice.
  • Pie charts show abundance and distribution of Foxp3, PD-1, and PD-L1 in the total CD3 + p16 High population (D). Data are mean.
  • E-F Different pattern of gene expression in the absence of p16 High cells.
  • Control and p16-Cre/R26-DTA (DTA) were treated with 2.5% DSS for 7 days and RNA from peritoneal cells (E) and liver (F) were isolated.
  • RNA was analyzed by quantitative SYBR-green based PCR (qPCR) to determine the level of expression of different genes. Data are mean +/- S.D. Difference between groups was analyzed using ANOVA test and Tukey post hoc test. *p ⁇ 0,05; **p ⁇ 0,01 and ***p ⁇ 0,001.
  • TLRs G-H. Specific TLRs induce p16 High state in vivo. 2-3 months p16-Cre/R26-mTmG mice were treated intraperitoneal (I.P) with a panel of agonist to different toll-like receptors (TLRs). 48 h later, peritoneal cells were stained with fluorescent-conjugated antibodies against F4/80 (G) and CD3 (H). The percentage of p16 High cells was determined by flow cytometry. Data are mean ⁇ SD. Statistical significance were determined using ANOVA plus Dunnett post hoc test. *p ⁇ 0,05 and ***p ⁇ 0,001. Figure 3: The BNT162b2 mRNA COVID-19 vaccine induces p16 High immune cells and disease tolerance to protect against severe inflammation and tissue damage
  • BNT162b2 mRNA COVID-19 vaccine induces p16 High cells in vivo. 2-3 month- old p16-Cre/R26-mTmG mice were treated intraperitoneal (I.P) with 5 pg of BNT162b2 mRNA COVID-19 vaccine. After 2 and 15 days the percentage of p16High cells was determined in different immune subsets in peritoneal cavity (A). Abundance and distribution of different p16 High cells in analyzed tissues. Each pie chart represents the total number of p16 High cells and how they are distributed in different cellular compartments (B). Data are mean ⁇ SD. Statistical significance was determined using ANOVA plus Dunnett post hoc test. **p ⁇ 0,01 and ***p ⁇ 0,001
  • D. BNT162b2 vaccine induces p16 High cells that are sensitive to senolytic treatment.
  • 2- 3 month-old p16-Cre/R26-mTmG mice were treated I.P with 5 pg of BNT162b2 vaccine. 5 days later, mice were either treated orally with a senolytic combination of dasatinib (5 mg/kg) and quercetin (50 mg/kg) or vehicle (Mock). After 24h mice were euthanized and single cell suspensions from peritoneal cavity and liver were stained with conjugated antibodies against F4/80 and CD3. Cells suspensions were analyzed by flow cytometry and the percentage of p16High cells in each population was determined.
  • E. Genetic ablation of p16 High cells modifies the expression of inflammatory genes after BNT162b2 treatment.
  • Wild type and p16-Cre/R26-DTA (DTA) were treated with 5 pg of BNT162b2 vaccine.
  • RNA from peritoneal cells was isolated.
  • RNA was analyzed by qPCR to determine the level of expression of inflammatory genes. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p ⁇ 0,05; **p ⁇ 0,01 and ***p ⁇ 0,001.
  • BNT162b2 vaccine induces p16 High cells in the lung.
  • 2-3 month-old p16-Cre/R26- mTmG mice were treated I.P with 5 pg of BNT162b2 vaccine.
  • Single cells suspensions from lung were stained with fluorescent conjugated-antibody against different markers of immune cells and analyzed by flow cytometry (H). Abundance and distribution of p16 High cells in the lung.
  • Each pie chart represents the total number of p16 High cells in the lung and how they are partitioned into different cellular compartments (I). Data are mean ⁇ SD. Statistical significance were determined using ANOVA plus Dunnett post hoc test. **p ⁇ 0,01 and ***p ⁇ 0,001.
  • J. BNT162b2 vaccine increases p16 High cells and induces Foxp3 + , PD-1 + and PD-L1 + in CD3 + CD4 + populations.
  • 2-3 month-old p16-Cre/R26-mTmG mice were treated I.P with 5 pg of BNT162b2 vaccine and after 5 days, single cell suspensions were stained with fluorescent- conjugated antibodies and the levels of CD3 + CD4 + PD1 + , CD3 + CD4 + PD-L1 + , and CD3 + CD4 + Foxp3 + populations and the percentage of p16 High cells were analyzed by flow cytometry. Data are mean ⁇ SD. Statistical significance were determined using ANOVA plus Dunnett post hoc test. ***p ⁇ 0,001.
  • mice were either treated with BNT162b2 vaccine or mock-treated and 3 days later were infected with a mouse-adopted MA10 virus. Analysis of probability of survival in experimental group as assessed, dead and mice that lost 70% and more body weight were considered as deceased. Difference between groups was analyzed using Gehan- Breslow-Wilcoxon test. *p ⁇ 0,05.
  • the BNT162b2 mRNA COVID-19 vaccine induces disease tolerance by activation of TLR7 and tonic STING response.
  • mice 2-3 months p16-Cre/R26-mTmG mice were treated with the BNT162b2 vaccine. One day before, in the same day, and day after treatment with BNT 162b2, some animals were treated I.P with TLR7 inhibitor (M5049, 1 mg/kg) or STING inhibitor (H151 , 10 mg/kg). 5 days after BNT162b2 treatment, animals were euthanized and single cells suspensions were analyzed by flow cytometry to determine the percentage of p16High cells on different immune subsets (left panel) and inside of Foxp3, PD-1 and PD-L1 (CD3 + CD4 + ) populations (right panel). Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p ⁇ 0,05; **p ⁇ 0,01 and ***p ⁇ 0,001.
  • mice F. 2-3 months wild type mice were treated I.P with the STING agonist DMXAA (10 mg/kg) one time (low dose) or 2 consecutive days (high dose). 5 days after first treatment, animals were euthanized and RNA from peritoneal cells (P.C), and liver was isolated. RNA was analyzed by qPCR to determine the level of expression of inflammatory genes. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test plus Dunnett post hoc test. *p ⁇ 0,05; and ***p ⁇ 0,001 .
  • Tonic STING activation promotes disease tolerance and tissue protection against severe inflammation.
  • Wild type mice were either treated with saline (Mock); BNT162b2 vaccine (5 pg by mouse); BNT162b2 vaccine plus H151 (10 mg/kg, 3 consecutive days starting one day before BNT162b2 treatment); and DMXAA (10 mg/kg) for one (low dose) or two consecutive days subcutaneously (high dose).
  • animals were injected with LPS 055: B5 30 mg/kg. Animals were euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow- Wilcoxon test. **p ⁇ 0,01 and ***p ⁇ 0,001.
  • NNMT gene is overexpressed in p16 High cells.
  • RNA-seq data reveals overexpression of NNMT gene in p16 High cells.
  • Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) from p16 High and p16 low populations were analyzed by t-test. Data are mean ⁇ SD. ***p ⁇ 0,001.
  • NNMT gene is induced after DSS treatment. 2-3 month-old control and p16- Cre/R26-DTA (DTA) were treated with 2.5% DSS for 7 days and RNA from peritoneal cells and liver was isolated on day 8. RNA was analyzed by a quantitative SYBR-green based PCR (qPCR) to determine the level of expression NNMT mRNA. Data are mean +/- S.D. Difference between groups was analyzed using ANOVA test and Tukey post hoc test. ***p ⁇ 0,001.
  • BNT162b2 vaccine-induced NNMT expression depends of p16 High cells. 2-3 month- old control and p16-Cre/R26-DTA (DTA) were treated with 5 pg of BNT162b2 vaccine. After 2 and 5 days, RNA from peritoneal cells, liver and lungs was isolated and analyzed by qPCR to determine the level of expression of NNMT mRNA. Data are mean +/- SD. Difference between groups was analyzed using ANOVA test and Tukey post hoc test. *p ⁇ 0,05; **p ⁇ 0,01 and ***p ⁇ 0,001.
  • G Percentage of p16 High positive cells in different T cell populations was determined during experiment show in E. Data are mean ⁇ SD. Statistical significance were determined using ANOVA plus T ukey post hoc test. ***p ⁇ 0,001 .
  • I. NNMT is necessary to protect against acute inflammation. 2-3 months wild type and p16/ NNMT-cKO mice were treated with 5 pg of BNT162b2 vaccine. After 5 days, animals were injected I.P with LPS 055: B540 mg/kg. Animals were euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. *p ⁇ 0,05.
  • CDKN2A' /_ (CDKN2A-KO) and CDKN2A +/+ (wild type) mice were either treated with saline (Mock) or BNT162b2 vaccine (5 pg by mouse).
  • Single cell suspensions were analyzed by flow cytometry to determine the percentage of cleaved-caspase-3 in CD45, F4/80 and CD3 populations. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p ⁇ 0,05; **p ⁇ 0,01 and ***p ⁇ 0,001.
  • Blocking adenosine receptor A2A increase survival during aging 2 year-old wild type mice were treated with 5 pg of BNT 162b2 vaccine, or BNT162b2 plus adenosine receptor A2A inhibitor (SCH58261 , twice by day, 3 mg/kg) starting one day before LPS treatment. 5 days after BNT162b2 treatment, animals were injected I. P with LPS O55:B520 mg/kg. Animals were euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. **p ⁇ 0,01
  • MDA5-KO mice 18-20 months MDA5 +/+ (wild type) and MDA5' /_ (MDA5-KO) littermates were treated with saline or STING inhibitor H151 (10 mg/kg). 48h before, animals were euthanized and mRNA from livers was isolated and analyzed by qPCR. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. **p ⁇ 0,01 and ***p ⁇ 0,001.
  • MDA5-KO mice exhibit reduced adenosine levels during aging. 3-4 months MDA5 +/+ (wild type); and 20 months MDA5 +/+ (wild type) MDA5' /_ (MDA5-KO) littermates were treated with saline or BNT162b2 vaccine (5 pg by mouse). 5 days after animals were euthanized and levels of adenosine were determined in peritoneal cells. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p ⁇ 0,05; **p ⁇ 0,01 and ***p ⁇ 0,001.
  • MDA5-KO mice shown less basal inflammation during aging. Liver RNA from 3-4 months MDA5 +/+ (wild type) and MDA5' /_ (MDA5-KO) littermates was isolated and analyzed by qPCR. The level of expression of inflammatory genes was determined. Data are mean +/-S.D.
  • F Immunofluorescent analysis of fibrosis marker aSMA in liver and muscle samples (same as in E). Graphs at the right represent mean ⁇ S.D of aSMA area between groups, Area was determined among 3 animals per group. Differences between groups were determined by unpaired, nonparametric Mann-Whitney test. *p ⁇ 0,05; and ***p ⁇ 0,001. Scale bars: 200 mm for liver, 100 mm for muscle.
  • G Muscle strength in 18 and 24 months old wild type and MDA5-KO littermates mice was determined with a grip test. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Dunnett post hoc test. **p ⁇ 0,01 and ***p ⁇ 0,001.
  • H. MDA5-KO mice show better physical fitness during natural aging. Frailty was assessed in 18 and 24 months old wild type and MDA5-KO littermates using an clinically relevant index for frailty during aging (see material and methods). Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Dunnett post hoc test.**p ⁇ 0,01 and ***p ⁇ 0,001.
  • the BNT162b2 vaccine induces p16 + immune subsets in humans that are significantly reduced in severe COVID-19 patients.
  • RNA were isolated from PBMCs and analyzed by quantitative-PCR. Data were analyzed as a bulk (left panel) and as matched samples (right panel), t-test and paired t- test were used to determine the differences between conditions. *p ⁇ 0,05 and **p ⁇ 0,01.
  • Flow cytometry data were also treated as matched samples. The level of p16 + cells inside singles populations are shown in each plot. Paired t-test was used to determine the differences before (Day 0) and after (Day 7 and day 30) the BNT162b2 vaccine treatment for CD45 + , granulocytes, Tregs and PD1 + . For monocytes and lymphocytes, Shapiro-Wilk test was used to determine normality of the data. ANOVA plus Dunnett's test or Friedman plus Dunn's test were used to determine difference before and after. *p ⁇ 0,05 and **p ⁇ 0,01 .
  • G.H One cohort of 40 unvaccinated patients older than 55 years were classified in negative for COVID-19, moderate COVID-19 and severe COVID-19.
  • RNA from PBMCs were isolated and analyzed by quantitative-PCR. Level of p16 (G) and NNMT (H) expression were determined. Differences among groups were determined using ANOVA test and Tukey post hoc test. *p ⁇ 0,05; **p ⁇ 0,01 and ***p ⁇ 0,001.
  • Figure 8 Schematic representation of disease tolerance induced by p16High immune cells
  • Figure 9 Percentage of tumor associated p16 high immune cells after treatments using BNT162b2 vaccine, a-PDL1 or combination.
  • Figure 10 Lesions counting in the tumorigenic KRAS G12D mouse model after treatments with BNT162b2 vaccine, a-PDL1 or a combination thereof.
  • the number of lesions was manually determined following the criteria: small ( ⁇ 1 mm), medium (between 2 and 3 mm) and large (more than 3 mm) lesions in a KRAS mouse model in which the p16 gene is not expressed (p16-cre/DTA/KRAS G12D mice). A reduction in the number of medium and large lesions was not observed in the animals treated with BNT162b2. By combining all size lesions, the ability of BNT162b2 treatment reducing the number of lesion is not significant (right panel).
  • Figure 14 Analysis of the proliferation of B16F10 cells expressing luciferase that have been intravenously injected in mice, with different treatments.
  • B Administration of the BNT162b2 in combination with anti PD-1 therapy halts tumor growth of melanoma.
  • (D) Tumor volume of B16F10 melanoma cells treated with BNT162b2 in combination with an anti PD-1 antibody, with or without a TLR7 inhibitor (M5049) or a STING inhibitor (H 151). 7 days after B16F10 injection, animals were treated with saline, single I.P injection of 5 ug of BNT162b2 by mouse plus 2 injections of 100 ug of anti-PD1 by mice (1 per week). A set of animals also was treated with TLR7 inhibitor (2 mg/kg of M5049) or STING inhibitor (10 mg/kg of H151) 12h before BNT162b2 treatment. Tumor size was measured along the experiment time frame using a caliper and the formula TV (W(2) x L)/2 where TV is Tumor Volume, W - width and L -length.
  • FIG. 15 Analysis of the effect of transplanted CD45 + cells in mice.
  • the protocol is detailed on (A). Briefly, CD45 + cells from donor mice (previously treated or not with the BNT162b2 vaccine) are isolated and administered to recipient mice prior to a LPS challenge. The results are provided in (B).
  • (C) describes the protocol for adoptive transfer of CD45 + immune cells from donor (wild-type (proficient for p16 High cells) or p16-cre/DTA (deficient for p16 Hi0h cells) mice) that were vaccinated with the BNT162b2 vaccine (“Vax”) into wild-type recipient mice that have been submitted to LPS-induced inflammation.
  • mice were euthanized by cervical dislocation. Tumors were collected and incubated with 1 mg/ml collagenase type A at 37°C during 45 min, sieved consecutively with two 100, and 30 pm strainers to obtain single cell suspensions. Red blood cells were removed using lysis buffer. Cells were then centrifuged at 1500 RPM for 5 minutes at 4°C. Cells were resuspended in cytometry blocking buffer (HBSS 1 % BSA, 4% FBS, 2 mM EDTA) and stained with fluorescent conjugated antibodies (see the list below) at 2 pg/mL during 25 minutes. Cells were washed 2 times with the cytometry buffer. Samples were analyzed by flow cytometry using CytoFlex system (Beckman-Coulter, California, United States). At least 3X104 events were recorded in singlets populations. Data were obtained and analyzed using CytExpert software KRAS Tissue analysis
  • KRAS mice were divided into three groups at the age of 2 months and injected with a drug of interest.
  • First group was a control group.
  • Second group was injected two times with BNT162b2 2 weeks apart and euthanized on week 8.
  • Third group was subjected to four alternating injections of BNT162b2 and a-PDL1 (200pg per mouse) throughout first 4 weeks and euthanized on week 8 as well.
  • Collected tissue samples (lungs) were fixed in 4% PFA overnight before alcohol dehydration, Number of tumor was determined manually by counting the number of lesions. After that tumors were embedded in paraffin. Samples were cut into 3 pm sections using Automated Rotary Microtome HistoCore AUTOCUT (Leica, France). Paraffin-embedded sections were deparaffinised in xylene and rehydrated in ethanol with increasing concentrations of water, stained with haematoxylin and eosin for visualization of cancerous lesions in lungs of KRAS mice.
  • mice were sacrificed and tissue resident CD45 + were isolated. 5x10 5 cells were injected into recipient mice. 24 h after cell injection, animals were challenged with a lethal dose of lipopolysaccharide (O55:B5) (35 mg/kg I.P) ( Figure 15A). Probability of survival was assessed. Differences between groups were calculated using Mantel-Cox test (p ⁇ 0,01).
  • Example 1 p16 high immune cells analysis in different tissues with age
  • TLR Toll-like receptor
  • TLR7 receptor activation induced regulatory p16 high subsets potentially creating disease tolerance by reducing the negative impact of the infection and tissue damage on host fitness, it has been focused on this signaling pathway.
  • TLR7 is located intracellularly primarily in endosomes and detects single-stranded RNA targets, including those from different viruses such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), and SARS-CoV-2 (Diebold SS., et al., 2004, Science 303, 1529-1531 and Mantovani S. et al., 2022, genes Immun. 23, 51-56).
  • HCV human immunodeficiency virus
  • HCV hepatitis C virus
  • SARS-CoV-2 Diebold SS., et al., 2004, Science 303, 1529-1531 and Mantovani S. et al., 2022, genes Immun. 23, 51-56.
  • mice were irradiated with 8 Gy on day 5 after treatment with saline or BNT162b2 and their survival was monitored. All saline-treated mice died by day 15, while 40% of the vaccinated control mice were alive and this effect was largely dependent on the presence of p16 high cells (Figure 3G).
  • the BNT162b2 vaccine-induced disease tolerance has a rapid and noticeable radioprotective effect that potentially could be improved by strategies that increase the presence of p16 high immune cells in the intestine.
  • Example 7 Effect of BNT162b2 vaccine in disease tolerance by early protection from lethal infection with SARS-CoV-2 virus
  • the BNT162b2mRNA COVID-19 vaccine has been proven to be very effective in the induction of neutralizing antibodies against a spike protein of the SARS-CoV-2 virus.
  • the antibody production starts around day 14 after the priming dose of vaccine both in humans and mice, reaching high levels only by day 21 or after a booster shot (Li C. et al., 2022, Nat. Immunol. 23, 543-555).
  • antigen-specific CD8+ T cells and increased levels of interferon-y were detected in mice only after the second dose of the vaccine (Li C. et al., 2022, Nat. Immunol. 23, 543-555).
  • mice For this purpose, a group of mice was pre-treated with BNT162b2 or PBS (mock) and on day 3, the mice were inoculated intratracheally with a lethal dose of the mouse-adapted SARS-CoV-2 strain MA10 (not shown).
  • mice started to lose weight at day 2 after inoculation and this dynamic continued in both groups until day 4, when a rapid recovery began in the BNT162b2 -treated mice, while the mock-treated animals continued to lose weight (not shown).
  • Analysis of the probability of survival showed a strong protection in the BNT162b2-treated group at 100% survival, while only 30% of the mock-treated mice were alive on day 14 (Figure 3K).
  • Example 8 both TLR7 and STING pathways are required for induction of p16 high immune subsets
  • TLR7- and STING-dependent pathways were focused on.
  • the use of TLR7 and STING inhibitors blocked the effect of BNT162b2 by reducing the percentage of p16 High immune subsets including specifically in Tregs as well as PD1 and PD-L1 -positive T cells in analyzed tissues (Figure 4).
  • an activated and phosphorylated at S366 form of STING was evaluated in p16 High cells.
  • a significant fraction of p16 High cells was positive for p-STING with most noticeable co-staining in F4/80 + macrophages (Figure 4C&D right panel and upper pie chart).
  • Example 9 The role of NNMT in the control of suppressive p16 high immune subsets
  • NNMT nicotinamide N-methyltransferase
  • Nnmt-cKO mice conditional knockout mice.
  • the first exon of the NNMT gene was flanked by LoxP sites to produce an inactive gene after excision with the Cre recombinase.
  • NNMT-cKO mice were crossed with mice expressing Cre under the control of the p16 promoter (p16-Cre). The resulting animals in which the removal of NNMT was induced specifically in p16 high cells were used for further experimentation.
  • Example 10 p16High immune cells protect from severe inflammation by reducing the levels of adenosine.
  • SAM S-Adenosyl Methionine
  • adenosine While the level of adenosine could be controlled via SAM, one of the major sources of adenosine, and specifically extracellular adenosine, is the ATP. ATP is released to the environment from apoptotic cells in damaged and inflamed tissues, including regulatory T cells. In turn, inhibition of the cell cycle, including by upregulation of p16, has been reported to have anti-apoptotic properties. In addition, p16 High cells commonly express the high level of anti- apoptotic protein Bcl2 (Schmitt, C.A., et al. (2002). Cell 109, 335-346). Because of that, it was hypothesized that p16 upregulation may protect immune cells from apoptosis which in turn could lead to reduction of extracellular adenosine.
  • Cdkn2a-KO mice 2-month-old wild type and Cdkn2a knockout mice were treated with BNT162b2 and 5 days later exposed them to sub-lethal doses of LPS.
  • the fourth dose of the vaccine (BNT162b2 only) was well- tolerated by all participants and did not induce an inflammatory syndrome (increased IL-1 [3 and IL-6), but produced a significant SARS-CoV2-specific T cell response 1 month later as shown in Table 1 below.
  • mice 1 million cells from B16F10 syngeneic melanoma line were injected subcutaneously in a 2 months p16-cre/R26-mTmG mice. After 9 days, and tumors establishment, mice were treated intraperitoneal as follows:
  • mice treated with a-PDL1 showed a slight increase in the number of p16 high
  • the use of a-PDL1 reduced the increase of p16 high when used in combination with the BNT162b2 vaccine in whole set of populations analyzed.
  • Example 14 Effect of BNT162b2 vaccine treatment on tumor burden in a murine model of constant expression of oncogenic KRAS gene
  • KRAS G12D is a life-threatening model of tumorigenesis. Constant oncogenic stress from KRAS G12D expression deteriorated health of mice early in life (between 4-6 months) until a terminal end point.
  • a set of mice overexpressing the oncogene form of KRAS G12D was divided in 3 groups. The group control was treated with vehicle (PBS), the second group was treated with the BNT 162b2 vaccine (5 g by mouse), and the third group with a combination of BNT162b2 and a-PDL1 (200 pg by mouse). Treatment started when the mice were 2 months old. Single treatments were applied 1 time a month for 2 consecutive months.
  • mice were euthanized at 4 months old. Lungs were fixed in 4% PFA for 24 h at 4 °C and then kept in 70% ethanol for 3 days. After that, the number of lesions was manually determined following the criteria: small ( ⁇ 1 mm), medium (between 2 and 3 mm) and large (more than 3 mm) ( Figure 10).
  • the p16-cre/DTA is a mouse model for trace and specifically eliminate cells that overexpress p16, since diphtheria toxin A expression is subrogate to p16 expression.
  • Animals carrying this genotype were crossed with the KRAS G12D mouse.
  • This new breeding p16- cre/DTA/KRAS G12D was used to compare the induction of tumorigenic lesion against the KRAS G12D line.
  • this mouse was treated with BNT162b2 (5 pg by mouse 1 time a month for 2 consecutive months) ( Figure 11).
  • B16F10 melanoma cells were injected intravenous as a model of metastatic growth. Before injection, cells were modified to express luciferase. To track tumor growth during time, a sample of blood was obtained and luciferase activity measured. Individual values at day 1 were used to draw the plot as fold change. Animals were treated with BNT162b2 (5pg/mouse) or saline (mock) at day 3. Differences were determined with t-test at day 10. p ⁇ 0,05. The results are shown on Figure 14. It is clearly observed that the proliferation of the B16F10 melanoma tumor cells is prevented during at least 10 days in mice treated with the BNT162b2 vaccine.
  • Example 16 Disease tolerance can be transplanted from mouse to mouse, even in conditions of severe inflammation.
  • BNT162b2 vaccine induce diseases tolerance against septic shock through the induction of p16 hi0h cells.
  • young mice were treated with BNT162b2 vaccine. 5 days later, CD45 + fraction from liver and blood were isolated and injected intravenously into young animals (5x10 5 cells from liver and 2,5x10 5 cells from blood) (Figure 15A). In parallel, another group of mice received the same number of cells, but from untreated donor mice.
  • Donor wild-type mice proficient for p16 High cells
  • p16-Cre/DTA mice deficient for p16 High cells
  • recipient mice received a dose of 32 mg/kg of LPS to induce severe sepsis.
  • recipient mice also received 5 x 10 5 freshly isolated CD45-positive immune cells from the livers of donor mice from different groups (as shown in Figure 15C), and their survival was assessed.
  • Example 17 Procedure for induction of p16 High cells in human peripheral blood mononuclear cells (PBMCs) in vitro.
  • PBMCs peripheral blood mononuclear cells
  • TLR7 and TLR7/8 agonists were used at 1 pg/ml (R837 and R848, respectively), and a STING agonist (DMXAA) was used at 10 pg/ml, either alone or in combination with anti-CD3, anti-CD28, and anti-CD2 stimulatory antibodies.
  • DMXAA STING agonist
  • TLR7/8 agonist R848
  • STING agonist DMXAA
  • CD3 antibody CD45-positive
  • RIG-I and MDA5 agonists were further tested as alternatives to the STING agonist but it was found that only a combination with the STING agonist worked in both total CD45-positive immune cells ( Figure 160) and T cells ( Figure 16D).
  • TLR7 and CD3 agonists are part of a routine test (QFM) used to evaluate interferon response and cytokine production.
  • This example describes a protocol for the in vitro induction of p16 Hi0h human immune cells, including within the CD3 and CD11 b populations, following simultaneous stimulation of the TLR7 and STING pathways.
  • Example 18 Adoptive transfer of p16-positive human PBMCs protects mice from severe sepsis.
  • mice were treated with the BNT162b2 vaccine to induce p16-positive immune cells.
  • blood was collected, and plasma was analyzed to determine the level of liver enzymes, aspartate transaminase (ASAT) and alanine transaminase (ALAT), as a readout of liver damage.
  • Animals pre-treated with the BNT162b2 vaccine showed less accumulation of both liver enzymes in plasma, suggesting a protective function of p16-positive immune cells on liver tissues during strong inflammation (Figure 18B).
  • mice were tested for gut leakage after sepsis induction. After 12 hours of LPS treatment, animals were orally gavaged with 12 mg of dextran-FITC (4 kDa), and 4 hours later (for a total of 16 hours), blood was collected and fluorescence was measured in the plasma. A significantly lower level of dextran-FITC was observed in the BNT162b2-treated animals, suggesting they had reduced gut permeability after sepsis induction and their intestines were better protected (Figure 18C).
  • hypothermia was investigated, which in mice is associated with poor prognosis under sepsis conditions. Sixteen hours after LPS treatment, it was observed that LPS-treated animals had significantly lower body temperatures than the animals pre-treated with the BNT162b2 vaccine (Figure 18D).
  • the process of aging represents the gradual deterioration of organism functions with time, which affects all tissues and organs.
  • aging is the most prevailing risk factor for the morbidity and mortality attributable to numerous acute and chronic diseases.
  • a defined set of mechanisms has evolved to limit the negative impact of tissue damage caused by multiple factors on homeostasis.
  • protective mechanisms that rely on the concerted action of both innate and adaptive immunity is an important but poorly understood defense strategy that limits the extent of tissue damage known as disease tolerance.
  • the concept of disease tolerance was originally introduced to explain additional to resistance mechanisms to fight inflammation by decreasing the host susceptibility to tissue damage without having a direct impact on the pathogens.

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Abstract

La présente invention concerne l'utilisation d'un agoniste de STING, d'un agoniste de TLR5 ou d'un agoniste de TLR7 pour améliorer le nombre de cellules immunitaires p16high in vitro ou chez un patient en ayant besoin, en particulier pour étendre la portée de santé et protéger les tissus contre le vieillissement. Il est également rapporté que les cellules immunitaires p16 high jouent de manière surprenante un rôle clé dans l'établissement de la tolérance aux maladies, et peuvent être utiles pour contrebalancer différentes conditions létales, y compris la septicémie induite par LPS, l'infection aiguë létale du SARS-CoV -2, le cancer et l'irradiation ionisante. La présente invention concerne ainsi une composition pharmaceutique comprenant des cellules immunitaires p16high autologues ou hétérologues telles que des cellules p16high CAR-T, et son utilisation à diverses fins thérapeutiques. Ces cellules sont de préférence obtenues par leur mise en contact avec un agoniste de STING, un agoniste de TLR5 et/ou un agoniste de TLR7.
PCT/EP2025/058170 2024-03-25 2025-03-25 Utilisation thérapeutique d'agonistes de sting et de tlrs pour induire l'expression de p16 dans des cellules immunitaires Pending WO2025202222A1 (fr)

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