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WO2018100410A1 - Modulateurs de sestrines - Google Patents

Modulateurs de sestrines Download PDF

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Publication number
WO2018100410A1
WO2018100410A1 PCT/IB2016/057209 IB2016057209W WO2018100410A1 WO 2018100410 A1 WO2018100410 A1 WO 2018100410A1 IB 2016057209 W IB2016057209 W IB 2016057209W WO 2018100410 A1 WO2018100410 A1 WO 2018100410A1
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cells
sestrin
use according
seq
modulators
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Alessio LANNA
Arne Nalpon Akbar
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Rejuviron Ltd
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Rejuviron Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention refers to modulators of Sestrins to be used for boosting the immune system response in an individual and/or for rejuvenating the immune system of an individual and/or for reverting the senescent immune system of an individual.
  • the Sestrin's modulators of the invention are useful for the treatments of conditions, preferably pathological conditions, associated to or caused by or related to the aging and/or the decline/impairment of the immune system, preferably the immune system response, preferably mediated by T cells.
  • Sestrins that in mammals are the products of the Sesnl, Sesn2 and Sesn3 genes, accumulate in stressed-cells, stimulate AMP-responsive protein kinase (AMPK) activation, and inhibit activation of the anabolic kinase mammalian target of rapamycin complex 1 (imTORCI ).
  • AMPK AMP-responsive protein kinase
  • MAPKs Mitogen activated protein kinases
  • ERK extracellular regulated protein kinase
  • JNK c-Jun N-terminal kinase
  • p38 extracellular regulated protein kinase
  • the present invention solves these needs by using modulators of Sestrins.
  • the Applicants have demonstrated for the first time that Sestrins show unrecognized pro-ageing activities in immune system cells, such as, T lymphocytes.
  • the Applicants have demonstrated that Sestrins are overexpressed in these cells during ageing, and that inhibitors of Sestrins allow rejuvenation of immune system cells, in particular, in a AMPK-MAPK dependent manner.
  • the Applicants have shown that, in senescent T cells, Sestrins work in a macromolecular signaling complex within which they simultaneously coordinate the AMPK-dependent activation of ERK, JNK and p38 MAPKs.
  • This Sestrin-coordinated MAPK signaling complex is only present in senescent T cells from young individuals. Instead, in older subjects, this complex is present in T cells at all stages of differentiation. The Applicants demonstrated that disruption of the Sestrin-coordinated MAPK signaling complex restored antigen-specific proliferation, cytokine production in T cells from old humans and enhanced responsiveness to influenza vaccination in old mice.
  • the Applicants identify a Sestrin-dependent immune-inhibitory complex that coordinates global MAPK activation and function in immune system cells, such as T cells. Interference with activation of the entire complex or its individual MAPK components allows either broad or selective reconstitution of immunity during ageing.
  • FIG. 1 shows representative surface CD27 and CD28 in human CD4 " T cells by flow-cytometry (A) and (B) immunobiots of endogenous Sesnl , Sesn2 and Sesn3 expression in CD4 'V T cells gated as in (A).
  • - Figure 2 shows results from 4 different donors as in Fig. 1 B (A). Moreover, it shows enhanced proliferative activity in senescent CD27- CD28- CD4+ T cells transduced with shRNAs to Sesnl, Sesn2 and Sesn3 genes (B).
  • Figure 3 shows enhanced telomerase activity in cells as in Fig.l B (A). Moreover, it shows clearance of DNA damage foci assessed by the DNA damage response marker ⁇ - ⁇ 2 ⁇ in cells as in Fig, 1 B (B), and the results from 3 separate experiments performed as in Fig, 3B (C).
  • FIG. 4 shows reconstitution of the TCR signalosome component LCK with pooled results from 3 separate donors (A-B) in senescent human T cells transduced as above. Moreover, it shows restored CD27 and CD28 co-stimulatory receptor expression in the same cells (C).
  • FIG. 5 shows the identification of an endogenous Sestrin-AMPK-MAPK complex in primary human CD27 " CD28 " CD4 + T cells.
  • FIG. 6 shows measurement of global MAPK activity assessed by in vitro kinase assays in A PK immunoprecipitates from senescent human CD27 " CD28 " CD4 + T cells transduced as indicated then treated with the AMPK agonist A-769662 (150 ⁇ , 80') 96h later.
  • Figure 7 shows increased ATP abundance in AMPK- ⁇ immunoprecipitates from CD27 " CD28 D4 ⁇ T cells whereby one or more Sestrins were silenced by shRNA (A). It also shows AMPK activity of the same cells transduced as above (B); immunob!ots representative of 3 separate donors. GAPDH, loading control.
  • FIG. 8 shows reconstitution of the Sestrin-MAPK complex in lysates from nonsenescent CD27 + CD28 + CD4 + T cells incubated with either GFP or recombinant human Sesnl protein for 1 h, followed by p-AMPK immunoprecipitation (A). It also shows that Sestrins coordinate global MAPK activation in the AMPK complex upstream of ⁇ - ⁇ ( ⁇ ). Finally, it shows that Sestrins do not significantly alter MAPK-AMPK binding in the complex (C).
  • FIG. 9 shows a model of Sestrin-MAPK complex function in primary human senescent T cells.
  • FIG. 1 0 shows the varicella zoster virus (VZV)-specific proliferation in CD27 + CD28 + and CD27 " CD28 ' CD4 + T cells from 3 young and 3 old donors (A).
  • Cells were challenged with autologous APCs pulsed with VZV lysates (antigen dilution 1 :1 00 ⁇ for 4 days, followed by Ki67 assays.
  • VZV lysates antigen dilution 1 :1 00 ⁇ for 4 days, followed by Ki67 assays.
  • VZV-specific sensitivity proliferation
  • modulator of Sestrin means any modulator of the biological activity of Sestrins.
  • said modulator can be a negative modulator, that is any molecule able to impair, to inhibit the activity of Sestrins.
  • the modulator can be a positive modulator, that is, any molecule able to improve, stimulate or boost the biological activity of Sestrins.
  • Sestrin activity can be modulated at protein level, meaning that the modulators act directly and/or indirectly on the protein.
  • the modulation of Sestrins is realized on the expression of the genes codifying these proteins.
  • the modulation of Sestrin is particularly referred to immune system cells, more preferably to B cells, Natural Killer (NK) cells, or T cells (T lymphocytes), still more preferably CD4+ and/or CD8+ T cells, still more preferably CD4+ CD27+ CD28+ cells, CD4+ CD27- CD28+ cells, CD4+ CD27- CD28- cells.
  • immune system cells more preferably to B cells, Natural Killer (NK) cells, or T cells (T lymphocytes), still more preferably CD4+ and/or CD8+ T cells, still more preferably CD4+ CD27+ CD28+ cells, CD4+ CD27- CD28+ cells, CD4+ CD27- CD28- cells.
  • NK Natural Killer
  • T lymphocytes T lymphocytes
  • CD4+ and/or CD8+ T cells still more preferably CD4+ CD27+ CD28+ cells, CD4+ CD27- CD28+ cells, CD4+ CD27- CD28- cells.
  • Sestrin is a p53 and/or FOXO regulated stress-protein.
  • Sestrin is from humans wherein it is encoded by the SESN genes.
  • SESN genes There are currently known three isoforms of Sestrin, namely Sestrin 1 , 2 and 3 that in humans are encoded by SESN 1, 2 and 3 genes respectively.
  • a “modulator” means preferably small molecules, peptides, polypeptides, antibodies, nucleic acids, preferably small RNAs, more preferably shRNAs, siRNAs or imiRNAs, drugs, organic compounds or any combination of these molecules.
  • the modulator is directed against the N-terminal domain of Sestrin proteins, preferably against the residues 1 -160 of Sestrins, preferably Sestrin 2. More preferably said modulator is directed against SEQ ID NO: 1 corresponding to aa 1 -160. These amino acid residues are sequences highly conserved across all three human Sestrins. It has been demonstrated that the truncated Sestrin 2 protein at 1 -160 sites fails to interact with (and thus to activate) AMPK.
  • the modulator is directed against amino acid residues D406 and/or D407 of Sestrin proteins (D means Aspartic acid).
  • D means Aspartic acid.
  • This double DD site is also conserved across the C-term domain of all three mammalian Sestrins.
  • the shRNA is selected from SEQ ID NO: 8-10 or combination thereof.
  • immunosenescence or senescence of immune system means a gradual deterioration of the immune system brought on by the natural age advancement. It involves both the host's capacity to respond to infections, and the development of long-term immune memory, especially by vaccination. It is considered a major contributory factor to the increased frequency of morbidity and/or mortality among the elderly. It is triggered by the age-dependent accumulation of senescent lymphocytes, especially T cells, with short telomeres, endogenous DNA damage, lack of telomerase activity and loss of responsiveness to antigen stimulation because of increased Sestrin expression.
  • boosting the immune-response means restoring immune system cells responsiveness, especially T lymphocytes, to antigen stimulation, preferably in old humans and mice.
  • "rejuvenate immune-system” means restoring telomere length, and/or telomerase activity, and/or immune cells responsiveness, preferably T cell receptor responsiveness and/or antigen-specific proliferation and/or cytokine production in, immune cells, preferably T cells from old humans and mice.
  • rejuvenation will mean restoring antigen-specific antibody production by B cells and CD8 + T cells and Natural Killer cell expansion.
  • Sestrin's modulators as defined above allow rejuvenating immune system in an AMPK-MAPK dependent manner.
  • the present invention therefore discloses modulators of Sestrins to be used for boosting the immune system response in an individual and/or for rejuvenating the immune system of an individual and/or for reverting the senescent immune system of an individual.
  • the Sestrin's modulators of the invention are also useful for the treatments of conditions, preferably pathological conditions, associated with, or caused by, or related to the aging and/or the decline/impairment of the immune system, responses that may be mediated by T cells, B cells and NK cells.
  • the Sestrin's modulators interfere, inhibit and/or disrupt Sestrin-coordinate MAPK signaling complex. Therefore, they are useful to interfere, inhibit and/or disrupt Sestrin-coordinate MAPK signaling complex.
  • the boost and/or the rejuvenation of the immune system and/or the reversion of the senescent immune system is (are) AMPK-MAPK dependent.
  • Sestrin's modulation preferably involves cellular modulation, more preferably the cells are immune system cells, preferably T cells, B cells or NK cells.
  • the T cells are CD4+ or CD8+ T cells, more preferably CD4+ CD27+ CD28+ cells, CD4+ CD27- CD28+ cells, CD4+ CD27- CD28- cells.
  • the most preferred cells in the context of the present invention are CD4+ CD27- CD28- and CD8+ CD27- CD28- T cells that are senescent T cells. Therefore, Sestrin's modulators interfere, inhibit and/or disrupt Sestrin- coordinate MAPK signaling complex preferably in these cells.
  • the Sestrin's modulators are able to induce or to restore antigen specific cells and/or cytokine production, preferably in immune system cells, more preferably in T cells, more preferably these cells are senescent cells.
  • the Sestrin's modulators are also able to improve or to enhance the responsiveness of the immune system to a vaccine in an individual, preferably a vaccine against viruses, more preferably influence viruses.
  • the individual is preferably an old individual, in whom senescent cells accumulate with age.
  • Preferably said individual belongs to humans, but said modulators can be used in any mammals or animals to obtain the purposes disclosed herein.
  • the immune system cells preferably the senescent cells, show preferably short telomeres, and/or endogenous DNA damage, and/or lack of telomerase activity and/or loss or reduced responsiveness to antigen stimulation.
  • said modulators are molecules able to modulate Sestrin proteins or their gene expression as defined above.
  • Sestrin protein is any isoform or fragments of the protein, preferably from mammals, more preferably from humans. More preferably the human Sestrin protein is selected from: SEQ ID NO: 1 -4. Any fragments of Sestrins derived from these sequences should be considered part of the present disclosure.
  • the Sestrin protein is codified by at least one Sesn gene or its hortholog/homolog.
  • said Sestrin's gene is selected from: Sesnl, Sesn2 and Sesn3, more preferably is selected from SEQ ID NO: 5-7 or fragments thereof. Any sequence having 80-99.9% identity should be considered part of the present disclosure.
  • the Sestrin's modulator is selected from: small molecules, peptides, polypeptides, antibodies, nucleic acids, drugs, organic compounds or any combination of these molecules. More preferably, the modulator is directed against the N-terminal domain of a Sestrin protein.
  • the modulator is directed against the amino acid residues 1 - 160 of Sestrins, preferably Sestrin 2, still more preferably against SEQ ID NO: 1 corresponding to amino acid residues 1 -160 of Sestrin 2
  • the Sestrin's modulator is directed against amino acid residues D406 and/or D407 of Sestrin proteins (D means Aspartic acid), preferably of Sestrin2.
  • D406 and/or D407 are marked as bold underlined on SEQ ID NO: 2, that is the full length sequence of Sestrin 2 (see Table I).
  • the nuclei acid is a single and/or a double stranded nucleic acid, more preferably it is an RNA molecule.
  • said RNA molecule is at least one small RNA molecule selected from: short hairpin RNA (shRNA) and/or a short interfering RNA (siRNA) and/or imiRNA or combination thereof.
  • the shRNA molecule is directed against at least a portion of the gene or the cDNA codifying Sestrin proteins, more preferably said shRNA molecule is selected from SEQ ID NO: 8-10 or combination thereof, wherein SEQ ID NO: 8 is preferably directed against Sestrin 1 cDNA, SEQ ID NO: 9 is preferably directed against Sestrin 2 cDNA, and SEQ ID NO: 10 is preferably directed against Sestrin 3 cDNA.
  • GLEALMSSGRVDNLAVVMGLHPDYFTSFWRLH correspond to SEQ ID YLLLHTDGPLASSWRHYIAIMAAARHQCSYLVG NO: 1 (aa residues 1 - SHMAEFLQTGGDPEWLLGLHRAPEKLRKLSEIN 160) while the aa KLLAHRPWLITKEHIQALLKTGEHTWSLAELIQAL residues in bold VLLTHCHSLSSFVFGCGILPEGDADGSPAPQAPT underlined are PPSEQSSPPSRDPLNNSGGFESARDVEALMERM D406/D407) QQLQESLLRDEGTSQEEMESRFELEKSESLLVTP
  • the modulator is expressed in a vector, preferably a lentiviral vector.
  • a further aspect refers to a vector, preferably an expression vector such as lentiviral vector comprising the modulator of Sestrin, a sequence codifying the modulator.
  • the vector comprising the modulator of Sestrin is used to transduce cells and therefore to allow the expression of the Sestrin modulator into the cells. Therefore, a further aspect of the invention refers to cells, preferably transduced cells comprising/expressing the modulator of the invention.
  • the cells are preferably the T cells as disclosed above.
  • the modulators of the invention can be used alone or in combination with further molecules.
  • a further aspect of the invention refers to a pharmaceutical composition comprising the modulators of the present invention and pharmaceutically acceptable excipients.
  • said modulators or said composition or said vector or said cells are used in the treatment of an aging-related disease, preferably increased age-dependent increase of opportunistic infections and/or cancer due to lack of T cell mediated immune responses.
  • said modulators or said composition or said vector or said cells are used, in an individual, preferably an old individual, to boost or restore, immune cells, preferably T cell mediated immune-responses during ageing; and/or an infection, preferably a chronic infection, more preferably a viral infection; and/or cancer.
  • the Sestrin's modulator is used as adjuvant for antigen-specific, immune cells, preferably T cell responses to vaccination in an individual, preferably an old individual. Said vaccination is preferably against a virus, preferably influenza viruses.
  • Heparinized peripheral blood samples were collected from 120 individuals (aged 20-85, male 55% and female 45%). Samples from young (aged: 20- 35) and old (aged: 65-80) donors were obtained with the approval of the Ethical Committee of Royal Free and University College Medical School and voluntary informed consent, in accordance with the Declaration of Helsinki. Donors did not have any co-morbidity, were not on any immunosuppressive drugs, and retained physical mobility and lifestyle independence.
  • Lysates from 2 x 10 6 cells were used for Western Blot analysis. Endogenous signaling studies were assessed in ex vivo purified CD4+ CD27/CD28 T cell subsets as described (Lanna et al Nature Immunology 2014)
  • Transduced CD4+ CD27- CD28- T cells were analyzed by immunoblotting one week after activation (see below).
  • Membranes were probed with antibodies to: Sestrinl (Genetex), Sestrin2 (Cell Signaling), Sestrin3 (Sigma), ERK, p-ERK, p-JNK, p-p38, AMPK-cc, ⁇ - ⁇ - ⁇ , ⁇ , S6K1 , MKK7, MKK4, p-MEK1 /2 and GAPDH (all from Cell Signaling).
  • Sestrinl Genetex
  • Sestrin2 Cell Signaling
  • Sestrin3 Sigma
  • ERK p-ERK
  • p-JNK p-JNK
  • p-p38 AMPK-cc
  • GAPDH all from Cell Signaling
  • cell-lysates were prepared using ice- cold HNGT buffer (50 mM HEPES, pH 7.5, 150 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium orthovanadate, 100 mM sodium fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride).
  • HNGT buffer 50 mM HEPES, pH 7.5, 150 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium orthovanadate, 100 mM sodium fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride).
  • Lysates from 20x 10 6 cells were used for immunoprecipitation analysis.
  • For the minor CD4+ CD28- CD27- subset cells from two separate individuals were pooled to obtain sufficient material for the assay.
  • Extracts were incubated with the indicated antibodies at 4 °C on a rotary shaker overnight, followed by incubation with protein A-G conjugated agarose beads (Santa Cruz Biotechnology) at 4 °C for 3 h.
  • Co-immunoprecipitated proteins were detected using Mouse Anti-rabbit IgG Conformation Specific (L27A9; Cell Signalling) or Mouse Anti-rabbit IgG light chain, followed by a secondary anti-mouse IgG antibody (all from Cell Signalling) and ECL Prime Western detection kit (GE Healthcare).
  • lysates from CD4+ CD27+ CD28+ or CD8+ CD27- CD28- T cells will be incubated with recombinant Sestrins for 1 hour, followed by phosphorylated or total AMPK immunoprecipitation.
  • AMPK complexes were washed extensively and global MAPK activation was measured by immunoblotting or in vitro kinase assays (see below). Absolute MAPK binding was determined using ELISA-based total ERK, JNK and p38 detection assays according to the manufacturer's instructions (Abeam; see below).
  • absolute MAPK binding was determined on endogenous Sestrin immunoprecipitates from CD4+ (or CD8+) CD27- CD28- T cells, 48h post-transfection.
  • MAPK binding assays are shown as proportional to fold increase at 450 nm absorbance emission of triplicate wells ⁇ s.e.m, and were normalized to the indicated 'bait' proteins i.e. Sestrinl (MBS9327570) or AMPK- a (ab151280) used in the immunoprecipitation assay.
  • Sestrin or AMPK Immunoprecipitates were washed twice in lysis buffer and twice in kinase buffer (all from Cell Signaling). Kinase reactions were incubated for 30 min at 30 °C either in the absence or in the presence of 200 ⁇ ATP (Cell Signaling), as indicated. MAPK activity was assessed using Phospho-Tracer ELISA Kits according to the manufacturer's instructions (Abeam; see below).
  • exogenous recombinant human Sestrin proteins (Genway; 1 ng/mL), the AMPK agonist A-769662 (150 ⁇ ), the ERK inhibitor FR18024 (20 ⁇ ); the JNK inhibitor SP-600125, (10 ⁇ ); or the p38 inhibitor SB-203580, (10 ⁇ ) were added directly to the in vitro kinase reaction itself for 30', as indicated.
  • In vitro kinase assays are shown as proportional to fold increase at 450 nm absorbance emission of triplicate wells ⁇ s.e.m, normalized to the total amounts of co-immunoprecipitated MAPKs.
  • an antibody mix was prepared by adding Capture Antibody Reagent(s) to MAPKs and Detection Antibody Reagent (both provided with the Phospho Tracer kit by Abeam), in a 1 :1 ratio, and 50 ⁇ of this antibody mix were added to the in vitro kinase/binding reaction product (50 ⁇ ), loaded in the Phospho Tracer micro-plates.
  • Antibody binding was then allowed for 1 hour at room temperature on a micro-plate shaker. Wells were rinsed three times with 200 ⁇ of 1 X washing buffer (provided with the kit). Meanwhile, a substrate mix was prepared, immediately before use, by diluting 1 :100 the HR-substrate 10- Acetyl-3, 7- dihydroxyphenoxazine (ADHP) with ADHP Dilution Buffer (both provided with the kit), a stabilized H 2 O 2 solution. Next, 100 ⁇ of this substrate mix were added to each well, and incubated 10' at room temperature on a micro-plate shaker for colour development. The reaction of conversion of ADHP into the fluorescent molecule Resorufin was then stopped by adding 10 ⁇ of stop solution to each well. Signal was read using an Elisa reader micro-plate. Background was calculated in parallel IgG control immunoprecipitation reactions.
  • T cell ⁇ - ⁇ ⁇ immunoprecipitates were analyzed for ATP content using an ATP determination kit, according to the manufacturer's instructions (Life Technologies). Total ⁇ - ⁇ 1 immunoprecipitates were quantified by Elisa (Lifespan Biosciences).
  • the pHIV1 -SIREN-GFP system used for knockdown of gene expression possesses a U6-shRNA cassette to drive shRNA expression and a GFP reporter gene which is controlled by a PGK promoter5.
  • the following siRNA sequences were used for gene knockdowns: CCAGGACCAATGGTAGACAAA (shSesnl - SEQ ID NO: 8);
  • CCGAAGAATGTACAACCTCTT (shSesn2 - SEQ ID NO: 9); CAGTTCTCTAGTGTCAAAGTT (shSesn3 - SEQ ID NO: 10);
  • the shAMPKcc sequence was described and validated in primary human CD4+ T cells according to (Lanna et al Nature Immunology 2014). VSV-g pseudotyped lentiviral particles were produced, concentrated and titrated in HEK 293 cells as described in (Escors et al Blood 2008). Cell cultures and lentiviral transduction of primary human T lymphocytes
  • Cells were cultured in RPMI 1640 medium supplemented with 10% heat- inactivated FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 pg/ml gentamicin, and 2 imM L-glutamine (all from Invitrogen) at 37°C in a humidified 5% CO 2 incubator.
  • Non-senescent CD4+ CD27+ CD28+ T cells were cultured and transduced as above after activation by plate-bound aCD3 (0.5 ⁇ g/mL) plus aCD28 (0.5 ⁇ g/mL).
  • CD27- CD28- CD4+ T cells were re- activated 10 days post-transduction using autologous antigen presenting cells (APCs) pre-loaded for 4 hours with various dilutions of Varicella zoster virus lysates (Zeptometrix Corporation), as indicated.
  • APCs autologous antigen presenting cells
  • Antigen presenting cells were obtained by depleting CD3 positive cells from autologous, fresh PBMC preparations and cultured with transduced T cells, in a 3:1 ratio. Antigen-specific responses are shown as fold increase normalized to transduced cells before re-stimulation, with control cells set as 1 , in triplicate experiments.
  • Human or mouse primary CD4+ T cells were transfected with siRNAs to ERK, JNK, p38, ⁇ - ⁇ , MEKK1 , MEKK4, MEKK7 or an irrelevant control siRNA sequence (all from Santa Cruz Biotechnology) using Nucleofector Kit according to the supplier's protocol (Amaxa, Lonza Walkersville; program V024), as indicated. Cells were then rested and analyzed 48 hours later for functional or signaling readouts.
  • Telomerase activity was determined using the TeloTAGGG telomerase ELISA kit from Roche according to the manufacturer's instructions from extracts of 2 x 103 viable CD4+ CD27- CD28- T cells. The absolute numbers of CD4+ CD27- CD28- T cells were enumerated by trypan blue (Sigma) and proliferation was determined by Ki67 staining as described 50. Telomerase activity is expressed as proportional to fold increase at 450 nm absorbance emission of triplicate wells ⁇ s.e.m.
  • mice Age-matched (20 month-old) mice were used for in vivo studies. Control Sesn1 +/- and Sesnl -/- null mice were subcutaneously injected with the seasonal influenza vaccine FLUAD (Novartis; 1 : 20 of the human dose). A saline solution (PBS) was used as control injection. Five days later, animals were euthanized in a CO2 chamber, spleens collected, and processed for splenocyte isolation. Mouse CD4+ T cells were obtained from splenocytes by immunomagnetic separation (Miltenyi Biotec) and immediately analyzed for phenotypic, signaling and functional profiles by either flow cytometry or Image-Stream, as indicated.
  • FLUAD seasonal influenza vaccine
  • CD4+ phenotypic analysis was performed by surface staining to CD62L and CD44.
  • control Sesnl +/- and Sesnl -/- null CD4+ T cells were re- challenged with control Sesnl +/- APCs pre-loaded with FLUAD (1 :100, 1 :50 and 1 :25 of the human dose), then IL-2+, IFN-y+ op Ki67+ T cells were analyzed 18-hours later by flow-cytometry.
  • Sesnl +/- and Sesnl -/- mice were vaccinated with FLUAD as above described. One week later, mice were sacrificed and blood samples were immediately collected by cardiac bleeding. Levels of circulating immunoglobulins (IgGs) were analyzed using a serum dilution of 1 :200 in 0.1 % non-fat dry milk 0.5% Tween-20/PBS, as described 33. Samples were incubated on Nunc Maxisorp plates pre-coated with FLUAD overnight (1 :40; for 18 hours at 4 °C), or PBS as background control. Plates were washed 3 times with 0.5% Tween-20/PBS and blocked with 200 ⁇ of 4% non-fat dry milk (GE-Healthcare).
  • IgGs immunoglobulins
  • Antigen-specific serum antibodies were detected using horseradish peroxidase (HRP) conjugated antibodies (anti-mouse IgG, Sigma Aldrich) at 1 :3000 dilution in 0.1 % nonfat dry milk 0.5% Tween-20/PBS, at room temperature. Substrate activity was detected using 100 ⁇ _ of tetramethylbenzidine (TMB) substrate (BD Biosciences) and stopped using 50 ⁇ _ 2N H2SO4 per well. Circulating vaccine-speciific IgG levels were determined by ELISA (absorbance emission at 450 nm) of triplicate wells ⁇ s.e.m.
  • mice Aged-matched mice (16 months) were injected i.p. with the ERK inhibitor FR18024 (25 mg/Kg); the JNK inhibitor SP-600125 (16 mg/Kg); and the p38 inhibitor SB-203580 (10 mg/Kg) individually or with all 3 inhibitors together.
  • Drug vehicle was DMSO.
  • mice Three hours later, mice were vaccinated with FLUAD as above. Triple and individual MAPK inhibition treatments were then repeated daily. Five days later, mice were culled, spleens collected and the impact of in vivo blocking MAPK signaling on immune-responsiveness was analysed in Sestrin2+ T and B cell populations, as indicated.
  • CD4+ T cells Primary human or mouse CD4+ T cells were fixed with 2% paraformaldehyde, permeabilized with methanol and stained for Sestrin2, p-ERK, p-JNK and p-p38 direclty ex vivo, as above described.
  • CD4+ T cells were separated into early-differentiated and highly differentiated populations according to their relative CD27/CD28 expression profile from either young (20-35 years) or old donors (70-85 years). All samples were run on an Amnis® Image Stream cytometer using INSPIRE® software, magnification 60x. Data were analyzed using IDEAS® v.6.1 software (Amnis). Co-localization signals were determined on a single cell basis using Bright Detail Similarity (BDS) Score analysis. Co-localization was considered with BDS of >2.0.
  • a Superdex 200 (GE Healthcare) gel filtration column was first equilibrated with 1 .5 CV of endotoxin free PBS (Hyclone) at room temperature. Sestrin2 complexes were injected at time 0 (500 ul) and eluted using a single isocratic wash, fractions collected, and analyzed by ELISA-based binding assays (absorbance at 450 nm) across the spectrum for the indicated proteins. Phosphorylated MAPKs were detected using Phospho- Tracer ELISA kits (Abeam) as above described. For detection of Mios (MBS9330396); RagA (MBS9334409); and RagB (MBS9318748). The column was calibrated with protein markers as described 15
  • Graphpad Prism was used to perform statistical analysis. For pairwise comparisons, a paired Student's t-test was used. For three matched groups, a one-way analysis of variance (ANOVA) for repeated measures with a Bonferroni post-test correction was used. * p ⁇ 0.05 , ** p ⁇ 0.01 and *** p ⁇ 0.001 throughout. For correlation studies, Pearson correlation test. RESULTS
  • Sestrins exhibit anti-ageing properties in muscle, however immune related functions of these molecules have not been studied.
  • a non-silencing shRNA lentiviral vector was used as a control. Silencing of each individual Sestrin resulted in broad functional reversal of senescence in CD27 " CD28 " CD4 + T cells, including enhanced cell proliferation (Fig. 2A-B) and telomerase activity (Fig. 3A), diminished formation of DNA damage foci (Fig. 3B-C) and re-constituted expression of the TCR signalosome components Lck and ZAP70 (Fig. 4A and data not shown) and CD27 and CD28 co-stimulatory receptors (Fig. 4B-C).
  • This global enhancement of functionality in senescent T cells was further accompanied by restored calcium flux and IL-2 synthesis after activation. Therefore, in contrast to their well documented anti-ageing properties in invertebrates, the Sestrins simultaneously induce multiple characteristics of senescence in T cells, via a reversible process.
  • Sestrin function including their anti-ageing effects, are largely imTORCI - dependent in Drosophila, mouse liver and mammalian cell lines.
  • Sestrin silencing restored imTORCI signalling in senescent T cells; however, imTORCI was nonessential for the senescence promoting activity of Sestrins.
  • Sestrin 1 immunoprecipitates were specifically enriched for activated species of ERK, JNK and p38 MAPKs (Fig. 5A).
  • AMPK a Sestrin activation target previously shown to activate p38, was also co- immunoprecipitated with Sestrinl (Fig. 5A). Similar results were obtained when we investigated the co- immunoprecipitation of Sestrin2 with activated MAPKs.
  • Sestrins might operate as non-canonical regulator of MAPK function.
  • Sestrins promote global MAPK activation via AMPK, with which they and MAPKs associate.
  • MAPK Thr-x-Tyr activation loop is not a direct AMPK substrate, we reasoned that Sestrin-bound MAPKs might undergo AMPK-dependent auto-phosphorylation (an event that relies on intrinsic MAPK activities).
  • Cellular AMPK exists as a heterotrimeric protein of catalytic a and regulatory ⁇ and ⁇ subunits that are activated in response to increased intracellular AMP:ATP ratio.
  • Sestrins promote AMPK activation through a poorly understood mechanism.
  • Sestrins may orchestrate global MAPK signaling by promoting ATP removal from the ⁇ - ⁇ subunit.
  • Sestrin-MAPK complex we performed 'reconstitution' experiments using lysates of nonsenescent CD27 + CD28 + CD4 + T cells that lack endogenous Sestrins. Using in vitro binding assays, we showed that recombinant Sestrinl protein triggered global activation of p-AMPK associated MAPKs even in lysates of nonsenescent T cells (Fig. 8A).
  • Sestrin-MAPK complex formed as a unique macro- molecular complex.
  • Sestrin-MAPK complex eluted with an estimated molecular mass of about 1000 kDA.
  • the known Sestrin-mTORCI -inhibitory complex which contains GATOR2 components and RAGA/B 1_3 , was significantly smaller (660 kDA).
  • Sestrin expression was strongly induced upon irradiation, and as expected triggered global MAPK activation.
  • the stress-sensing capacity of Sestrins may be important for the manifestation of senescence in T cells.
  • each MAPK once activated, regulates unique hallmarks of the senescent state.
  • Sestrin2 expression enhanced telomerase activity, T cell activation as measured by increased calcium flux and decreased formation of DNA damage foci. Reinstating AMPK activation with the agonist reversed all the functional changes that occurred upon Sestrin2 silencing, suggesting that the Sestrins act upstream to and via AMPK to inhibit functional responses in CD27 " CD28 " CD4 + T cells. Importantly, inhibition of either p38, JNK or ERK in Sestrin2-depleted cells treated with the AMPK agonist significantly restored telomerase activity and TCR activation and reduced DNA damage foci, respectively. Similar observations were made when silencing Sestrinl . Thus, each individual MAPK regulates a distinct functional response. In contrast, since the Sestrins act upstream to all three MAPKs, their silencing restores all of the different MAPK-inhibited responses observed in senescent T cells, in an AMPK-dependent process (Fig. 9, proposed model).
  • the proliferation marker Ki67 was also up-regulated in CD4 + T cells from vaccinated Sesn1 ' mice relative to Sesn1 +/ ⁇ controls (Fig. 11 C).
  • Sesn1 ' null B cells showed enhanced IgG isotypic switch compared to controls after vaccination (a process that requires CD4 + T cell help and that declines with age) B cell proportions, however, were slightly reduced in the spleen of vaccinated Sesnl mice compared to vaccinated Sesnl controls.
  • the inhibition of Sestrins may be a strategy to boost vaccine responsiveness during ageing.
  • Sestrinl deficiency did not affect MAPK signalling in the minor Sestrin2 " subset. Because global disruption of Sestrin-MAPK signaling and enhanced T cell responsiveness to vaccination correlated directly, we reasoned that simultaneous inhibition of MAPKs would also boost vaccine responsiveness in aged mice, phenocopying Sestrin-deficiency.
  • ERK, JNK and p38 inhibitors i.p. either individually or simultaneously to 16-month old mice vaccinated with FLUAD, and evaluated the responsiveness to vaccination within the Sestrin2 + subset of T cells where MAPKs are Sestrin-responsive (see above).
  • mice simultaneously treated with all three MAPK inhibitors developed splenomegaly compared to controls, while those receiving each individual MAPK inhibitor did not.
  • triple (but not individual) MAPKi boosted responsiveness to vaccination of Sestrin2 + T cells, as measured by IFN- ⁇ synthesis. As per Sestrin deficient animals, the enhanced responsiveness to vaccination was not restricted to T cells.
  • Sestrin determines the impact of MAPKi on immune function in vivo.
  • simultaneous disruption of Sestrin-MAPK signalling is needed to enhance vaccine responsiveness during ageing, and phenocopies Sestrin deficiency.
  • Sestrin-MAPK complex is conserved between humans and mice. Our results suggested that the Sestrin-MAPK signalling complex might be conserved between mice and humans.
  • Sestrin2 complexes obtained from Sesn1 ' null CD4 + T cells exhibited much less ERK, JNK and p38 phosphorylation than control cells. Sestrini deficiency did not significantly alter total MAPK recruitment in the Sestrin2 complex instead.

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Abstract

La présente invention concerne des modulateurs de sestrines destinés à être utilisés pour renforcer la réponse du système immunitaire chez un individu et/ou pour régénérer le système immunitaire d'un individu et/ou pour inverser la sénescence du système immunitaire d'un individu. En outre, les modulateurs de sestrines de l'invention sont également avantageux pour le traitement d'états, de préférence d'états pathologiques, associés à, liés à ou provoqués par le vieillissement et/ou le déclin/la dégradation du système immunitaire, de préférence une réponse du système immunitaire de préférence médiée par des lymphocytes T.
PCT/IB2016/057209 2016-11-30 2016-11-30 Modulateurs de sestrines Ceased WO2018100410A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021069543A1 (fr) * 2019-10-08 2021-04-15 Luxembourg Institute Of Health (Lih) Inhibiteur de dj-1 destiné à être utilisé dans le traitement de l'immunosénescence
WO2023119337A1 (fr) * 2021-12-24 2023-06-29 Sentcell Ltd Inhibiteurs de complexes sestrine-mapk
WO2024256826A2 (fr) 2023-06-13 2024-12-19 SentCell Limited Activateurs complexes de sestrine-mapk

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WO2016040824A2 (fr) * 2014-09-12 2016-03-17 Whitehead Institute For Biomedical Research Procédés d'identification de modulateurs de l'interaction sestrine-gator2 et utilisation de ceux-ci pour moduler le mtorc1
WO2016116768A1 (fr) * 2015-01-23 2016-07-28 Imperial Innovations Ltd Agents neurothérapeutiques

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WO2016040824A2 (fr) * 2014-09-12 2016-03-17 Whitehead Institute For Biomedical Research Procédés d'identification de modulateurs de l'interaction sestrine-gator2 et utilisation de ceux-ci pour moduler le mtorc1
WO2016116768A1 (fr) * 2015-01-23 2016-07-28 Imperial Innovations Ltd Agents neurothérapeutiques

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J. H. LEE ET AL: "Sestrin as a Feedback Inhibitor of TOR That Prevents Age-Related Pathologies", SCIENCE, vol. 327, no. 5970, 5 March 2010 (2010-03-05), pages 1223 - 1228, XP055012738, ISSN: 0036-8075, DOI: 10.1126/science.1182228 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021069543A1 (fr) * 2019-10-08 2021-04-15 Luxembourg Institute Of Health (Lih) Inhibiteur de dj-1 destiné à être utilisé dans le traitement de l'immunosénescence
WO2023119337A1 (fr) * 2021-12-24 2023-06-29 Sentcell Ltd Inhibiteurs de complexes sestrine-mapk
WO2024256826A2 (fr) 2023-06-13 2024-12-19 SentCell Limited Activateurs complexes de sestrine-mapk

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