Sestrin-mapk complex activators

Abstract

The invention provides a linear or cyclic polypeptide mobiliser of sestrin from GATOR / mTOR to sMAC in non-senescent cells, a derivative or analogue thereof, comprising or consisting of an amino acid sequence derived from sestrin, or a truncation thereof. The polypeptides can be used in the treatment of conditions that require immediate interventions such as acute disorders, sepsis due to pathogen infections, or allergic reactions such as anaphylactic shock, as immunotherapeutic agents for cancer treatment, and in autoimmune disorders.

Description

[0001]Sestrin-MAPK Complex Activators The present invention relates to activator of sestrin-MAPK complexes (sMAC), herein also referred to as mobilisers of sMAC (“MOS”). The invention also relates to the treatment of conditions that require immediate interventions such as acute disorders, sepsis due to pathogen infections, or allergic reactions such as anaphylactic shock. The invention further relates to the use of MOS compounds as immunotherapeutic agents for cancer treatment, since they can trigger a potent and specific immune response against tumour antigens, especially metastatic untreatable cancer. More specifically, given the delayed effect of the compounds on promoting regulatory T cell generation and suppressing function, the invention relates to slow release MOS formulations which have utility in autoimmune disorders. The invention also extends to pharmaceutical compositions comprising the MOS compounds, and to a process for making a pharmaceutical composition. Every year sepsis affects between 47 and 50 million people in the world, with a low rate of survival (at least 11 million die – one death every 2.8 seconds) (global-sepsis-alliance data). The best approach for sepsis is currently rapid intervention followed by antibiotic treatment (Marx G et al. Chirurg.2005). In many cases, patients encounter antibiotic resistance problems encompassed by the use of alternative therapeutic agents with unsatisfactory results obtained so far (John Turnidge; Scand J Infect Dis2003). In addition to this, a major problem remains that many survivors to sepsis suffer from delayed consequences of sepsis for the rest of their lives (Shankar-Hari et al. Curr Infect Dis Rep.2016). Similarly to sepsis, anaphylactic shocks (serious allergic reactions) are another worldwide cause of hospitalisation and death (40 to 500 per million person-years) (Cardona V. et al. W.A.O. Journal 2020). Here, a prompt injection of adrenaline is crucial to restrain such immune reaction. Recently, the increased rate of autoimmune disorders due to nonspecific and abnormal responses of the immune system against the self has gained much attention. At the moment, there are more than 80 types of autoimmune diseases encompassing as defined herein inflammatory bowel diseases, rheumatoid arthritis, type 1 diabetes and multiple sclerosis. It has been reported that these conditions affect around 10% of the global population; the majority of these cases are incurable and require lifelong treatment mainly of immune- suppressive nature (Conrad et al; Lancet Journal 2023). Neurodegenerative diseases also affect millions of people worldwide; with major incidence for Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis (herein included as “neurodegenerative diseases”). The direct effect of neurodegeneration, with progressive dysfunction and loss of neurons and axons in the central nervous system, constitutes the primary pathological feature; the intervention of the immune system in this context constitute an additional aggravating factor. Several immunotherapeutic approaches have been used so far to contrast inflammation with serious unwanted side effects (Amor S. et al. Immunology 2010). In addition to the above-mentioned conditions, another worldwide health emergency is cancer; about ten million people die from cancer every year (Max Roser and Hannah Ritchie; OurWorldindata 2019). It has been well reported that in many cases cancer cells are able to evade the immune system to grow and invade tissues (S. Keun Kim & S. Wook Cho; Front. Pharmacol, 2022). This is possible by presenting on their surface proteins that dampen the ability of immune cells to react against them. Recent therapeutic approaches are based on blocking the interactions of these cancer surface proteins with the immune cells (immune checkpoint inhibition) (Yavar Shiravand et al; Curr Oncol.2022). Although this approach confers high expectation for the cancer treatment, adverse effects linked to the acute clinical toxicities of these agents are still under consideration (Johson et al.; Nature Reviews Clinical Oncology); furthermore, escape mechanisms make cancer cells resistant to immune therapy or other chemotherapy (Wang X et al; Cancer Drug Resist 2019). There is, therefore, a great need for improved therapies that can directly target, awaken and quickly boost the immune response, in particular CD4 and CD8 T cell response to resolve pathogen sepsis and to arm a response against cancer cells. There is also a great need for treatments able to boost proliferation and regulatory activity of Treg cells; these includes autoimmune and neurodegerative disorders. The inventors have previously shown that sestrin-MAPK complexes (sMAC) accumulate in immunological cells (e.g. T cells) during aging or during hyper-inflammation status (Lanna et al, Nature Immunology, 2014 and 2017; WO / 2018 / 100410). sMAC is a large complex composed of stress-molecules known as sestrins, of heterotrimeric protein AMPK, a large complex of immune-inhibitory proteins, and of mitogen-activated protein kinase (MAPK). Sestrins bind to, and then activate AMPK that triggers the following activation through auto- phosphorylation of the MAPKs, ERK, JNK and p38 (Lanna et al; Nature Immunology 2017). These three MAPKs have been reported to regulate different cellular processes: ERK induces DNA damage, JNK inhibits TCR-CD28 signalling and p38 blocks telomerase activity. The inventors have previously shown that a new pharmaceutical class of compounds, called “DOS” (disruptors of the sMAC), are able to inhibit the sMAC complex that accumulates in senescent cells. Disruption of the sMAC complex triggers a long-term rejuvenation cascade in the targeted immune cells becoming able to mount a specific and strong immune response to vaccines or to future pathogen insults. Possible sMAC inhibitory approaches including shRNA and sestrin-based peptide inhibitory technologies have been described. However, in the alternative there are situations where the accumulation of the sMAC in specific immune cells may be beneficial. As is described herein, the inventors have identified a novel role for the sMAC in triggering robust T cell proliferation through activation of mTOR: novel sestrin-derived peptides have an unexpected opposite effect in unleashing sestrin from GATOR / mTOR complexes resulting in sMAC activation. The compounds, referred to herein as “MOS” (mobiliser of sMAC), initially activate mTOR and telomerase activity in T cells resulting in potent T cell proliferation and effector T cell expansion. Paradoxically, these compounds also simultaneously activate the sMAC, which is a well- known inhibitor of telomerase, through p38 MAPK. There is therefore, for a short time (e.g. 12-24 hours), coexistence of both activated sMAC and mTOR complexes in the same effector T cell, at the same time, when the MOS compounds are administered which is surprising from previous knowledge of sMAC and mTOR. Subsequently (e.g.3 days after treatment), sMAC activation prevails, mTORC1 becomes inactive, and regulatory T cells are formed. This paradoxical pathway operates in young T cells that possess a GATOR / mTOR regulatory system, most preferentially in CD27+ / CD28+ and / or CD27+ / Cd28- and / or CD45RA+ and CD45RO+, but not in highly differentiated senescent T cells (CD27- / CD28-) that possess sMAC but not mTOR. Therefore, T cells treated with MOS can become responsive to stimulus very quickly although this will drive them to senescence. Subsequently, they can turn into regulatory T cells that suppress excessive inflammation, or autoimmunity. A specific formulation that is either fast or long lasting (e.g. slow release) may alter or favour one process over the other, respectively. Furthermore, at the moment, there are no drugs for all those medical emergencies where immediate immune response is imperative. There is, therefore, a great need to develop a therapeutic approach that can directly target sestrin-dependent sMAC activation in early stage of immune response, in those conditions where T cell senescence may be a reasonable cost to sustain immediate T cell responses in life-threatening conditions such as sepsis, anaphylactic shocks or in case of unresponsive metastatic tumors. In this context, the MOS approach could provide a valid and resolutive approach. As described above, the present inventors previously reported inhibitory sMAC peptide compounds (“DOS”) for immune rejuvenation. During the course of further assessing peptide activity to minimal sMAC in vitro, it was unexpectedly found that certain peptides of identical lengths to DOS (pentameric), derived from the same portion of the sestrin protein, displayed opposite sMAC stimulating activity. These compounds are referred to as “MOS” (mobiliser of sMAC). Accordingly, the MOS compounds mobilise the sestrin from GATOR to the sMAC complex. In the GATOR-mTOR complex two related proteins, GATOR1 and GATOR2, control mTOR activity; GATOR1 is a known inhibitor of mTOR and GATOR2 is an inhibitor of GATOR1 thereby producing positive regulation of mTOR signalling instead. Moreover, sestrins bind to and provide additional negative regulation of GATOR2 suppressing mTOR function. As such, when sestrins dissociate from GATOR2 in response to physiological stimuli such as leucine uptake in T cells, mTOR activation occurs. However, the fate of the dissociated sestrin has not been studied. The present inventors found that MOS quickly disrupt the binding between GATOR2 and sestrins in T cells. The resulting MOS effect is therefore potent mTOR activation. There is an initial equilibrium between mTOR activation (e.g. when sestrin dissociate from GATOR2) and generation of the sMAC (e.g. when sestrin move to the ER and bind AMPK- MAPK complex). Then, sMAC signals prevail and mTOR is inhibited by p38 in the sMAC. Therefore, when not in the GATOR, sestrins are found in the sMAC instead (Figure 1). The present invention provides MOS, the first class of compounds that modulate this balance between mTOR activation and sMAC generation, suitable for emergency boosting of the immune response, or delayed control of autoimmunity. Thus, according to a first aspect of the invention, there is provided a linear or cyclic polypeptide mobiliser of sestrin from GATOR / mTOR to sMAC in non-senescent cells, a derivative or analogue thereof, comprising or consisting of an amino acid sequence derived from sestrin, or a truncation thereof. The term “derivative or analogue thereof” includes polypeptides within which amino acid residues are replaced by residues (whether natural amino acids, non-natural amino acids or amino acid mimics) with similar side chains, peptide backbone properties or chemical nature. For instance, serine present in MOS SEQ ID No 6, discussed below, may be substituted with a chemical analogue (polar) of this amino acid, such as tyrosine, deriving a similar MOS compound with an analogue effect. Additionally, “derivative or analogue thereof” includes polypeptides composed of amino acids that due to the “degeneracy of the genetic code” derive from a different DNA sequence. Therefore, the introduction of silent changes of the third nucleotide of the triplet in the DNA sequences can produce an identical MOS peptide sequence. In addition, the terminals of such peptides may be protected by N- and / or C-terminal protecting groups with similar properties to acetyl or amide groups. Preferably, the linear or cyclic polypeptide, derivative or analogue thereof comprises or consists of 5 amino acid residues. In one embodiment, the linear or cyclic polypeptide, derivative or analogue thereof comprises or consists of an amino acid sequence as set out in any one of SEQ ID Nos: 4 to 11, as follows: SEQ ID No Amino acid Compound SEQ ID No Amino acid Compound sequence name sequence name 4 LAVVM MOSα 8 GSHMA MOSε 5 AVVMG MOSβ 9 SHMAE MOSζ 6 YFTSF MOSγ 10 TGGDP MOSη 7 IAIMA MOSδ 11 GLHRA MOSθ The present invention also extends to polypeptides having a similarity of more preferably at least 80% identity to the sequences disclosed herein, preferably more than 60% identity. Human cells express three sestrin proteins, sestrin 1, sestrin 2 and sestrin 3 (Lee et al., 2013), coded by the genes Sesn1, Sesn2, and Sesn3, respectively. Accordingly, in an embodiment, the linear or cyclic polypeptide, derivative or analogue thereof, comprises or consists of an amino acid sequence derived from sestrin 2, or a truncation thereof. More preferably, the linear or cyclic polypeptide, derivative or analogue thereof, comprises or consists of an amino acid sequence derived from SEQ ID No: 1. Thus, the 8 MOS compounds of SEQ ID Nos: 4 to 11 fully match the sestrin 2 protein sequence (Gen Bank: EAX07703.1) as showed below in the SEQ ID No: 1; (in bold are the conserved amino acids): MOSγ ------------------------------------------------------------ 5 MOSβ ------------------------------------------------------------ 5 MOSα ------------------------------------------------------------ 5 sestrin2 arhqcsylvgshmaeflqtggdpewllglhrapeklrklseinkllahrpwlitkehiqa 180 [SEQ ID No: 1] In another embodiment the 8 MOS compounds result to partially match the human sestrin 1 sequence (Gen Bank: AAI12037.1; full-length form). Thus, the linear or cyclic polypeptide, derivative or analogue thereof, may comprises or consists of an amino acid sequence derived from SEQ ID No: 2. MOS matches with SEQ ID No: 2 shown below, are as follows; (in bold are the conserved amino acids): MOSα ------------------------------------------------------------ 0 MOSβ ------------------------------------------------------------ 0 MOSε ------------------------------------------------------------ 0 MOSζ ------------------------------------------------------------ 0 MOSθ ------------------------------------------------------------ 5 MOSγ ------------------------------------------------------------ 5 MOSη ------------------------------------------------------------ 5 [SEQ ID No: 2] Alternatively, in another embodiment MOS compounds show a slight match with the sestrin 3 protein sequence (Gen Bank: KAI2562389.1; full-length form). Thus, the linear or cyclic polypeptide, derivative or analogue thereof, may comprises or consists of an amino acid sequence derived from SEQ ID No: 3. MOS matches with SEQ ID No: 3 shown below, are as follows (in bold are the conserved amino acids): MOSα ------------------------------------------------------------ 0 MOSβ ------------------------------------------------------------ 0 MOSε ------------------------------------------------------------ 0 MOSζ ------------------------------------------------------------ 0 MOSθ ------------------------------------------------------------ 0 MOSη ---TGGDP---------------------------------------------------- 5 MOSγ ------------------------------------------------------------ 0 MOSδ ------------------------------------------------------------ 0 sestrin3 mnrgggspsaaanyllctncrkvlrkdkrirvsqpltrgpsafipekevvqantvdertn 60 [SEQ ID No: 3] Therefore, MOS compounds most preferably show specificity to sestrin2, preferably to sestrin1, and less preferably to sestrin 3. Of note, only sestrin 2 and sestrin 1 are reported to be mobile elements in the GATOR / mTOR complex (e.g. in response to leucine uptake), whereas sestrin 3 may be tonically bound (Wolfson R. L. et al; Science 2016). In turn, this mobilises sestrins from the GATOR-mTOR complex to the sMAC. In one embodiment, the most preferred compounds are MOSγ and MOSη. In another embodiment, preferred compounds are MOSε, MOSα and MOSζ. Additionally, in another embodiment less preferred compounds are MOSθ, MOSβ and MOSδ. The most preferred MOS compounds are those that induce 60-100% of sMAC activity; preferred MOS compounds are those with >50% of sMAC activity, and less preferred MOS compounds are those with <50% of sMAC activity. The linear or cyclic polypeptide, derivative or analogue thereof of the present invention is preferably for use in therapy. The present invention also provides a nucleic acid encoding the linear or cyclic polypeptide, derivative or analogue thereof of the present invention. The nucleic acid is preferably for use in therapy. The linear or cyclic polypeptide, derivative or analogue thereof or nucleic acid of the present invention are preferably for use in suppressing inflammation, boosting immune response, and / or delaying control of autoimmunity. The linear or cyclic polypeptide, derivative or analogue thereof or nucleic acid of the present invention preferably target sestrin-dependent sMAC activation in immune response. The linear or cyclic polypeptide, derivative or analogue thereof or nucleic of the present invention are preferably for use in the treatment, prevention or amelioration of acute disorders, sepsis, or allergic reactions. The linear or cyclic polypeptide, derivative or analogue thereof or nucleic acid of the present invention are preferably for use in the treatment, prevention or amelioration of anaphylactic shock. The linear or cyclic polypeptide, derivative or analogue thereof or nucleic acid of the present invention are preferably for use in the treatment, prevention or amelioration of autoimmune disorders and / or neurodegenerative diseases. The linear or cyclic polypeptide, derivative or analogue thereof or nucleic acid of the present invention are preferably for use as an immunotherapeutic agent in treating, preventing or ameliorating cancer. The linear or cyclic polypeptide, derivative or analogue thereof or nucleic acid of the present invention are preferably for use in the proliferation and regulatory activity of Treg cells. The linear or cyclic polypeptide, derivative or analogue thereof or nucleic acid of the present invention are preferably for use as a bactericidal agent, for example in a bactericidal or antibiotic composition. The present invention also provides a pharmaceutical composition comprising a linear or cyclic polypeptide, derivative or analogue thereof or nucleic acid according to the present invention and optionally a pharmaceutically acceptable vehicle. The pharmaceutical composition may comprise a sustained or controlled or delayed release dosage form. The present invention further provides a process for making a pharmaceutical composition according to the present invention, the process comprising combining a therapeutically effective amount of a linear or cyclic polypeptide, derivative or analogue thereof or a nucleic acid according to the present invention with a pharmaceutically acceptable vehicle. For a better understanding of the invention, and to show how embodiments of it may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which: Figure 1 shows a graphical representation of the mechanism of action of MOS compounds. In the first instance the binding of MOS to sestrin triggering its detachment from the GATOR complex leads to mTOR activation. This activation results in cell proliferation with T effector induction; also teomerase activity is induced at this stage. There is then an intermediate status when mTOR is still active and sMAC starts to be formed. The latest step is instead characterised by switch-off of mTOR and sMAC activation that triggers robust Treg induction and senescence formation. mTOR inactivation in this latest phase is directly due to p38 MAPK activation within the sMAC. Figure 2 in (A) demonstrates MOS disruption of sestrin-GATOR2 (MIOS) binding. The dissociated is now bound to the sMAC (p38). (B), direct biotinylated MOS binding to Gator- sestrin complexes in human CD4+ T cells. MOS compounds were directly reacted with T cell extracts for 18 hours prior to assay. In (C) reported increase formation of the sMAC in human CD4+ T cells treated with MOS compounds (MOS γ, MOS η, MOS ε) compared to an untreated control. On the right, a quantitative plot summarizing generation of the sMAC. Figure 3 in (A) shows the induction of senescence by MOS compounds in T cells. On the left, representative population phenotype; on the right quantitative plot of induction of senescence levels. Quantification of (B) reduction of stemness; and (C) increase of progenitors in cells treated with MOS. (D) Triggered cytokine response, specifically TNFα and IFNγ, under MOS treatment among effector senescent prone T cells. In (E) are reported increased levels of Treg in CD28+ cells when treated with MOS. (F) Augmented Fox p3 expression and suppressive function of human Tregs overnight pre-treated with MOS ( ^ or η) and then co-cultured with CTV labelled conventional CD4+ T cells for one week. Figure 4 shows in (A) the high proliferative activity that MOS compounds trigger in T cells. The proliferation is investigated by following the levels of the nuclear protein Ki67. On the left a quantitative plot with Ki67 levels; while on the right a representative FACS plot is shown. In (B) it is reported the increased telomerase activity found when MOS is used. In (C) the levels of pS6k support the mTOR activation induced by MOS; while (D) a time course of mTOR and sMAC activation under MOS treatment is presented. Figure 5 presents disruption of telomere transfer from APCs to MOS treated T cells compared to the DOS compound earlier reported to stimulate this process. Figure 6 (A) shows levels of MCF7 cancer cell killing induced by the co-culture with MOS treated T cells after the overnight culture. (B) shows that tumour infiltrating Tregs can be transformed into potent effector killers upon short treatment with MOS (48 hours). Human CD4+ T cells were exposed or not to neuroblastomas in culture for one week, their Tregs derived, and then exposed or not to MOS ^ (1 ^M), prior to Treg cancer re-exposure. Specific Treg-tumour killing was measured as neuroblastoma CTV dilution in co-culture assays. Data are shown as fold change to Tregs that had not been treated with MOS, with or without cancer pre-exposure. Figure 7 shows bactericidal effects of MOS on bacterial growth. CD3- cells (APCs) were treated or not to MOS ^ (1 ^ ^) ^ and exposed to E. Coli 18 hours later. Supernatants were then diluted and bacterial were seeded on agar plates, with or without Ampicillin. MOS treated APCs killed bacteria both in the presence or in the absence of antibiotic and with or without resistant bacterial strains. Table 1 summarizes the sMAC activity modulation (fold change) of 8 preferred MOS compounds vs DOS compounds known to decrease the sMAC activity. Table 2 summarizes all the MOS properties in comparison to the earlier reported DOS compounds. Materials and Methods In vitro screening inhibition of AMPK1 stimulation by Sestrin1 using the peptide library 1. Recombinant protein AMPK1 (340 ng) and DOS peptide (20 µM) were incubated for 30 minutes at RT in a 384 well plate; 2. Sestrin1 / ATP / AMP / SAMsite (1ug) were added and incubate for 1 hour at room temperature; 3. 20 µl ADP-Glo reagent (Promega) were added and incubated for 40 min at room temperature; 4. Were then added 40 µl of kinase detection reagent and incubated for 30 min at RT room temperature 5. Results were acquired at the ELISA reader. Cell isolation and culture 1. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (17-1440-03; GE Healthcare) density gradient centrifugation from blood of healthy volunteers. 2. CD4+T cells were isolated using CD4+T Cell Isolation Kit (130-096-533; Miltenyi); 3. CD3- cells (thereafter APCs) were instead isolated by using human CD3 MicroBeads (130-050-101; Miltenyi). 4. Cells were cultured in RPMI1640 (R2405; Sigma-Aldrich) supplemented with 10% FBS (F9665; Sigma-Aldrich) and 1% Pen-Strep (DE17-602E; Lonza). 5. Cells were activated with Dynabeads Human T-Activator CD3 / CD28 (11161D; Gibco) in an incubator (37 ⁰C and 5% of Co2). In vitro MOS disruption of sestrin-GATOR2 interaction 1. Purified total CD4+ T cells were treated with MOS ^ ( ^ ^ ^M) overnight. 2. After 18 hours, cells were lysed in IP buffer (Hepes (20 mM), NaCl (100 mM), KCl (100 mM), MgCl2 (1 mM), Glycerol (10%) and NP-40 (0.5%) and protease inhibitor (1X)) for 30 minutes on ice followed by centrifugation at 20000g for 30 minutes. 3. The supernatant was then incubated with anti-Sestrin 2 antibody overnight (5 µg). 4. The following day, protein A / G agarose beads (20422; Thermo Scientific) were incubated together with the lysate with anti-sesn2 antibody for 3 hours at 4°C on a rotor. 5. The resulting immunoprecipitate was then eluted by addition of Laemmli buffer 1x, and assessed by western-blot assay. In vitro pull down with biotinylated-MOS 1. Purified CD4+ T were lysed in IP buffer as above described and the lysate was in vitro treated with biotinylated MOS ^ or MOS ^ (10 ^M) overnight. 2. After 18 hours the lysate was incubated with streptavidin agarose beads (20353; Thermo Scientific) 3 hours at 4°C on a rotor. 3. Streptavidin beads were then washed with IP buffer and proteins were eluted by addition of Laemmli buffer 1x, followed western-blot assay. Western blotting 1. Protein lysates, prepared as above described, were denatured with Lemmli buffer + β- Mercaptoethanol. 2. Proteins were separated on polyacrylamide gel and then transferred on nitrocellulose membrane by the Trans-Blot dry system (Biorad). 3. Membranes were blocked for 1 hour at room temperature with milk (5%) or BSA (5%) depending on if the protein to detect were phosphorylated or not. 4. After blocking, membranes were incubated overnight at 4 °C with primary antibodies (1:1000; anti-MIOS; #13557, Cell Signaling; anti-Sestrin2; ab178518, abcam; anti-p38; #8690, Cell Signaling; anti-WDR59; #53385, Cell Signaling). 5. Membranes were then washed in TBS-tween (0.1 %) and incubated with secondary antibody IgG-HRP conjugated antibody (1:5000) 1 hour at room temperature. Cell treatment with MOS 1. Purified CD4+T cells were electroporated with linear MOS compounds (two different concentration 10µM and 400nM) according to the Amaxa (Lonza) protocol for human lymphocytes. 2. Cells were left to rest overnight or left in culture for three days with supplemented RPMI media. Immunephenotype validation 1. CD4+T cells treated with MOS or left untreated were stained for 30 min at room temperature with antibodies against canonical surface proteins (CD4, CD8, CD3, CD45RA, CD28, CD62, CD95, CD25). All antibodies were diluted 1:100. 2. Followed cell washes with PBS. 3. Cells were then fixed with PFA 4% for 15 mins at 37C 4. Permeabilized with cold methanol 90% for 30 mins at 4C 5. Cells were then stained for 1hr at room temperature, for the intracellular proteins sestrin2 and phospho-p38 with primary unconjugated antibodies diluted 1:100. 6. Followed incubation with secondary antibodies conjugated (diluted 1:250). 7. Cells were washed and analysed at the cytometer Cytoflex (Beckman-Coulter). Antibody Supplier Clone CD3 PE Miltenyi REA613 CD4 Alexa Fluor 700 BioLegend SK3 CD28 APC Miltenyi REA612 CD45RA BV510 BioLegend HI100 CD62L PE-Vio 770 Miltenyi 145 / 15 Ki-67 PE Miltenyi REA183 KLRG1 VioBlue Miltenyi REA261 CD95 APCVio770 Miltenyi REA570 CD25 APC Miltenyi REA570 FOXP3 PE Miltenyi REA1253 Phospho-p38 CellSignaling D3F9 (unconjugated) Sestrin2 (unconjugated) Abcam EPR18907 p-S6 (unconjugated) CellSignaling 2211 Cytokine production validation by flow-cytometry 1. CD4+ T cells treated with linear MOS at 10µM or 400nM or left untreated were co- cultured with autologous APCs for three days in presence or not of M-M-RvaxPro vaccine (1:50; Merck Sharp & Dohme B.V). 2. After this time, co-cultured cells were treated with brefaldin A (1:1000) for 4hr. 3. Cells were then stained with antibody against conventional T cell surface proteins 4. Followed intracellular staining with antibodies against IFNγ, TNFα 5. Cells were then assayed at the flow-cytometer. Investigation of Tregs suppressive function 1. Purified CD4+CD25+ were labelled with CFSE (1:1000) and treated 2 hours with MOS ^ or MOS ^ (1uM). 2. Cells were then washed and co-cultured for 7 days with CD4+CD25- Tconv cells, prior labelled with CTV (1:1000). 3. After one week, FOXP3 expression and Treg suppressive function on Tconv cells were assessed by flow-cytometry. Telomere live transfer 1. Purified APCs were left to adhere to the plate and then labelled with the telomere probe (TelC Cy3-PNA; Panagene) 2. In parallel, autologous CD4+ T cells were electroporated with MOS compounds and left to rest. 3. Stained APCs and treated CD4+T cells were then co-cultured in a ratio of 2:1, in presence or not of the CMV and EBV antigen mix (1:50). 4. Cells were left in co-culture overnight 5. The day after, cells were stained for the conventional T cell surface proteins and analyzed at the flow-cytometer. Telomerase activity assay 1. Purified PBMCs were electroporated with MOS compounds or left untreated. 2. Cells were then stimulated Dynabeads Human T-Activator CD3 / CD28 and left in culture overnight. 3. After this time, cells were pelleted and assayed for telomerase activity according to the TeloTAGGG Telomerase PCR ELISA Kit (11854666910; Roche). mTOR activation through pS6 detection 1. Purified CD4+ T cells were electroporated with linear MOS compounds or left untreated. 2. Cells were then stimulated with Dynabeads Human T-Activator CD3 / CD28 (1:100) and treated with rapamycin (100 nM; Sigma Aldrich) or with p38 inhibitor (10 µM; SB203580-Invivogen) or with a combination of both. Untreated cells were used as control. 3. After the overnight incubation CD4+T cells were stained for the conventional T cell surface proteins and for the intracellular phosphorylated form of the protein S6. 4. Cells were then investigated for the pS6 levels at the flow-cytometer. Assess the ability of MOS treated conventional CD4+ T cells to kill tumor cells 1. Purified CD4+ T cells were treated with linear MOS compounds at 10µM 2. In parallel, MCF7 human breast cancer cell line were detached and counted 3. Two millions of MCF7 cells were then stained with CTV dye (ThermoFisher) 4. 10*4 stained tumor cells / well were then plated in a pre-treated 96 well plate 5. 10*5 / well treated CD4+ T cells were then added to the plated tumor cells 6. The co-cultured was left for 18 and 48 hr 7. Followed staining of surface T cell proteins and cyto-fluorimeter analysis Generation of anti-cancer activity among tumour infiltrating regulatory T cells 1. Purified CD4+ T cells were co-cultured with SK-N-SH human neuroblastoma cells to generate TILs (tumour infiltrating lymphocytes), or activated with anti-CD3 / CD28, for one week, as a control. 2. Human Tregs were purified one week later and exposed or not to cyclic MOSγ ( ^ ^ ^), for 48 hours prior to re-exposure to SK-N-SH neuroblastomas that had been pre- labelled with CTV dyes. 3. Forty-eight hours later, specific Treg driven cancer lysis was detected by flow- cytometry, as described (Pereira et al., Nature Immunology 2020). Bactericidal effects of MOS on bacterial growth 1. CD3- cells (APCs) were purified from total PBMCs and were treated or not with cyclic MOS ^ ( ^ ^ ^) overnight at 37 °C. 2. After 18 hours, cells were co-cultured with E. Coli (MOI 10) in RPMI with 10% FBS (with no addition of Penicillin-Streptomycin) for 1:30 hours. 3. Supernatants were then diluted with sequential dilutions (1:10) and bacteria were seeded on agar plates, in the presence or in the absence of ampicillin (8 ^g / ml) ^ 4. Plates were incubated overnight at 37 °C followed by bacterial colony counts. Example 1 – Identification of MOS compounds The inventors, during the development of the DOS compounds previously reported, tested 156 polypeptides for AMPK activity in an in vitro assay with bacterially-purified proteins (sestrin 1 and sestrin 2 and AMPK) by ATP level detection. On this screening it was found that 8 polypeptides were not able to abrogate AMPK activity in response to both sestrin 1 and 2. Rather they had the opposite effect (potent sestrin-driven AMPK activation). Nevertheless these polypeptides with different sequences, were rationally designed on the same region essential for sestrin-AMPK interaction (residues 1-160 of sestrin 2) that allowed identification of sMAC inhibitory compounds (DOS). Their length of five amino acids is considered to be the limit length to generate cell penetrating peptides with inhibitory functions as previously reported. However, nothing in the structure of the compounds suggested an opposite regulatory effect on the sMAC (activation rather than inhibition). Example 2 – Ability of MOS compounds to activate sMAC Among the 8 MOS compounds tested, the inventors identified MOSγ, MOSη and MOSε as candidates to test the sMAC activation; for this reason the following studies are focused on these compounds as matter of exemplification. Biochemical studies demonstrate disruption of sestrin-GATOR2 binding in immunoprecipitate sestrin2 from not senescent CD4+T cells and a co-current binding of the dissociated sestrin to the sMAC (Figure 2A), and direct MOS binding to the GATOR-sestrin complex in T cells (Figure 2B). Titration studies of the MOS compounds in total CD4+ T cells revealed that they are effective at nanomolar amounts by increasing the generation of the sMAC (400 nM), evident as the increase in p38 phosphorylation and sestrin 2 expression as show in Figure 2C and Table 1. These effects are comparable with the physiological induction of sMAC in highly differentiated senescent T cells of the elderly (Lanna et al.; Nature Immunology 2017). Table 1 Example 3 – Ability of MOS compounds to induce senescent and regulatory T cells among immune cells Investigation of the immune-phenotype of immune conjugated between T cells and APCs pulsed with antigen mix (M-M-RvaxPro vaccine) treated with MOS compounds for 3 days show a significant increase of senescent CD4+T cells evident as prominent increase of both effector memory and EMRA human T cell subsets (Figure 3A). In parallel, MOS compounds reduce the formation of stem like cells (CD62L+ CD95+ CD4+T cells among naïve cells) as reported in Figure 3B. Contamination of the CD45RA- CD4+T cell memory compartment with CD62L- KLRG1+ cells named progenitors was also observed (Figure 3C). In addition to this scenario, treatment with MOS compounds triggers cells to be hyper-responsive in terms of cytokine production. This is reported in Figure 3D, where levels of IFNγ and TNFα are increased among senescent effector CD4+T cells under treatment. By contrast, Treg generation was also observed in non-senescent cells (Figure 3E). There is therefore a delayed effect evident starting from 3 days in culture whereby both senescent T cells and regulatory T cells are formed. Formulations that favour one process over the other can have exciting clinical use. Correspondingly, MOS treated Tregs demonstrated enhanced suppressing properties, evident as augmented FOXp3 expression and inhibition of conventional T cell proliferation in co-culture assays (Figure 3F). Example 4 – MOS compounds trigger mTOR activation in CD4+ T Assessment of cell proliferation in CD4+T cell treated with MOS for 3 days reveal a significant increase of ki67 levels, a nuclear protein expressed in cells under active division (Figure 4A). This robust cell proliferation supports the fact that MOS compounds directly act on mTOR activation. As previously reported, activation of mTOR triggers an increase of the telomerase activity (Dogan et al. Gene 2018). In fact, CD4+ T cells treated with MOS compounds present with amplified activity of the telomerase enzyme. (Figure 4B). The direct effect of MOS compounds on mTOR complex is further confirmed by the increased phosphorylation levels of the downstream protein S6 kinase, a surrogate marker of mTORC1 activation. In addition, the block of p38 combined with MOS treatment result to a further mTOR activation, revealing a direct role of p38 on the mTOR regulation (Figure 4C). Time studies of MOS treatment reveals that mTOR activation happens already in the first 4 hr of treatment then decreases 18hr after treatment; in contrast, sMAC activation appears later and keeps forming steadily after MOS treatment (Figure 4D). Example 5- MOS treatment affects telomere transfer The phenomena of transferring vesicles filled with telomere from APCs to metabolic fit T cells has been described by the inventors earlier and it has been connected to stem like generation (Lanna et al. Nature Cell Biology 2022). Co-culture of stimulated APCs with CD4+T cells treated with DOS compounds revealed an amplification of telomere transfer likely due to increased fatty acid oxidation and improved fitness of DOS treated T cells with consequent rejuvenation (Lanna et al. preprint bioRxiv 2024). Because MOS work oppositely to DOS compounds, an impairment of the telomere vesicle transfer was found and is demonstrated in Figure 5 as predicted. Example 6- MOS potentiate the anti-cancer response The fact that MOS mobilise sestrins from GATOR-mTOR complex to the sMAC with subsequent activation of mTOR and accumulation of sMAC complex, lead immune cells, especially effector T cells, to quickly expand and respond to external stimuli. These properties that MOS treated T cells exert might be beneficial to contrast highly resistant tumours. To investigate this aspect, an in-vitro system with the metastatic human cell line MCF-7 that it is known to be poorly immunogenic and unresponsive to chemo and radio- therapy was set up MOS treatment lead CD3+T cells to acquire potent killing capacity versus the cancer cells after just an overnight co-culture (Figure 6A). Furthermore, strikingly, tumour infiltrating Tregs briefly exposed to the MOS (e.g. up to 2 days) gained potent killing activity against the same cancer itself (Figure 6B). Thus, MOS treatment can induce potent effector function in both conventional and regulatory T cells that pays the way to exciting new types of immunotherapy whereby the cancer cells are suppressed by the same regulatory T cells that are formed in the tumour micro-environment. In turn, this hitherto unknown immunotherapy approach breaks tumour resistance. Also, CD3- cells (containing natural-killers among other antigen presenting cells) under MOS treatment showed increased ability to kill malignant cells. Therefore, immediate action of MOS can be used for emergency treatment of otherwise resistant cancers. Similar principles can also have utility in sepsis where an immediate response is warranted. In fact, MOS treated APCs potently killed bacteria, even those resistant strains that has exceptional medical utility to fight antibiotic resistance (Figure 7). By contrast, later Treg generation can be of use in autoimmunity and neurodegenerative disorders, or function to self-limit the impact of the effector T cells that had been generated by the MOS in the early phases of treatment (e.g. up to 3 days) during the emergency attack in case of sepsis or aggressive cancer eradication. In conclusion, the inventors have identified mobilisers of the sMAC that specifically target mTOR expressing T cells. Once in these cells, MOS compounds quickly act by detaching sestrins from the GATOR-mTOR complex and shuttling it to the sMAC complex. This movement of sestrins triggers a potent mTOR activation in the very early instance leading to cell proliferation together with activation of the telomerase. As soon as sestrin is detached from GATOR following mTOR activation the sMAC accumulates in these cells, and senescence is induced with consequent reduction of the telomere transfer ability, and inhibition of both telomerase and mTOR. This process culminates with senescent and regulatory T cell generation. In summary, all the properties exerted by the MOS compounds are the opposite of these of DOS compounds, as reported in Table 2. Table 2-Summary of the MOS properties DOS MOS Activity Rejuvenating Pro-ageing Cellular lifespan Extended Arrested sMAC expression Reduced Increased Requirement for mTOR No Yes presence Telomere transfer Increased Reduced Kinetics Long acting Short acting Immune phenotype Stemness Senescence Targeted cells Senescent cells Naïve like

Claims

Claims 1. A linear or cyclic polypeptide mobiliser of sestrin from GATOR / mTOR to sMAC in non-senescent cells, a derivative or analogue thereof, comprising or consisting of an amino acid sequence derived from sestrin, or a truncation thereof.

2. A linear or cyclic polypeptide, derivative or analogue thereof according to any preceding claim which comprises or consists of 5 amino acid residues.

3. A linear or cyclic polypeptide, derivative or analogue thereof according to claim 1 or 2 which comprises or consists of an amino acid sequence as set out in any one of SEQ ID Nos: 4 to 11 or a functional variant or fragment thereof.

4. A linear or cyclic polypeptide, derivative or analogue thereof according to any preceding claim which comprises or consists of an amino acid sequence as set out in SEQ ID No: 6 or a functional variant or fragment thereof.

5. A linear or cyclic polypeptide, derivative or analogue thereof according to any preceding claim, which comprises or consists of an amino acid sequence as set out in SEQ ID No: 10 or a functional variant or fragment thereof.

6. A linear or cyclic polypeptide, derivative or analogue thereof according to any preceding claim, which comprises or consists of an amino acid sequence as set out in SEQ ID No: 8 or a functional variant or fragment thereof.

7. A linear or cyclic polypeptide, derivative or analogue thereof according to any preceding claim, which comprises or consists of an amino acid sequence as set out in SEQ ID No: 4 or a functional variant or fragment thereof.

8. A linear or cyclic polypeptide, derivative or analogue thereof according to any preceding claim, which comprises or consists of an amino acid sequence as set out in SEQ ID No: 9 or a functional variant or fragment thereof.

9. A linear or cyclic polypeptide, derivative or analogue thereof according to any preceding claim, which comprises or consists of an amino acid sequence as set out in SEQ ID No: 11 or a functional variant or fragment thereof.

10. A linear or cyclic polypeptide, derivative or analogue thereof according to any preceding claim, which comprises or consists of an amino acid sequence as set out in SEQ ID No: 5 or a functional variant or fragment thereof.

11. A linear or cyclic polypeptide, derivative or analogue thereof according to any preceding claim, which comprises or consists of an amino acid sequence as set out in SEQ ID No: 7 or a functional variant or fragment thereof.

12. A linear or cyclic polypeptide, derivative or analogue thereof which has a similarity of at least 60% identity with a linear or cyclic polypeptide, derivative or analogue thereof according to any preceding claim.

13. A linear or cyclic polypeptide, derivative or analogue thereof which has a similarity of at least 80% identity with a linear or cyclic polypeptide, derivative or analogue thereof according to any preceding claim.

14. A linear or cyclic polypeptide, derivative or analogue thereof according to any preceding claim which induces 40-100% of sMAC activity.

15. A linear or cyclic polypeptide, derivative or analogue thereof according to any preceding claim, for use in emergency therapies, such as sepsis or uncurable cancers.

16. A nucleic acid encoding the linear or cyclic polypeptide, derivative or analogue thereof according to any preceding claim.

17. A nucleic acid according to claim 26 for use in emergency therapies, such as sepsis or uncurable cancers.

18. A linear or cyclic polypeptide, derivative or analogue thereof according to any one of claims 1 to 15 or a nucleic acid according to claim 16 or 17 which suppresses inflammation, boosts immune response, and / or delays control of autoimmunity.

19. A linear or cyclic polypeptide, derivative or analogue thereof according to any one of claims 1 to 15 or a nucleic acid according to claim 16 or 17 which targets sestrin-dependent sMAC activation in immune response.

20. A linear or cyclic polypeptide, derivative or analogue thereof according to any one of claims 1 to 15 or a nucleic acid according to claim 16 or 17 for use in the treatment, prevention or amelioration of acute disorders, sepsis, or allergic reactions.

21. A linear or cyclic polypeptide, derivative or analogue thereof according to any one of claims 1 to 15 or a nucleic acid according to claim 16 or 17 for use in the treatment, prevention or amelioration of anaphylactic shock.

22. A linear or cyclic polypeptide, derivative or analogue thereof according to any one of claims 1 to 15 or a nucleic acid according to claim 16 or 17 for use in the treatment, prevention or amelioration of autoimmune disorders and / or neurodegenerative diseases.

23. A linear or cyclic polypeptide, derivative or analogue thereof according to any one of claims 1 to 15 or a nucleic acid according to claim 16 or 17 for use as an immunotherapeutic agent in treating, preventing or ameliorating cancer.

24. A linear or cyclic polypeptide, derivative or analogue thereof according to any one of claims 1 to 15 or a nucleic acid according to claim 16 or 17 for use in the proliferation and regulatory activity of Treg cells.

25. A linear or cyclic polypeptide, derivative or analogue thereof according to any one of claims 1 to 15 or a nucleic acid according to claim 16 or 17 for use as a bactericidal agent, most preferably with a bacteria resistant strain.

26. A pharmaceutical composition comprising a linear or cyclic polypeptide, derivative or analogue thereof according to any one of claims 1 to 15 or a nucleic acid according to claim 16 or 17 and optionally a pharmaceutically acceptable vehicle.

27. A pharmaceutical composition according to claim 26 which comprises a sustained or controlled or delayed release dosage form.

28. A process for making a pharmaceutical composition according to claim 26 or 27, the process comprising combining a therapeutically effective amount of a linear or cyclic polypeptide, derivative or analogue thereof according to any one of claims 1 to 15 or a nucleic acid according to claim 16 or 17 with a pharmaceutically acceptable vehicle.

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