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WO2025134119A1 - Cellules tueuses naturelles dysfonctionnelles reprogrammées et utilisations associées - Google Patents

Cellules tueuses naturelles dysfonctionnelles reprogrammées et utilisations associées Download PDF

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WO2025134119A1
WO2025134119A1 PCT/IL2024/051199 IL2024051199W WO2025134119A1 WO 2025134119 A1 WO2025134119 A1 WO 2025134119A1 IL 2024051199 W IL2024051199 W IL 2024051199W WO 2025134119 A1 WO2025134119 A1 WO 2025134119A1
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cell
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dgk
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Mira Barda-Saad
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Bar Ilan University
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Definitions

  • the present disclosure relates to the field of immune modulation. More specifically, the present disclosure relates to reprogramming dysfunctional natural killer (NK) cells, and uses thereof.
  • NK natural killer
  • Natural killer (NK) cells are lymphocytes of the innate immune system, providing the first line of immunosurveillance against viral infections and tumor growth. Most human NK cells express inhibitory receptors, such as the killer cell immunoglobulin-like receptors (KIRs) in humans and the heterodimeric inhibitory receptor CD94-NKG2A (hereafter referred to as NKG2A) in humans and mice, all of which recognize major histocompatibility complex class-I (MHC-I) molecules.
  • KIRs killer cell immunoglobulin-like receptors
  • NKG2A heterodimeric inhibitory receptor CD94-NKG2A
  • MHC-I major histocompatibility complex class-I
  • the immune system employs intricate regulatory mechanisms to ensure that immune cells distinguish foreign invaders from healthy tissues. NK cells eliminate target cells lacking the expression of MHC-I molecules, whereas MHC-I-expressing cells are unaffected by NK cells, accounting for NK cell tolerance.
  • NK cells are mainly tuned by target cells expressing MHC-I molecules in their surroundings (Long et al, 2013, Annu Rev Immunol 31 :227- 258). This process of self-tuning, calibrated through “education” allows NK cells to acquire functional competence and host-specific adaptations (Orr and Lanier, 2010, Cell 142:847-856). Overall, NK cell education is determined by coordinating inhibitory, activating, and adhesion signals, through which inhibition and activation of NK cells are functionally linked (He and Tian, 2017, Cell Mol Immunol 14:321-330). This allows NK cells to assess the balance of activating versus inhibitory signals they receive.
  • NK education models including the licensing, disarming, rheostat, and confining models, propose that classical killing inhibitory receptors (KIR) play an instructive role in NK cell responsiveness (Boudreau and Hsu, 2018, Trends Immunol 39:222; Zhang et al, 2019, Nat Commun 10:5010).
  • KIR killing inhibitory receptors
  • ITIM immune-receptor tyrosinebased inhibitory motifs
  • PTP protein tyrosine phosphatases
  • SH2 Src homology region-2 domain
  • SHIP1 SH2 domain-containing inositol polyphosphate 5-phosphatase 1
  • NK cell reactivity increases with the number of different self- MHC-I-specific inhibitory receptors expressed (Thomas et al, 2013, J Immunol 191:3981-3985; Jaeger and Vivier, 2012, J Clin Investig 122:3053). If the target cell lacks MHC-I or expresses low levels of this surface marker, it results in NK cell activation and clearance of the target.
  • NK cell dysfunction reflects different states, including anergy and exhaustion, each with a distinct etiology.
  • “Anergic” NK cells are naturally unresponsive peripheral blood cells, constituting ⁇ 13 ⁇ 6% of the entire NK cell population (Raulet et al, 2003, Annu Rev Immunol 19:291-330; Fernandez et al, 2005, Blood 105:4416-4423).
  • NK cells arise from chronic viral infections, inflammation, or cancer, because of their overstimulation by their targets.
  • NK cells play a complementary role to T cells in tumor immunity by recognizing tumors that downregulate MHC-I expression and escape CD8+ T cell-mediated tumor clearance (Fruci et al, 2013, J Transl Med 11 : 1M-; Eanier, 2008. Nat Immunol 9:495-502). Although the anti-tumor role of NK cells has been described in hematological malignancies, their role in the solid tumor milieu remains unclear due to their lack of activity in the tumor microenvironment (TME) (Kumblel et al, 2012, Cancer Res 72:4311-4317). Clinical observations highlight NK cells as a critical component in the anti-tumor response (Remark et al, 2013.
  • EGR Early Growth Response
  • Egr2 and Egr3 were shown to be important for preventing autoimmunity activity of immune cells [1].
  • Symonds et al [2] have shown that deficiency of Egr2 and Egr3 in T cells resulted in enhanced tumor growth and fewer tumor infiltrating T cells (TILs) in mouse models, thereby demonstrating the critical role of Egr2 and Egr3 in sustaining the anti-tumor activity of exhausted CD8+ TILs.
  • TILs tumor infiltrating T cells
  • the present disclosure provides herein key intrinsic checkpoints in NK “anergy”, including early growth response (EGR)-2 and diacylglycerol kinase (DGK)-a , and reveal functional, phenotypic and transcriptional similarity with NK cell “exhaustion”.
  • EGR early growth response
  • DGK diacylglycerol kinase
  • the inventors further demonstrate in situ reprogramming of “anergic” and “exhausted” NK cells by modulation of these key intrinsic regulators, DGKa and Egr2, via a nanoparticle (NP)-based drug delivery platform (Biber et al, 2021, EMBO Mol Med 14:el4073), both in vitro and in vivo, revealing that the identified markers are critical to reprogram both the dysfunctional states.
  • NP nanoparticle
  • the inventors' in vivo model of NK cell exhaustion reveals that gene silencing of Egr2 empowers NK cells to effectively control tumor growth and enhance NK cell effector functions for improved tumor lysis and clearance.
  • the inventors' data suggest that “anergy” and “exhaustion” are not simply intrinsic non-responsive states, but that these newly identified targets can potentially enable these dysfunctional NK cells to be reprogrammed in their native environment, to become functional in diverse contexts, including cancer and viral infections.
  • a first aspect of the preset disclosure relates to a re -programming agent (also referred to herein as a modulator and/or re -programmer or an activator), comprising at least one compound that specifically inhibits the expression, activity and/or stability of at least one of: (i), at least one member of the Early Growth Response (EGR) family of transcription factors; and (ii), at least one member of the Diacylglycerol Kinase (DGK) family.
  • EGR Early Growth Response
  • DGK Diacylglycerol Kinase
  • the disclosed reprogramming agent may provided in a nano- or micro-particle, micellar formulation, vehicle, matrix, or a composition and thus the present disclosure further encompasses any nano- or microparticle, micellar formulation, vehicle, matrix, or composition comprising the disclosed reprogramming agent.
  • the disclosed re-programming agent activates, rewires and/or activates at least one lymphocyte.
  • the disclosed reprogramming agent rewires at least one dysfunctional lymphocyte.
  • the disclosed lymphocytes may be NK cells, specifically, dysfunctional NK cells.
  • the disclosed lymphocytes may be T cells, specifically, dysfunctional T cells.
  • the disclosed re -programming agent activates dysfunctional natural killer (NK) cells.
  • the present disclosure relates to a re-programming agent that comprises an effective amount of the at least one compound that specifically inhibits the expression, activity and/or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family.
  • the effective amount as used herein is the amount sufficient for activation and/or re-wiring of the dysfunctional NK cells.
  • a further aspect of the present disclosure relates to at least one nano- or micro-particle, micellar formulation, vehicle or matrix comprising at least one reprogramming agent comprising at least one compound that specifically inhibit the expression, activity and/or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and/or (ii), at least one member of the DGK family; wherein said reprogramming agent activates dysfunctional NK cells.
  • the at least one nano- or micro-particle, micellar formulation, vehicle or matrix disclosed herein may comprise an effective amount of the disclosed nano- or micro-particle, micellar formulation, vehicle or matrix activators.
  • the amount of the re-programming agent in the disclosed nano- or micro-particle, micellar formulation, vehicle or matrix is the amount effective for activation of NK cells, specifically, activation of dysfunctional NK cells.
  • a further aspect of the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of at least one re-programming agent comprising at least one compound that specifically inhibit the expression, activity and/or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family; and/or any nano- or micro-particle, micellar formulation, vehicle, matrix comprising the re-programming agent. More specifically, the re-programming agent of the disclosed composition activates dysfunctional NK cells.
  • the composition further comprises at least one of pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s. It should be understood that in some embodiments, the disclosed composition comprises an effective amount of the disclosed re -programming agent that is sufficient for and effectively activate and/or for rewiring NK cells, specifically, dysfunctional NK cells.
  • a further aspect of the present disclosure relates to a method for activating and/or re-wiring at least one dysfunctional NK cell.
  • the method comprising the step of contacting the dysfunctional NK cell with an activating effective amount of at least one re-programming agent comprising at least one compound that specifically inhibit the expression, activity and/or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family; and/or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising the re-programming agent.
  • the re-programming agent activates and/or re-wires dysfunctional NK cells.
  • a further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an immune-related disorder in a subject in need thereof.
  • the method comprising the step of administering to the subject a therapeutically effective amount of at least one re-programming agent comprising at least one compound that specifically inhibit the expression, activity and/or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family; and/or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising the re-programming agent, wherein said re -programming agent activates dysfunctional NK cells.
  • a further aspect of the present disclosure relates to at least one re-programming agent or any nano- or micro-particle, micellar formulation, vehicle, matrix, cell or composition comprising said reprogramming agent, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an immune-related disorder in a subject in need thereof.
  • the re-programming agent used herein comprises at least one compound that specifically inhibit the expression, activity and/or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and/or (ii) at least one member of the DGK family; and/or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising said reprogramming agent, wherein said re -programming agent activates dysfunctional NK cells.
  • a further aspect of the present disclosure relates to a kit comprising: (I), at least one reprogramming agent comprising at least one compound that specifically inhibits the expression, activity and/or stability of at least one of: (i), at least one member of the EGR family of transcription factors, in a first dosage form; and/or (ii), at least one member of the DGK family, in a second dosage form; and/or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising said re -programming agent, wherein said re-programming agent activates dysfunctional natural killer (NK) cells.
  • the disclosed kit may optionally further comprise: (II), at least one additional immunomodulatory therapeutic agent, in a third dosage form.
  • a further aspect of the present disclosure relates to a method for determining a personalized treatment regimen for a subject suffering from an immune-related disorder, and optionally for monitoring the effectiveness of a treatment regimen in the subject.
  • the method comprising the steps of: In step (a), determining the levels of at least one of ERG2 and DGKa in at least one NK cell of at least one biological sample of said subject, to obtain the level value of said at least one of ERG2 and DGKa.
  • step (b) classifying said subject as: (i), a subject displaying a decreased NK cell functioning, (or having dysfunctional NK cells), if the level value determined in (a), for the at least one of: ERG2 and/or DGKa in the at least one sample is higher as compared to a predetermined standard value.
  • the subject is classified as (ii), a subject displaying functioning NK cells, if the level value determined in (a) for the at least one of ERG2 and DGKa in the at least one sample is equal or lower as compared to a predetermined standard value.
  • the next step (c) involves selecting for a subject displaying a decreased NK cell functioning (or dysfunctional NK cells), an activating treatment regimen that decreases the levels of at least one of ERG2 and DGKa in at least one NK cell of said subject.
  • RNA seq analysis gene and pathway enrichment
  • Fig. 1A Graphical presentation of NK isolation process -gating strategy.
  • Primary human NK cells were isolated from PBMCs using negative selection; following purity check by CD3-CD56+ expression, the cells were stained for NKG2A PE and panKIR PE and further sorted to anergic and responsive NK cell subsets based on PE (Phycoerythrin) expression (NKG2A-panKIR- (PE negative) anergic subset; NKG2A+ panKIR-i- (PE positive) responsive subset).
  • PE Physicalerythrin
  • Fig. 1B-1C Purified NK (pNK) anergic and responsive cells were subjected to incubation with 721.221 No HLA cell lines (E:T - 1:3) for 5 h at 37°C.
  • pNK Purified NK
  • MFI mean fluorescence intensity
  • Fig. 1F-1G Metascape analysis of key genes and the enriched terms annotated to pathways from various datasets associated with the top-hit significant genes (Fig. IF), and the transcription factors (TFs) reflecting the top-significant genes upregulated in the anergic subset, obtained via enrichment analysis in TRRUST (Fig. 1G).
  • Fig. 1H The interactome cluster of the enriched terms annotated to pathways obtained from the significant Differentially Expressed Genes (DEGs).
  • FIG. 1A-2E Gene expression profiling and analysis of anergic and responsive populations
  • Fig. 2A PC A performed using DESeq2 on the anergic vs. responsive subset VST normalized RNA-seq data.
  • the variance is depicted on the X and Y axes, and each individual donor is indicated.
  • the anergic and responsive populations demonstrate high transcriptional differences reflected by a variance of 81%.
  • Fig. 2B Heatmaps (transcripts per kilobase million normalization with Z score) showing expression of representative genes in anergic vs responsive populations from individual donors. The heatmaps illustrate subtle distinctions among the donors with noteworthy similarities.
  • Fig. 2D-2E The transcript levels of key genes expressed in the anergic, and responsive populations were normalized to log2 and are presented as line dot graphs. The P value was calculated using a two-paired tailed t test and is indicated within the graph (NC normalized counts).
  • Figure 3A-3I Naturally induced “anergic” NK cells share a transcriptional program similar to “exhausted” NK/T cells with an overlap of key regulators
  • Fig. 3B Transcript and protein levels of DNAM-1 , 2B4, and CD160.
  • the P value was calculated using a two-tailed paired t test and is indicated above the graph.
  • panels represent (from left to right): RNA transcript normalized count, relative MFI, raw MFI, and percentage of parent.
  • Fig. 3D Gene Sequence Enrichment Analysis (GSEA) using gene sets from exhausted NK cells obtained from HB V-infected individuals vs healthy donors compared to the anergic and responsive population (Marotel et al, 2021, eLife 10: 1 - 47).
  • GSEA Gene Sequence Enrichment Analysis
  • the statistical tests for the GSEA (Fig. 2D-2G) were performed using the weighted Kolmogorov-Smirnov test.
  • FIG. 4A-4D RNA seq analysis and DGKa expression levels
  • the P value was calculated using a two-tailed t test, and the number of repeats is indicated within the graph presented as means ⁇ SEM.
  • Figure 5A-5F In vivo model exhibits shared intrinsic regulators governing tumor infiltrating “exhausted” NK cells and those controlling NK cell “anergy”
  • Fig. 5A Experimental protocol. Timeline of in vivo experiment depicting tumor engraftment, effector administration, tumor excision, and ex vivo analysis.
  • the P values were calculated using a two-tailed t test with pairing and are indicated within the graph (mean ⁇ SEM) along with the number of experimental repeats. Each symbol represents an individual mouse.
  • FIG. 6A-6C Flow cytometry gating strategy
  • NK cells were distinguished from the target PANC-1 cells according to FSC and SSC.
  • CD45 antibody specifically recognizing human (h)CD45
  • h human
  • NK cells which are CD45+ (95.9%) referred as tumor infiltrating NK cells (TINK).
  • Fig. 6B Flow cytometry analysis of intracellular staining was performed to measure pNK expression on cells from the mice with tumor-only (no NK administration) control and distinguished based on CD45 expression.
  • the NK cells were distinguished from the target cells based on their volume and density (FSC and SSC) and were re-gated to CD45+ subsets.
  • DGKa and Egr2 expressions were measured on the CD45+ gated population. Histogram offsets and MFI were used for graphical presentation. Graphs show fluorescence intensity.
  • the Y axis indicates relative cell number, and the X axis indicates the MFI.
  • Fig. 6C Representative histograms and contour plots including outliers for the data in Fig. 5C-5F.
  • Figure 7A-7I Knockdown of the intrinsic regulators reveals underlying molecular mechanism to reverse NK cell dysfunction
  • Fig. 7F Anergic cells were treated with either Egr2 or NS siRNA, and responsive cells were treated with NS siRNA. The cells were stained with Indo-1 AM, activated by PMA/ionomycin activation cocktail, and intracellular calcium flux was measured as the ratio between the Ca2+ bound/unbound over 22 min.
  • Fig. 8B Gating strategy employed for the analysis of results in Fig. 7.
  • the NK cells and the 221 HLA-Cw7 cells were differentiated based on size and granularity (FSC-SSC) and gated for PE to distinguish the anergic (PE-) versus the responsive population (PE+). They were subsequently gated for CD 107a, as indicated on the overlaid histograms.
  • Fig. 8C-8E Purified responsive and anergic NK cells were stimulated with 721.221 target cells, lysed and subjected to FACS analysis with anti-pSHP-l(S591) (Fig. 8C), anti-pPLCyl (Y783) (Fig. 8D), and anti-pPLCy2 (Y1217) (Fig. 8E) antibodies.
  • FIG. 8F Representative overlaid histograms for Fig. 7G-7I.
  • FIGS Figure 9A-9J. Targeting Egr2 in situ using 3D organotypic spheroid (OTS) culture and in vivo model as a potential therapeutic approach
  • Fig. 9A-9B Correlation of NK-DGKA-EGR2 signatures with the overall survival of AML patients (Fig. 9A), and glioma patients (Fig. 9B) comparing high and low quartiles.
  • Left panels Kaplan Meier curves are presented showing patient survival (obtained from Survival Genie (Dwivedi et al, 2022)) along with the P values.
  • Right panels show the signature of immune cell infiltration in AML and glioma; and red squares represent positive correlation to immune cells observed in the cancer dataset.
  • Bottom panels indicate the correlation of the survival score to the NK cell signatures.
  • P values (Fig. 9A, 9B) were calculated using Log-rank t test.
  • Fig. 9C Left panel: Schematic representation of the 3D OTS model using the NP delivery platform to reprogram anergic NK cells in a tumor milieu.
  • Primary NK cells were used to obtain anergic and responsive subsets and were then seeded to preconstructed 3D cultures and administered with NP encapsulating Egr2 or NS siRNA. Next, the pNKs were subjected to an Incucyte-based killing assay.
  • OTS 3D domes were established using Matrigel and cultured for 48 h in OTS media consisting of RPMI media supplemented with 1 pg/mL fibroblast growth factor (FGF), 0.18 pg/mL epidermal growth factor (EGF) and 500 lU/mL transforming growth factor (TGFP).
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • TGFP transforming growth factor
  • Fig. 9D Timeline of the in vivo experiment.
  • PDAC-1 -xenograft NRG mice were established as previously described and received a single infusion of l.l*10 7 human pNK from four healthy donors on day 9 once the tumors reached -250-300 cm2; “tumor only” control did not receive NK cells but received NS siRNA encapsulated NPs.
  • NPs encapsulating Egr2 siRNA or NS siRNA were administered i.v. from day 12 for every 3 days until day 27.
  • Fig. 9E Tumor volume (in mm3) was monitored and measured daily throughout the experiment.
  • the pNK injection is depicted by a black arrow in a stripes-shaded region, and the NP injections are indicated by black arrows.
  • the bold lines indicate the average, and the dashed lines represent individual mice within their respective experimental groups.
  • Fig. 9F Tumor sizes (mm3) measured during the indicated days.
  • Black graph represents the control group with tumor only.
  • the gray and the white graphs represent groups of mice that received treatment with NP encapsulating NS siRNA or Egr2 siRNA, respectively.
  • Tumor sizes at specific time points are indicated: Day 8 (one day before pNK injection) and Day 11 (2 days after pNK injection) are tumor sizes prior to NP injection; day 15 (6 days after pNK injection), day 19 (10 days after pNK injection) and day 27 (18 days after pNK injection and final day before tumor excision) represent tumor sizes following NP injection.
  • Data are presented as mean ⁇ SEM.
  • P values are calculated using one-way ANOVA accompanied by a Tukeys’ post hoc multiple comparison test individually for each day presented and are indicated within the graph.
  • Fig. 9H-9I Ex vivo analysis.
  • the pNK were distinguished based on hCD45 expression and NP incorporation (PE positive). Fluorescence is represented as both relative MFI (left panels) and percentage of pNK (right panel). P values were calculated using a two-tailed paired t test and are represented within the graph presented as means ⁇ SEM.
  • Fig. 9J Scheme depicting the proposed signaling pathway of anergic cells in accordance with the transcriptome and protein level profiling; PA phosphatidic acid, PLC phospholipase Cyl/2, DAG diacylglycerol, DGK diacylglycerol kinase, Egr early growth response, MAPK mitogenactivated protein kinase, NF AT nuclear factor of activated T cells, PD-1 programmed cell death protein 1, PKC0 protein kinase C0, SHP-1 Src homology 2 domain-containing protein tyrosine phosphatase 1, pS591 phospho - S591.
  • “Anergic” cells exhibit elevated EGR2 expression, which subsequently triggers an increase in DGKa.
  • Anergic and responsive cells were transfected with EGR2 siRNA or NS siRNA, seeded with 721.221 HLA-Cw7 expressing mCherry cells at an E:T ratio of 10:1, and subjected to Incucyte imaging and analysis; the decrease in fluorescence intensity was measured, and images from the indicated time points are shown. The values were normalized to 721.221 Cherry only — no effector control.
  • Right panel Images showing cell fluorescence at different time points (scale bar: 200 pm).
  • Fig. 10B Human CML OTS prepared using K562 CFP cells in Matrigel (1 : l-v/v%), were seeded with anergic or responsive pNK cells at an E:T ratio of 5:1.
  • NPs encapsulating Egr2 siRNA or NS siRNA were added to the OTS, and the decrease in fluorescence intensity was monitored and measured; the images along with the fluorescence intensity at the respective time points are shown (numbers on top right of each frame).
  • the decrease in fluorescence over time shows the enhanced cytotoxic activity of anergic cells following Egr2 siRNA treatment (left panel: middle) similar to the responsive cells treated with NS siRNA (left panel: bottom).
  • Whole well images of the OTS at 48 h are presented.
  • Right panel Images showing cell fluorescence at different time points (Scale bar: 200 pm).
  • Fig. 10C The tumors were excised on day 27, a single-cell suspension was made, and cells were stained for intracellular Egr2.
  • the pNK cells were distinguished based on FSC vs SSC, and PE content, reflecting nanoparticle incorporation. Representative histogram showing Egr2 expression levels, black line shows the EGR2 levels in the group treated with control NP (NS siRNA), and the dotted line shows the group receiving Egr2 siRNA- NP, as indicated in the graph.
  • Fig. 10D The dissociated tumors were then subjected to Annexin V staining for apoptotic cells. Representative histogram on the left panel shows the apoptosis of the tumors from each group (one mouse representative of three independent repeats).
  • the right panel shows a graph summarizing the tumors obtained from three different mice. Data are presented as mean ⁇ SEM. P values were calculated using one-way ANOVA with Tukeys post hoc test after normalization of the values to the “tumor only” group, which served as the control.
  • NK cells serve as the first line of immune defense against cancer cells, however, increasing evidence shows emergence of dysfunctional phenotypes.
  • the inventors performed a thorough characterization and uncovered the underlying etiologies of the “anergic” and “exhausted” dysfunctional states.
  • many surface markers have been suggested (Judge et al, 2020, Front Cell Infect Microbiol 10:49), the molecular and transcriptional wiring remains unexplored.
  • the inventors demonstrate that the naturally existing “anergic” NK cell population shares phenotypic, transcriptomic, and functional similarity with the canonical NK cell “exhaustion” state arising from the tumor microenvironment (TME).
  • TEE tumor microenvironment
  • DGKa and Egr2 were identified as key intrinsic regulators governing NK cell dysfunctional states of “anergy” and “exhaustion”.
  • the inventors' findings indicate that Egr2 serves as the primary transcription factor accounting for the intrinsic non-functionality of these cells.
  • the transcriptome analysis reveals sharing of key genes between anergic/exhausted NK cells, and exhausted CD8+ T cells (Fig. 3D-3G), including NFAT2, EGR2, PDCD1, and TOX2, revealing transcriptional commonalities.
  • TILs tumor-infiltrating lymphocytes
  • CAR chimeric antigen receptors
  • IB immune checkpoint blockade
  • NK cells Adoptive transfer of NK cells, a common approach, also leads to reduced cytotoxic potential due to loss of NK activity during ex vivo expansion of NK cells (Judge et al, 2020, Front Cell Infect Microbiol 10:49; Gill et al, 2012, Blood 119:5758).
  • ICB therapies target one or more checkpoints such as the surface markers PD- 1, CTLA4, TIGIT, or NKG2A (Leach et al, 1996, Science 271:1734-1736; Ott et al, 2013, Clin Cancer Res 19:5300-5309; Pan et al, 2023, Adv Mater 35:2211370).
  • DGKa facilitates conversion of DAG to PA, leading to impaired signaling cascades through the recruitment of the phosphatase SHP-1 to the cell membrane, resulting in dephosphorylation of its targets, e.g., PLCyl/2 (Fig. EV4C-E), LAT, and ZAP70, suppressing the signaling cascade, and resulting in the dysfunctional phenotype.
  • PLCyl/2 Fig. EV4C-E
  • LAT LAT
  • ZAP70 ZAP70
  • the inventors' data implies that EGR2 gene silencing downregulates SHP-1 activity, potentially via enhancing DAG availability by reducing DGKa levels. This, in turn, initiates the activation of PKC0, which modulates SHP-1 activity through serine 591, supported by the inventors' recent data (Ben-Shmuel et al, 2022, eLife l l:e73282).
  • RNA sequencing analysis and the in vivo model shown here suggest that Egr2 potentially cooperates with other TFs, presumably NFAT2, to initiate the dysfunction-based transcriptional program along with high mobility group (HMG)-box TFs such as TOX2, and members of the NR4a family, which are validated targets of calcium-calcineurin regulated NFAT2 in the absence of partners such as AP-1 and NFkB (Wagle et al, 2021, Nat Commun 12:2782; Seo et al, 2019; Sekine et al, 2020, Sci Immunol 5:eaba7918).
  • HMG high mobility group
  • Egr2 and potentially NFAT2/ TOX2/other TFs in the terminal regulation of anergy highlights the complexity of the transcriptional program associated with dysfunctional NK cells. This finding establishes a functional link between the naturally induced “anergy” and the “exhausted” state of NK cells within the TME.
  • Egr2 the intrinsic regulators of dysfunctional NK cells
  • leads to the rewiring of NK cell functional circuitry overcomes the dysfunction-associated transcriptional imprint, and potentially enhances the efficiency of immunotherapies.
  • depletion of dysfunctional cells from the TME was suggested to improve therapeutic efficacy.
  • the inventors study provides a novel strategy for in situ reprogramming of these dysfunctional cells rather than depleting them (Marcucci and Rumio, 2021, Cells 10:872; Cook and Whitmire, 2013, J Immunol 190:641-649).
  • the inventors describe a potential platform for this approach using an NP drug delivery system (Biber et al, 2021, EMBO Mol Med 14:el4073) (Fig. 5C).
  • the in vitro 3D OTS CLL model and in vivo PDAC model of NK cell exhaustion demonstrate that EGR2 silencing, not only restores the functionality of anergic cells but also enables these NK cells to potentially control solid tumor growth and influence disease outcomes.
  • NK cells This potentially serves as proof of concept for the possibilities of reprogramming dysfunctional NK cells in situ, bypassing the need for adoptive transfer. While the majority of studies on anergic NK cells have focused on their presence in peripheral blood, evidence suggests that NK cells develop and mature in different tissues including spleen, liver, bone marrow, lymph node, and lung (Dogra et al, 2020, Cell 180:749; Hashemi and Malarkannan, 2020, Cancers 12: 1-23). This leads to the notion that the NK cells undergo education in the respective niche, resulting in the potential emergence of anergic phenotype. The characterization and the functional implications of anergic NK cells arising in different tissue contexts are still unknown.
  • the inventors' study not only elucidates the molecular framework of NK cell anergy but also highlights its functional, phenotypical, and transcriptional parallels with canonical NK cell exhaustion.
  • the inventors identified shared intrinsic factors that can be targeted and reversed through immunotherapy.
  • the inventors' results provide valuable insights into the mechanisms and signaling pathways responsible for NK cell dysfunctional states. Focusing on the regulation of NK cell anergy and exhaustion will potentially lead to the development of strategies to modulate the anergic state in settings of autoimmunity and transplantation or to enhance NK cell function in cancer and chronic viral infection.
  • the present disclosure provides in one aspect thereof re-programming agent (or modulator and/or re-programmer) of lymphocytes, for example, lymphocytes of the T lineage.
  • the disclosed modulator and/or re-programmer activates dysfunctional lymphocytes, specifically, dysfunctional lymphocytes of the T lineage.
  • the disclosed reprogramming agents at least one of: (i), at least one member of the Early Growth Response (EGR) family of transcription factors; and (ii), at least one member of the Diacylglycerol Kinase (DGK) family.
  • EGR Early Growth Response
  • DGK Diacylglycerol Kinase
  • the disclosed re-programming agent activates, rewires and/or activates at least one lymphocyte.
  • the disclosed re-programming agent rewires at least one dysfunctional lymphocyte.
  • the disclosed re-programming agent rewires dysfunctional lymphocytes residing in a microenvironment of a diseased tissue.
  • Such diseased tissue may be in some embodiment, a tumor tissue.
  • the dysfunctional lymphocytes reprogramed and/or rewired and/or activated by the disclosed re-programming agent may be infiltrating lymphocytes of a tumor microenvironment.
  • the disclosed lymphocytes may be NK cells, specifically, dysfunctional NK cells.
  • the disclosed reprogramming agent re-programs and/or rewires at least one dysfunctional NK cell.
  • the disclosed re-programming agent rewires and/or activates, and/or reprograms at least one NK cells that infiltrates into the tumor tissue, specifically, a least one TINK (tumor infiltrating NK cells).
  • the disclosed lymphocytes may be T cells, specifically, dysfunctional T cells.
  • the disclosed re-programming agent re-program and/or rewires at least one dysfunctional T cell.
  • the disclosed re-programming agent rewires and/or activates, and/or reprograms at least one T cells that infiltrates into the tumor tissue, specifically, at lest one TIL (tumor infiltrating lymphocytes). More specifically, Tumor-Infiltrating Natural Killer (TINK) cells, refer to a subset of natural killer (NK) cells that are present within the tumor microenvironment and exhibit the ability to infiltrate solid tumors. These cells are a component of the innate immune system and play a crucial role in recognizing and eliminating malignant cells through mechanisms such as cytotoxic activity and cytokine production.
  • TIL tumor infiltrating lymphocytes
  • TINKs are often phenotypically and functionally distinct from circulating NK cells, as they are influenced by the immunosuppressive tumor milieu, which can alter their activation status, cytotoxicity, and cytokine secretion profiles. Notably, TINKs may exhibit markers of exhaustion or dysfunction, reflecting tumor-induced immune evasion, while certain subsets retain potent anti-tumor activity.
  • the tumor microenvironment refers to the complex and dynamic ecosystem surrounding a tumor, composed of cellular and non-cellular components that interact with the malignant cells to influence tumor progression, metastasis, and therapeutic response.
  • TME tumor necrosis factor
  • B cells e.g., T cells, B cells, macrophages, and NK cells
  • fibroblasts e.g., T cells, B cells, macrophages, and NK cells
  • non-cellular components encompass extracellular matrix (ECM) proteins, signaling molecules, cytokines, chemokines, and growth factors.
  • ECM extracellular matrix
  • the TME is characterized by hypoxia, altered pH, and nutrient deprivation, which promote tumor survival, immune evasion, and resistance to therapies.
  • the interactions within the TME can lead to immunosuppressive conditions, enabling tumor growth and progression by impairing antitumor immune responses.
  • a first aspect of the preset disclosure relates to a re -programming agent (also referred to herein as a modulator and/or re-programmer) comprising at least one compound that specifically inhibits the expression, activity and/or stability of at least one of: (i), at least one member of the Early Growth Response (EGR) family of transcription factors; and (ii), at least one member of the Diacylglycerol Kinase (DGK) family.
  • EGR Early Growth Response
  • DGK Diacylglycerol Kinase
  • the disclosed re-programming agent may provided in a nano- or micro-particle, micellar formulation, vehicle, matrix, or a composition and thus the present disclosure further encompasses any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising the disclosed re-programming agent/s. More specifically, the disclosed re-programming agent activates and/or functionalize dysfunctional natural killer (NK) cells.
  • the present disclosure relates to a re-programming agent that comprises an effective amount of the at least one compound that specifically inhibits the expression, activity and/or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family.
  • the effective amount as used herein is the amount sufficient for activation and/or re-wiring of the dysfunctional NK cells.
  • the present disclosure provides re -programming agents of NK cells, specifically of dysfunctional NK cells.
  • a "re-programming agent' also referred to herein as a modulator and/or re-programmer), as used herein, is defined as a compound, substance, material, or agent that reprograms, induces or enhances a specific biological, chemical, or physical effect in a targeted system.
  • the modulators and/or re-programmers disclosed herein act in functionalizing NK cells, and more specifically, in functionalizing dysfunctional NK cells.
  • the re-programming agents of the present disclosure act in rewiring the NK cells or reprograming the NK cells such that the re-wired, functionated, activated or reprogramed NK cells are functional NK cells, or even in some embodiments, displaying improved functionality.
  • Rewiring or reprogramming of natural killer (NK) cells refers herein to the process of altering the gene expression profiles, signaling pathways and/or metabolic state of NK cells to enhance their activity, particularly in the context of immune responses against cancer, infections, or other diseases.
  • Such rewiring or reprogramming improve NK cell functions, which may include for example cytotoxicity, cytokine production, and tumor infiltration, while overcoming immunosuppressive signals within the tumor microenvironment.
  • this process involves inhibiting the expression, activity and/or stability of at least one member of EGR family and/or at least one member of the DGK family.
  • functionalizing dysfunctional NK cells refers to the process of restoring, enhancing, or even in some embodiments modifying (specifically enhancing), the functional capabilities of natural killer (NK) cells that have lost or reduced, specifically restoring the ability of the cells to perform their natural biological functions.
  • NK cells that exhibit impaired activity, which can arise due to intrinsic factors (e.g., genetic or epigenetic defects, e.g., leading to anergy), external suppression by a diseased (tumor) microenvironment, or chronic stimulation leading to exhaustion.
  • Functionalizing such cells by the disclosed re -programming agent encompass any action that rejuvenate or improve their activity, including, but not limited to Restoring cytotoxicity, enhancing proliferation and survival and/or reversing exhaustion.
  • the NK natural biological functions are as defined in more detail herein after by the present disclosure and are encompassed by the present aspect.
  • re-programming agent of the present disclosure thus may be interchangeably referred to herein as “ re-programming agent ", "NK re-programming agent”, “NK cell/s re-programming agent”, “dysfunctional NK cell/s re -programming agent”, and/or “re-programming agent of dysfunctional NK cells”, “modulator and/or re -programmer”, “NK modulator”, “NK cell/s modulator “, “dysfunctional NK cell/s modulator “, and/or “ modulator of dysfunctional NK cells”, “re -programmer”, “NK re-programmer”, “NK cell/s re-programmer”, “dysfunctional NK cell/s reprogrammer”, and/or " re-programmer of dysfunctional NK cells”.
  • the disclosed modulator and/or re-programmer also acting as an activator or a reactivator promotes, initiates, or increases the activity, efficacy, or functionality of the NK cells. More specifically, the disclosed reprogramming agents provide activation of NK cells, as herein defined, as a process by which NK cells are stimulated to exert their immune functions, including cytotoxicity and cytokine production. More specifically, activation of dysfunctional NK cells involves restoring or enhancing their cytolytic potential and immunomodulatory functions.
  • NK cells or activated NK cells may be characterized for example by altered receptor expression (e.g., activating receptors like NKG2D or CD 16) on the surface of NK cells, increased cytokine production, increased degranulation (e.g. increase in CD107), increased calcium flux and/or increased cytotoxic activity (lysis of target cells) as compared to a reference standard or a control sample.
  • the reference standard or the control sample may comprise for example dysfunctional non-treated NK cells or NK cells of a healthy subject, that also naturally includes dysfunctional NK cells (e.g., anergic NK cells).
  • the functionality or activity of NK cells may also be evaluated by improved disease parameters (e.g., reduced tumor size in animal models).
  • the disclosed re-programming agents increase the functionality of the NK cells or any population thereof in about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, as compared to NK cells, specifically, dysfunctional NK cells that were not activated or reprogramed or re- wired or functionated by the disclosed re-programming agents.
  • Measurable indicators reflecting functionality of NK cells specifically, that NK cells are functionally stimulated and engaged in their immune responses. These parameters can be assessed through various laboratory techniques, including flow cytometry, cytokine assays, and cytotoxicity tests.
  • NK cells can be assessed through a variety of parameters that reflect their functional and physiological changes.
  • Surface marker expression such as upregulation of early activation markers like CD69, prolonged activation markers like CD25, and degranulation indicators like CD107a (LAMP-1), are key indicators of activation. These parameters reflect normal functioning of NK cells.
  • Downregulation of inhibitory receptors e.g., KIRs, NKG2A
  • normal function of NK cells e.g., NKG2A
  • NKIS inhibitory immunological synapse
  • Activated NK cells also produce key cytokines, including interferon-gamma (IFN-y), tumor necrosis factor-alpha (TNF-a), and granulocyte-macrophage colony-stimulating factor (GM- CSF), which enhance immune responses and recruit other immune cells.
  • IFN-y interferon-gamma
  • TNF-a tumor necrosis factor-alpha
  • GM- CSF granulocyte-macrophage colony-stimulating factor
  • Their cytotoxic activity is reflected in their ability to lyse target cells, measured through assays like chromium release or fluorescent dye staining, as well as the release of cytotoxic molecules such as perforin and granzyme B.
  • Intracellular signaling pathways including phosphorylation of proteins like STAT4, STATS, PI3K, and MAPK, as well as increased calcium flux, indicate activation at the molecular level.
  • NK cells undergo a shift to increased glycolysis and exhibit enhanced mitochondrial activity to meet the energy demands of activation.
  • Gene expression changes such as upregulation of genes like IFNG, GZMB, PRF1, and TNF, provide a molecular signature of activation and can be measured through PCR or RNA sequencing. Activation also drives NK cell proliferation, particularly in response to cytokines like IL-2 and IL-15, which can be tracked through cell counting or division assays.
  • cytotoxic granules perforin, granzyme B
  • Fas ligand Fas ligand
  • NK cells Upon recognition of activating signals on the surface of target cells, NK cells form an immunological synapse, leading to the polarization and directed exocytosis of lytic granules toward the target cell membrane. Perforin creates pores in the target cell membrane, facilitating the entry of granzymes, which trigger apoptosis through caspase activation and other pathways. Degranulation is a hallmark of NK cell cytotoxic function and can be measured by the surface expression of markers such as CD107a (LAMP-1), which translocate to the NK cell membrane during granule release.
  • LAMP-1 markers
  • CD107 may be used as a marker for NK cell degranulation.
  • CD107 (also known as LAMP-1, or lysosome-associated membrane protein-1) is a transmembrane glycoprotein found in the membranes of cytolytic granules within natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). During degranulation, CD107 is transported to the cell surface as the cytolytic granules fuse with the plasma membrane, making it a key marker for evaluating the activation and cytotoxic activity of these immune cells. The surface expression of CD107a correlates with the release of effector molecules, such as perforin and granzymes, which are essential for target cell lysis. Its detection, commonly performed using flow cytometry, provides a reliable measure of immune cell degranulation and functional activity. Consequently, CD 107 plays a vital role as a biomarker in immunological research, particularly in studies of cancer immunotherapy, viral infections, and cell-mediated immune responses.
  • Calcium influx or intracellular calcium flux refers to the process by which calcium ions (Ca 2+ ) enter the cytoplasm of a cell, typically through calcium-permeable channels located in the plasma membrane or from intracellular stores such as the endoplasmic reticulum. This influx is a critical signaling event that regulates a wide range of cellular processes, including activation, proliferation, secretion, and apoptosis.
  • NK natural killer
  • T lymphocytes calcium influx is essential for initiating downstream signaling pathways that trigger degranulation, cytokine production, and cytotoxic activity.
  • the entry of Ca 2+ often occurs following receptor stimulation, leading to the opening of store-operated calcium channels (e.g., CRAC channels) or voltage-gated calcium channels, resulting in a rise in intracellular calcium levels.
  • activation of NK cells by the disclosed reprogramming agents may be evaluated by increased degranulation, that may be reflected in some embodiments by a measured increase in CD 107, increased calcium flux and/or increased lysis of target cells as compared to non-treated NK cells, specifically, cells that were not exposed or incubated with the re-programmer and/or modulator of the preset disclosure, to inactivated NK cells, to dysfunctional NK cells (e.g., exhausted or anergic NK cells as defined herein), or NK cells of a healthy subject that naturally contain a population of dysfunctional (e.g., anergic NK cells).
  • dysfunctional NK cells e.g., exhausted or anergic NK cells as defined herein
  • measurable parameters in immune-related disorders e.g., reduced tumor size in animal models (of at least 10% or more, 20% or more of tumor size, volume or weight). More specifically, an increase as referred to herein is meant any increased elevated, enhanced, amplified, boosted, expanded, augmented, heightened, escalated, strengthened, intensified, maximized measurement in at least one of the indicated parameters.
  • Activation, reactivation and functionalization of dysfunctional NK cells involves restoring or enhancing their cytolytic potential and immunomodulatory functions.
  • NK cells or activated NK cells may be characterized for example by altered receptor expression (e.g., activating receptors like NKG2D or CD 16) on the surface of NK cells, increased cytokine production, increased degranulation (e.g. increase in CD107), increased calcium flux and/or increased cytotoxic activity (lysis of target cells) as compared to a reference standard or a control sample, as disclosed herein after.
  • the reference standard or the control sample may comprise for example dysfunctional nontreated NK cells or NK cells of a healthy subject (that also includes dysfunctional cells (e.g., anergic cells).
  • the activity of NK cells may also be evaluated by improved disease parameters (e.g., reduced tumor size in animal models).
  • Increase as used herein encompasses any increase of about 1% to about 100%. More specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, as compared with dysfunctional functional NK cells that were not treated, contacted and/or exposed to the disclosed re-programming agent.
  • 10%, 50%, 120%, 500%, etc. are interchangeable with "fold change” values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively.
  • 10%, 50%, 120%, 500%, etc. are interchangeable with "fold change” values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively.
  • NK cells that exhibit impaired functionality, deviating from their normal cytotoxic and immunoregulatory roles in the immune system. These cells typically show diminished ability to recognize and eliminate aberrant cells, such as tumor cells or virus-infected cells, due to deficiencies in key molecular pathways.
  • the dysfunction may result from intrinsic defects in activation or signaling pathways, such as reduced expression of activating receptors (e.g., NKG2D, NKp46), impaired signal transduction downstream of these receptors, or defective exocytosis of lytic granules containing perforin and granzymes.
  • Extrinsic factors can also contribute to NK cell dysfunction, including an immunosuppressive microenvironment, such as in tumors where cytokines (e.g., TGF-P) and metabolic stressors alter NK cell metabolism and suppress their activity.
  • cytokines e.g., TGF-P
  • metabolic stressors alter NK cell metabolism and suppress their activity.
  • dysfunctional NK cells may exhibit phenotypic markers of exhaustion, such as increased expression of inhibitory receptors (e.g., PD-1, TIM-3), and a skewed cytokine profile that reduces their efficacy.
  • dysfunction is typically assessed through assays measuring cytotoxic activity, cytokine production, and/or receptor expression profiles.
  • dysfunctional NK cells display reduced activity, and in more specific embodiments reduced, attenuated, decreased, or inhibited activity of about 1% to 99.9%, as compared with functional NK cells. More specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, as compared with functional NK cells.
  • NK cells display diminished activity, abolished activity, impaired activity, or no activity, specifically, when compared to functional NK cells, specifically, NK cells that display the natural functions of NK cells specified herein above.
  • the disclosed modulators comprise at least one compound that inhibits or reduces the levels, expression, stability, and/or activity of at least one member of the Early Growth Response (EGR) family of transcription factors.
  • EGR Early Growth Response
  • the Early Growth Response (EGR) family of transcription factors constitutes a group of zinc finger proteins that play a pivotal role in regulating gene expression in response to a variety of extracellular and intracellular stimuli, including growth factors, stress signals, and neuronal activity.
  • Members of this family, including EGR1, EGR2, EGR3, and EGR4 are characterized by the presence of highly conserved Cys2-His2-type zinc finger motifs within their DNA-binding domains, which facilitate specific interaction with GC-rich promoter regions of target genes.
  • EGR proteins act as molecular switches, modulating the transcriptional activation or repression of genes implicated in critical cellular processes such as proliferation, differentiation, apoptosis, and immune responses.
  • EGR proteins are rapidly inducible, often categorized as immediate-early genes, and their expression is tightly regulated at transcriptional and post- translational levels.
  • the disclosed modulators and/or reprogrammers comprise at least one compound that inhibits or reduces the levels, expression, stability, and/or activity of at least one member of the Diacylglycerol Kinase (DGK) family.
  • the Diacylglycerol Kinase (DGK) family comprises a group of enzymes that catalyze the phosphorylation of diacylglycerol (DAG), a lipid second messenger, to produce phosphatidic acid (PA). Both DAG and PA serve as critical signaling molecules that regulate various cellular processes, including proliferation, differentiation, metabolism, and immune responses.
  • DGKs play a central role in modulating the balance between DAG and PA, thereby influencing downstream signaling pathways mediated by protein kinase C (PKC) and RasGRP proteins, which depend on DAG, as well as PA-dependent pathways.
  • PLC protein kinase C
  • RasGRP proteins which depend on DAG, as well as PA-dependent pathways.
  • the DGK family is highly conserved across species and is classified into ten isoforms (DGKa through DGKK) in mammals, each characterized by unique structural domains and regulatory properties.
  • isoforms share a conserved catalytic domain and two cysteine-rich Cl domains, which mediate DAG binding, but they differ in auxiliary domains such as EF-hand motifs, pleckstrin homology (PH) domains, and sterile alpha motifs (SAM), which confer specificity in their subcellular localization, lipid substrate preference, and interaction with other proteins.
  • auxiliary domains such as EF-hand motifs, pleckstrin homology (PH) domains, and sterile alpha motifs (SAM), which confer specificity in their subcellular localization, lipid substrate preference, and interaction with other proteins.
  • the compounds used in the re -programming agents of the present disclosure specifically inhibit the expression, stability and/or activity of at least one member of the EGR family and/or at least one member of the DGK family.
  • “Expression”, as used herein generally refers to the process by which gene-encoded information is converted into the structures present and operating in the cell. Therefore, according to the disclosure “expression” of a gene, specifically, may refer to transcription into a polynucleotide (also referred to herein as a transcript), and/or translation into a protein, or even posttranslational modification of the protein.
  • Protein and/or transcript "stability”, as used herein, refers to the physical (thermodynamic) stability, and chemical stability of the protein and/or transcript and relates to the net balance of forces, which determine whether a protein will be in its native folded conformation or a denatured state. More specifically, the levels of proteins and/or transcripts within cells are determined not only by rates of synthesis as discussed above, but also by rates of degradation and the half-lives of proteins and/or transcripts within cells that vary widely, from minutes to several days. In eukaryotic cells, two major pathways mediate protein degradation, the ubiquitin-proteasome pathway and lysosomal proteolysis.
  • the compounds used in the re -programming agents of the present disclosure and any compositions, kits and methods thereof inhibit and/or reduce the expression, level, stability and/or activity of at least one of: at least one member of the EGR family and/or at least one member of the DGK family.
  • the terms “inhibition”, “moderation”, “reduction” or “attenuation” as referred to herein, relate to the retardation, restraining or reduction of the expression, levels, stability and/or activity of at least one EGR protein and/or at least one member of the DGK family by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%.
  • inhibit or decrease refers to an inhibition of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 folds or more.
  • the disclosed re-programming agent activates and/or functionalize dysfunctional NK cells that comprise anergic NK cells and/or exhausted NK cells. More specifically, in some embodiments, the dysfunctional cells may be anergic cells. In yet some embodiments the dysfunctional NK cells may be exhausted cells. In some embodiments, the dysfunctional NK cells may be exhausted cells and anergic cells.
  • the dysfunctional NK cell referred to by the present disclosure may be anergic cells.
  • “Anergic NK cells” refer herein to a subset of non-responsive peripheral NK cells or dysfunctional NK cells that fail to engage inhibitory receptors during development, due to lack of inhibitory receptor expression on the NK cell. Anergy results primarily from insufficient or inappropriate activation signals during the priming of NK cells, often due to a lack of co- stimulatory cues or the dominance of inhibitory signals.
  • functionally anergic NK cells that form a portion of about 13%+/-6, or the entire NK cell population in a healthy subject
  • these cells may be further structurally characterized.
  • anergic NK cells comprise a phenotype of NKG2A“panKIR“. More specifically, anergic NK cell do not express the NKG2A protein, and do not express the panKIR. In some other embodiments, anergic NK cells do not express or express only undetectable levels of the NKG2A protein, and the panKIR proteins. More specifically, "panKIR” as used herein, refers to molecules that target Killer-cell Immunoglobulin-like Receptors (KIRs) broadly across their subtypes. The KIR family consists of multiple inhibitory and activating receptors, which interact with MHC class I molecules to regulate NK cell activity.
  • KIR family is composed of multiple inhibitory and activating receptors, which interact with MHC class I molecules to regulate NK cell activity.
  • KIR proteins considered under the scope of panKIR include but are not limited to activating KIRs such as KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1 as well as to inhibitory KIRs such as KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3. It should be thus understood that according to some embodiments, anergic NK cells are NK cells that do not express at least one of the specified KIRs.
  • anergic NK cell is characterized in not expressing the NKG2A. More specifically, NKG2A is an inhibitory receptor expressed on NK cells and CD8+ T cells that interacts with HLA-E on target cells to suppress immune cell activation. NKG2A is a C-type lectin-like inhibitory receptor that pairs with CD94 to form a heterodimeric receptor complex known as CD94/NKG2A.
  • anergic NK cells as used herein are cells that do not express the NKG2A, and any of the KIRs as specified above, and any combinations thereof.
  • the dysfunctional NK cell referred to by the present disclosure may be exhausted NK cells. More specifically, “Exhausted” NK cells, refer herein to another subset of dysfunctional NK cells that arise because of chronic stimulation by their targets. This state is typically observed in settings such as chronic infections, cancer, or persistent inflammatory conditions, where continuous antigen exposure or signaling from inhibitory receptors leads to a dysregulated immune response.
  • Exhausted NK cells exhibit a distinct phenotypic profile marked by the upregulation of inhibitory receptors (e.g., PD-1, TIM-3, LAG-3, TIGIT, and in some embodiments, NKG2A and KIRs) and downregulation of activating receptors (e.g., NKG2D, NKp30). Additionally, they often display altered transcriptional and epigenetic signatures that reinforce their dysfunctional state. As opposed to anergic NK cells, exhausted NK cells may comprise a phenotype of NKG2A + panKIR + ' Unlike anergic NK cells, exhausted NK cells have a history of hyperactivation before transitioning to a dysfunctional state, whereas anergic cells may never achieve full activation. Still further, in some embodiments anergic cells do not achieve full activation.
  • inhibitory receptors e.g., PD-1, TIM-3, LAG-3, TIGIT, and in some embodiments, NKG2A and KIRs
  • activating receptors e
  • the at least one compound that specifically inhibits the expression, activity and/or stability of at least one of at least one member of the EGR family of transcription factors; and/or at least one member of the DGK family comprises at least one nucleic acid molecule, at least one amino acid-based molecule, and/or at least one chemical inhibitor, each specific for one of: (i) at least one member of the EGR family of transcription factors; and/or (ii) at least one member of the DGK family.
  • the disclosed re-programming agent may comprise several compounds, however, each of the compounds is specific for one target, for example, for one member of the ERG family or for one member of the DGK family.
  • the disclosed re-programming agent may comprise more than one compound (can be two or more different compounds specific for the same target), each specific for one member of the ERG family or for one member of the DGK family.
  • the re-programming agent (or modulator and/or re -programmer) of the present disclosure comprises at least one compound that inhibits the expression, activity and/or stability of at least one member of the EGR family, or of at least one member of the DGK family.
  • Such compound is also referred to herein as an "inhibitory compound”. It should be therefore understood that the inhibitory action of the compound is specific for the target EGR family member or the target DGK family member, while it acts as a re -programming agent for NK cells, and specifically for dysfunctional NK cells.
  • a "Compound” is used herein to refer to any substance, agent (e.g., molecule), supramolecular complex, material, or combination or mixture thereof.
  • a compound may be any agent that can be represented by a chemical formula, chemical structure, or sequence.
  • Example of compounds applicable for the present disclosure include, e.g., nucleic acid molecules (e.g., RNAi agents, antisense oligonucleotide, gRNAs, aptamers), small molecules, amino acid based molecules, for example, polypeptides, peptides, antibodies specific for EGR and/or DGK, lipids, polysaccharides, etc., that inhibit or disturb the activity thereof.
  • nucleic acid molecules e.g., RNAi agents, antisense oligonucleotide, gRNAs, aptamers
  • small molecules e.g., amino acid based molecules, for example, polypeptides, peptides, antibodies specific for EGR and/or DGK, lipids, polysaccharides, etc., that inhibit or disturb the activity thereof.
  • amino acid based molecules for example, polypeptides, peptides,
  • a compound may be obtained using any suitable method known in the art. The ordinary skilled artisan will select an appropriate method based, e.g., on the nature of the compound.
  • a compound may be at least partly purified.
  • a compound may be provided as part of a composition, which may contain, e.g., a counter-ion, aqueous or non-aqueous diluent or carrier, buffer, preservative, or other ingredient, in addition to the compound, in various embodiments.
  • a compound may be provided as a salt, ester, hydrate, or solvate.
  • a compound is cell-permeable, e.g., within the range of typical compounds that are taken up by cells and acts intracellularly, e.g., within mammalian cells, to produce a biological effect.
  • Certain compounds may exist in particular geometric or stereoisomeric forms. Such compounds, including cis- and trans-isomers, E- and Z- isomers, R- and S -enantiomers, diastereomers, (D)-isomers, (L)-isomers, (-) - and (+)-isomers, racemic mixtures thereof, and other mixtures thereof are encompassed by this disclosure in various embodiments unless otherwise indicated.
  • Certain compounds may exist in a variety or protonation states, may have a variety of configurations, may exist as solvates (e.g., with water (i.e. hydrates) or common solvents) and/or may have different crystalline forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by the present disclosure where applicable.
  • the inhibitory compound may be an antibody specifically directed against at least one member of the EGR family or at least one member of the DGK family, and affects, specifically, reduces the amount, stability and function of at least one member of the EGR family or at least one member of the DGK family. It should be noted that specific definition of the term antibodies as defined herein after in connection with other embodiments of the disclosure, is also relevant for these embodiments as well.
  • candidate compounds that specifically inhibit at least one of, the expression, activity and stability of at least one member of the EGR family, or of at least one member of the DGK family can be screened from large libraries of synthetic or natural compounds.
  • a compound to be tested may be referred to as a test compound or a candidate compound. Any compound may be used as a test compound in various embodiments.
  • a library of FDA approved compounds that can be used by humans may be used.
  • Compound libraries are commercially available from a number of companies including but not limited to Maybridge Chemical Co.
  • a library useful in the present disclosure may comprise at least 10,000 compounds, at least 50,000 compounds, at least 100,000 compounds, at least 250,000 compounds, or more.
  • the compound used for the re-programming agents of the present disclosure may be a small molecule, and as indicated above, may be referred to herein as an inhibitory compound.
  • a "small molecule” as used herein is an organic molecule that is less than about 2 kilodaltons (kDa) in mass. In some embodiments, the small molecule is less than about 1.5 kDa, or less than about 1 kDa.
  • the small molecule is less than about 800 daltons (Da), 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Often, a small molecule has a mass of at least 50 Da. In some embodiments, a small molecule is non-polymeric. In some embodiments, a small molecule is not an amino acid. In some embodiments, a small molecule is not a nucleotide. In some embodiments, a small molecule is not a saccharide.
  • a small molecule contains multiple carbon-carbon bonds and can comprise one or more heteroatoms and/ or one or more functional groups important for structural interaction with proteins (e.g., hydrogen bonding), e.g., an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodiments at least one functional groups.
  • proteins e.g., hydrogen bonding
  • Small molecules often comprise one or more cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups.
  • the re -programming agents of the present disclosure may comprise at least one compound that may comprise at least one nucleic acid molecule.
  • each nucleic acid molecule is specifically directed against, or is specific for at least one of: (i) at least one of EGR-2 and/or EGR-4; and (ii) at least one of DGK-a and/or DGK- ⁇ .
  • nucleic acid' refers to polymers of nucleotides, and includes but is not limited to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA/RNA hybrids including polynucleotide chains of regularly and/or irregularly alternating deoxyribosyl moieties and ribosyl moieties (i.e., wherein alternate nucleotide units have an —OH, then and — H, then an —OH, then an — H, and so on at the 2' position of a sugar moiety), and modifications of these kinds of polynucleotides, wherein the attachment of various entities or moieties to the nucleotide units at any position are included.
  • RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double- stranded polynucleotides. Preparation of nucleic acids is well known in the art. It should be appreciated that the disclosure may further refer to polyribonucleotide.
  • polyribonucleotide refers to a polynucleotide comprising one or more modified or unmodified ribonucleotides and/or their analogs.
  • polyribonucleotide is used interchangeably with the term “oligoribonucleotide”.
  • a nucleic acid molecule according to the disclosure may be an iRNA molecule, more specifically, dsRNA molecule.
  • the nucleic acid molecule of the disclosed re-programming agent is a ribonucleic acid (RNA) molecule, or any nucleic acid sequence encoding such RNA molecule. More specifically, in some embodiments, the disclosed re-programming agent comprises at least one RNA molecule that may be at least one of: a doublestranded RNA (dsRNA), an antisense RNA, a single-stranded RNA (ssRNA), guide RNA (gRNA) and a Ribozyme.
  • dsRNA doublestranded RNA
  • ssRNA antisense RNA
  • gRNA guide RNA
  • Ribozyme Ribozyme
  • the re-programming agent of the present disclosure may comprise at least one dsRNA molecule.
  • dsRNA molecule is at least one of: a small interfering RNA (siRNA), endoribonuclease-prepared short interfering RNA (esiRNA), a MicroRNA (miRNA), a short hairpin RNA (shRNA) and a PIWI interacting RNAs (piRNAs).
  • siRNA small interfering RNA
  • esiRNA endoribonuclease-prepared short interfering RNA
  • miRNA MicroRNA
  • shRNA short hairpin RNA
  • piRNAs PIWI interacting RNAs
  • the re-programming agents of the present disclosure may comprise at least one dsRNA molecules, specifically, each directed against, or specific for, at least one of: (i) at least one of EGR-1 and EGR-2; and (ii) at least one of DGK-b, c-DGK and DGK-3.
  • dsRNA molecules may be at least one of small interfering RNA (siRNA), MicroRNA (miRNA), short hairpin RNA (shRNA) and PIWI interacting RNAs (piRNAs).
  • the at least one compounds of the disclosure may be nucleic acid molecules that may comprise at least one of a small interfering RNA (siRNA), a short hairpin RNA (shRNA), microRNA (miRNA), antisense oligonucleotide (ASO), locked nucleic acid (LNA), as well as other nucleic acids derivatives.
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • miRNA microRNA
  • ASO antisense oligonucleotide
  • LNA locked nucleic acid
  • the at least one compounds of the disclosure may be dsRNA molecules participating in RNA interference. More specifically, the dsRNA encompassed by the disclosure may be selected from the group consisting of small interfering RNA (siRNA), MicroRNA (miRNA), short hairpin RNA (shRNA), PIWI interacting RNAs (piRNAs).
  • RNA interference is a general conserved eukaryotic pathway which down regulates gene expression in a sequence specific manner. It is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by siRNA that is homologous in its duplex region to the sequence of the silenced gene.
  • RNAi is a multistep process. In a first step, there is cleavage of large dsRNAs into 21-23 ribonucleotides-long double-stranded effector molecules called “small interfering RNAs” or “short interfering RNAs” (siRNAs). These siRNAs duplexes then associate with an endonuclease-containing complex, known as RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the RISC specifically recognizes and cleaves the endogenous mRNAs/RNAs containing a sequence complementary to one of the siRNA strands.
  • One of the strands of the double-stranded siRNA molecule (the “guide” strand) comprises a nucleotide sequence that is complementary to a nucleotide sequence of the target gene, or a portion thereof
  • the second strand of the doublestranded siRNA molecule (the passenger” strand) comprises a nucleotide sequence substantially similar to the nucleotide sequence of the target gene, or a portion thereof.
  • the guide strand After binding to RISC, the guide strand is directed to the target mRNA cleaved between bases 10 and 11 relative to the 5' end of the siRNA guide strand by the cleavage enzyme Argonaute-2 (AGO2).
  • AGO2 cleavage enzyme Argonaute-2
  • the process of mRNA translation can be interrupted by siRNA.
  • the re-programming agent (or modulator and/or reprogrammer) of the present disclosure may comprise at least one siRNA molecule that specifically target at least one member of the EGR family, and at least one siRNA molecule that specifically target at least one member of the DGK family.
  • siRNAs comprise a duplex, or double-stranded region, of about 5-50 or more, 10-50 or more, 15-50 or more, 5-45, 10-45, 15-45, 5-40, 10-40, 15-40, 5-35, 10-35, 15-35, 5-30, 10-30 and 15-30 or more nucleotides long.
  • the siRNAs of the disclosure comprise a nucleic acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides.
  • siRNAs contain from about two to four unpaired nucleotides at the 3' end of each strand. At least a portion of one strand of the duplex or double- stranded region of a siRNA is substantially homologous to or substantially complementary to a target sequence within the gene product (i.e. RNA) molecule as herein defined.
  • the strand complementary to a target RNA molecule is the “antisense guide strand”
  • the strand homologous to the target RNA molecule is the “sense passenger strand” (which is also complementary to the siRNA antisense guide strand).
  • siRNAs may also be contained within structured such as miRNA and shRNA which has additional sequences such as loops, linking sequences as well as stems and other folded structures.
  • a double-stranded interfering RNA e.g., siRNA
  • a hairpin or stem-loop structure e.g., shRNA
  • shRNA short hairpin RNA
  • the compounds according to the present disclosure may be a micro-RNA (miRNA).
  • miRNAs are small RNAs made from genes encoding primary transcripts of various sizes. They have been identified in both animals and plants.
  • the primary transcript (termed the “pri-miRNA") is processed through various nucleolytic steps to a shorter precursor miRNA, or "pre-miRNA.”
  • the pre-miRNA is present in a folded form so that the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA.
  • the pre-miRNA is a substrate for a form of dicer that removes the miRNA duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex.
  • miRNAs bind to transcript sequences with only partial complementarity and usually repress translation without affecting steady-state RNA levels.
  • Both miRNAs and siRNAs are processed by Dicer and associate with components of the RNA-induced silencing complex (RISC). More specific embodiments relate to the compounds of the re-programming agent (or modulator and/or re-programmer) of the present disclosure that may be at least one shRNA molecule.
  • RISC RNA-induced silencing complex
  • shRNA refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence. The degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions.
  • the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop.
  • an “antisense RNA” is a single strand RNA (ssRNA) molecule that is complementary to an mRNA strand of a specific target gene product. Antisense RNA may inhibit the translation of a complementary mRNA by base-pairing to it and physically obstructing the translation machinery.
  • complementary it is meant the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands.
  • Complementary polynucleotide strands can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes.
  • the inhibitory compound/s of the re-programming agent (or modulator and/or re-programmer) of the present disclosure may comprise an antisense oligonucleotide, or any derivatives thereof.
  • such oligonucleotide is an antisense oligonucleotide (ASO).
  • ASO antisense oligonucleotide
  • oligonucleotide means a compound comprising a plurality of linked nucleosides.
  • an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.
  • modified oligonucleotide means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
  • Antisense oligonucleotide (AON, or ASO/s as used herein interchangeably) means an oligomeric compound, at least a portion of which is at least partially complementary to a target nucleic acid to which it hybridizes, for example, a target sequence within the nucleic acid sequence encoding at least one of, at least one EGR protein, and at least one DGK protein. Such hybridization results in at least one antisense activity.
  • the present disclosure provides antisense oligonucleotides of any of a variety of ranges of lengths.
  • the present disclosure provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths.
  • the disclosure provides oligomeric compounds or oligonucleotides consisting of X to Y linked nucleosides or nucleotides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range. In certain such embodiments, X and
  • Y are each independently selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 and more nucleosides or nucleotides; provided that X ⁇ Y.
  • the oligonucleotides provided and used by the disclosure may comprise DNA, RNA, any derivatives thereof or any combinations thereof.
  • the currently used antisense oligonucleotides are rarely regular RNA or DNA oligonucleotide, as alternative antisense oligonucleotide chemistries have been developed to improve affinity, boost stability in the circulation and in target cells, and enhance cell penetration and nuclear accumulation.
  • the non-bridging oxygen in the phosphate backbone may be replaced with a sulfur atom, generating phosphorothioate (PS) AONs. This modification enhances cellular uptake and improves resistance to nucleases but reduces the affinity of the AON to the target RNA.
  • PS phosphorothioate
  • MOE or “2'-M0E” or “2'-OCH2CH2OCH3” or “2'-O-methoxyethyl” each means a nucleoside comprising a sugar comprising a — OCH2CH2OCH3 group at the 2' position of the sugar ring.
  • a methylene bridge connects the 2'-0 and the 4'-C of the ribose, forcing the nucleotide in the “endo” conformation, in what has been dubbed “locked nucleic acid” (LNA). This modification leads to a very high affinity for the target nucleic acid.
  • two more oligonucleotide chemistries may be used in attempts to inhibit at least one of the activity, expression and stability of at least one of, at least one EGR protein and at least one DGK protein, in accordance with some embodiments of the disclosure, specifically, peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs).
  • PNAs peptide nucleic acids
  • PMOs phosphorodiamidate morpholino oligomers
  • the inhibitory compounds acting as NK activating and/or functionalizing re -programming agent of the present disclosure may comprise at least one ribozyme.
  • Ribozymes ribonucleic acid enzymes
  • ribozyme refers to a catalytically active RNA molecule capable of site-specific cleavage of target mRNA.
  • a ribozyme is a Varkud satellite ribozyme, a hairpin ribozyme, a hammerhead ribozyme, or a hepatitis delta ribozyme.
  • the inhibitory compounds used in the NK cell re-programming agent of the present disclosure may be based on any gene editing system, specifically programmable system, that is specifically directed against nucleic acid sequences comprised within the nucleic acid sequence encoding at least one of, at least one EGR protein and at least one DGK protein.
  • the inhibitory compound of the reprogramming agent of the present disclosure may comprise at least one nucleic acid sequence that targets a modifier protein, for example, a nuclease or any fusion proteins thereof, to a target sequence within the nucleic acid sequence encoding at least one of, at least one EGR protein and at least one DGK protein.
  • a targeting molecule such as a specific guide RNA
  • targeted manipulation e.g., cleavage or any other modification
  • the inhibitory compound is at least one guide RNA that guides at least one programmable engineered nucleases (PEN) to the target nucleic acid sequence as specified herein.
  • the PEN comprises at least one clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR associated (cas) protein.
  • CRISPR clustered regulatory interspaced short palindromic repeat
  • the inhibitory compound used by the disclosure comprises: first (a), at least one nucleic acid sequence comprising at least one gRNA, or any nucleic acid sequence encoding the gRNA; or any kit, composition, vector or vehicle comprising the gRNA or nucleic acid sequence encoding the gRNA.
  • the inhibitory compound may further comprise (b), at least one CRISPR/cas protein, or any nucleic acid molecule encoding the Cas protein, or any kit, composition, vector or vehicle comprising the CRISPR/cas protein or nucleic acid sequence encoding the CRISPR/cas protein, or any nucleic acid sequence encoding said gRNA; or any kit, composition or vehicle comprising at least one of (a) and (b).
  • the Cas protein and the specific gRNA may be provided to and/or contacted with the target cell (e.g., hematopoietic cell, such as NK or T cell), or administered to the treated subject, either as a protein and gRNA, or alternatively, as nucleic acid sequences encoding these two elements, either in two separate nucleic acid molecules (e.g., two separate constructs), or in one nucleic acid molecule (e.g., a construct encoding both).
  • PEN programmable engineered nucleases
  • the at least one PEN target and cut specific genomic sequences (recognition sequences) such as DNA sequences.
  • the at least one PEN may be derived from natural occurring nucleases or may be an artificial enzyme, all involved in DNA repair of double strand DNA lesions and enabling direct genome editing.
  • the inhibitory compound according with the present disclosure encompasses also any nucleic acid molecule comprising at least one nucleic acid sequence encoding the PEN or any kit, composition or vehicle comprising the at least one PEN, or any nucleic acid sequence encoding the PEN.
  • nucleases may include RNA guided nucleases such as CRISPR-Cas.
  • RNA guided nucleases such as CRISPR-Cas.
  • other nucleases such as ZFN, TALEN, Homing endonuclease, Meganuclease, Mega-TALEN may be used by the methods of the disclosure for targeting at least one target nucleic acid sequence comprised within the nucleic acid sequence that encodes at least one of: at least one EGR protein and at least one DGK protein.
  • the at least one PEN may be at least one of a mega nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a clustered regularly interspaced short palindromic repeats (CRISPR/Cas) system.
  • the at least one PEN may be a mega nuclease.
  • Mega nucleases are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs); such that this site generally occurs only once in any given genome. Meganucleases are specific naturally occurring restriction enzymes and include among others, the LAGLID ADG family of homing endonucleases, mostly found in the mitochondria and chloroplasts of eukaryotic unicellular organisms.
  • the at least one PEN may be a megaTAL.
  • MegaTALs are fusion proteins that combine homing endonucleases, such as LAGLIDADG family, with the modular DNA binding domains of TALENs.
  • the at least one PEN may be a zinc finger nuclease (ZFN).
  • ZFNs are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA- cleavage domain.
  • Zinc finger domains can be engineered to target specific desired DNA sequences, enabling ZFN to target the target sequences within the target transcripts specified by the disclosure, thereby inhibiting the expression, activity and/or stability of at least one of, at least one EGR protein and at least one DGK protein.
  • the at least one PEN may be a transcription activator-like effectorbased nuclease (TALEN).
  • TALEN are restriction enzymes that can be engineered to cut specific sequences of DNA.
  • TALEN are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands).
  • the targeting of the target nucleic acid sequence that is comprised within the nucleic acid sequence that encodes at least one of, at least one EGR protein and at least one DGK protein may be mediated by a PEN that may comprise at least one clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR associated (cas) protein system.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • cas CRISPR associated
  • Class 1 may be divided into types I, III, and IV and class 2 may be divided into types II, V, and VI (that target mRNA targets).
  • the Cas protein may be a member of at least one of CRISPR-associated system of Class 1 and Class 2.
  • the cas protein may be a member of at least one of CRISPR-associated system of any one of type II, type I, type III, type IV, type V and type VI from E. coli, Mycobacterium tuberculosis, Haloferax mediterranei, Methanocaldococcus jannaschii, Thermotoga maritima and other bacteria and archaea.
  • the CRISPR-Cas system targets DNA molecules based on short homologous DNA sequences, called spacers that exist between repeats. These spacers guide CRISPR-associated (Cas) proteins to matching sequences within the target DNA, called proto-spacers, which are subsequently cleaved.
  • the spacers can be rationally designed to form guide RNAs (gRNAs) that target any target DNA sequence, for example, the target sequence within the nucleic acid sequence that encodes at least one of, at least one EGR protein and/or at least one DGK protein.
  • inhibitory compounds of the NK re-programming agent of the present disclosure may comprise in some embodiments at least one gRNA targeted against at least one nucleic acid target that is comprised within at least one nucleic acid sequence that encodes at least one of, at least one EGR protein and at least one DGK protein.
  • the inhibitory compound acting as NK activating and/or functionalizing re-programming agent of the present disclosure may comprise any nucleic acid sequence encoding such gRNA.
  • the RNA guided DNA binding protein nuclease used by the disclosure may be a CRISPR Class 2 system.
  • class 2 system may be a CRISPR type II system.
  • the type II CRISPR-Cas systems include the ' HNH’-typc system (Streptococcus-like; also known as the Nmeni subtype, for Neisseria meningitidis serogroup A str. Z2491, or CASS4), in which Cas9, a single, very large protein, seems to be sufficient for generating crRNA and cleaving the target DNA, or mRNA, in addition to the ubiquitous Casl and Cas2.
  • Cas9 contains at least two nuclease domains, a RuvC-like nuclease domain near the amino terminus and the HNH (or McrA-like) nuclease domain in the middle of the protein, but the function of these domains remains to be elucidated.
  • HNH or McrA-like nuclease domain
  • HNH or McrA-like nuclease domain in the middle of the protein
  • HNH nuclease domain is abundant in restriction enzymes and possesses endonuclease activity responsible for target cleavage. It should be appreciated that any type II CRISPR-Cas systems may be applicable in the present disclosure, specifically, any one of type II-A, typell-B or typell-C.
  • At least one cas protein of type II CRISPR system used by the disclosure may be the cas9 protein, or any fragments, mutants, fusion proteins, variants or derivatives thereof (e.g., Cas9/Cpfl/CTc(l/2/3), SpCas9, SaCas9, engineered Cas9, and any mutants or fusion proteins thereof, for example, dCas9-Fokl, and the like).
  • the CRISPR- associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA:DNA complementarity to a target site (proto-spacer).
  • CRISPR type II system requires the inclusion of two essential components: a “guide” RNA (gRNA), that is comprised within the inhibitory compound/s of the NK re-programming agent (or modulator and/or re-programmer) of the present disclosure, and a non-specific CRISPR-associated endonuclease (Cas9).
  • gRNA guide RNA
  • Cas9 non-specific CRISPR-associated endonuclease
  • gRNA Guide RNA
  • crRNA targeting sequence
  • the gRNA of the disclosure may comprise between about 15 to about 50 nucleotides, specifically, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides.
  • spacers, or gRNA may comprise between about 20-35 nucleotides.
  • the gRNA that specifically targets EGR2 comprises the nucleic acid sequence as denoted as SEQ ID NO: 15 and/or SEQ ID NO: 16.
  • the gRNA that specifically targets DGKa comprises a nucleic acid sequence as denoted as SEQ ID NO: 17 and/or SEQ ID NO: 18.
  • inhibitory compounds of the re-programming agent of the present disclosure comprise at least one nucleic acid sequence encoding the gRNA
  • such encoding sequence may be designed to target at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • target protospacers within the at least one nucleic acid sequence that encodes at least one of, at least one EGR protein and at least one DGK protein.
  • PAM protospacer adjacent motif sequence recognition
  • the PAM is absolutely necessary for target binding and the exact sequence is dependent upon the species of Cas9 used.
  • Cas9 from S. pyogenes may be used in the methods, cells, compositions, and kits of the disclosure. Nevertheless, it should be appreciated that any known Cas9 may be applicable.
  • Non-limiting examples for Cas9 useful in the present disclosure include but are not limited to Streptococcus pyogenes (SP), also indicated herein as SpCas9, Staphylococcus aureus (SA), also indicated herein as SaCas9, Neisseria meningitidis (NM), also indicated herein as NmCas9, Streptococcus thermophilus (ST), also indicated herein as StCas9 and Treponema denticola (TD), also indicated herein as TdCas9.
  • SP Streptococcus pyogenes
  • SA Staphylococcus aureus
  • NM Neisseria meningitidis
  • ST Streptococcus thermophilus
  • TD Treponema denticola
  • type V CRISPR/Cas including Casl2a, Cpfl (type VI), C2C1 (type V-B), Casl3 (type VI), specifically, C2C2 and CasRx and CasX, as well as any variants or fusion proteins thereof, is also applicable in the methods of the disclosure.
  • the gRNA comprised within the inhibitory compounds acting as NK activating and/or functionalizing re -programming agent of the present disclosure targets the specific target sequence as disclosed by the disclosure and guides the CRISPR/Cas-protein, specifically, Cas9 to cleave, or perform other modification in the target site.
  • the end result of Cas9-mediated DNA cleavage is a double strand break (DSB) within the target DNA.
  • the resulting DSB may be then repaired by one of two general repair pathways, the efficient but error-prone Non-Homologous End Joining (NHEJ) pathway and the less efficient but high-fidelity Homology Directed Repair (HDR) pathway.
  • NHEJ Non-Homologous End Joining
  • HDR Homology Directed Repair
  • the targeted nucleic acid sequences specified above are repaired through the NHEJ pathway, resulting in most cases in alteration of the target sequence (deletions/insertions/non-sense mutations etc.), thereby inhibiting the expression, activity and/or stability of at least one of, at least one EGR protein and at least one DGK protein.
  • the DGK ⁇ as referred to herein relates to the human DGKq.
  • the human DGK ⁇ is as denoted by genebank accession number NM_001105540.
  • the DGK ⁇ as used herein refers to a protein comprising the amino acid sequence as denoted by SEQ ID NO: 7, or any variants or isoforms thereof.
  • the DGK ⁇ as used herein refers to a protein encoded by a nucleic acid molecule comprising the nucleic acid sequence as denoted by SEQ ID NO: 8, or any variants or isoforms thereof.
  • the DGK family comprises at least one of DGKa, DGKq, DGK5, DGK0 and DGKy.
  • the disclosed re -programming agent may comprise at least one compound, each specific for one of DGKa, DGK ⁇ , DGK5, DGK0 and DGKy.
  • the disclosed re-programming agent may comprise at least one siRNA molecule specific for DGKa and/or at least one siRNA molecule specific for DG Kq and/or at least one siRNA molecule specific for DGK5, and/or at least one siRNA molecule specific for DGK0 and/or at least one siRNA molecule specific for DGK5, and/or at least one siRNA molecule specific for DGKy.
  • the disclosed re-programming agent comprises at least one compound that inhibits the expression and/or the stability and/or the activity of at least on member of the EGR family, with the proviso that such EGR family member is not the EGR3.
  • the at least one member of the EGR family of transcription factors comprises at least one of EGR2 and EGR4; and the at least one member of the DGK family comprises at least one of DGKa and DGK ⁇ .
  • the disclosed NK re-programming agent may comprise at least one compound, each specific for one of EGR2 and EGR4, and/or at least one compound, each specific for one of DGKa and DGK ⁇ .
  • the disclosed re-programming agent may comprise at least one siRNA molecule specific for EGR2, and/or at least one siRNA molecule specific for EGR4, and/or at least one siRNA molecule specific for DGKa, and/or at least one siRNA molecule specific for DGK ⁇ .
  • at least one siRNA molecule specific for EGR4 and at least one siRNA molecule specific for DGKa may comprise at least one siRNA molecule specific for EGR2, and/or at least one siRNA molecule specific for EGR4, and/or at least one siRNA molecule specific for DGKa, and/or at least one siRNA molecule specific for DGK ⁇ .
  • the disclosed re-programming agent may comprise at least one siRNA molecule specific for DGKa, and at least one siRNA molecule specific for EGR2. In yet some further embodiments, the disclosed re-programming agent may comprise at least one siRNA molecule specific for EGR2 and at least one siRNA molecule specific for DGK ⁇ . Still further, in some embodiments, the disclosed re-programming agent may comprise at least one siRNA molecule specific for DGK ⁇ and at least one siRNA molecule specific for EGR4. Still further, in some embodiments, the disclosed re-programming agent may comprise at least one siRNA molecule specific for EGR4, and at least one siRNA molecule specific for EGR2 and at least one siRNA molecule specific for DGKa.
  • the disclosed re-programming agent may comprise at least one siRNA molecule specific for EGR4, and at least one siRNA molecule specific for EGR2 and at least one siRNA molecule specific for DGK ⁇ . In some embodiments, the disclosed re-programming agent may comprise at least one siRNA molecule specific for DGKa, and at least one siRNA molecule specific for DGK ⁇ . In some other embodiments, the disclosed re -programming agent may comprise at least one siRNA molecule specific for DGKa, and at least one siRNA molecule specific for DGK ⁇ , and at least one siRNA molecule specific for EGR2.
  • the disclosed re-programming agent may comprise at least one siRNA molecule specific for DGKa, and at least one siRNA molecule specific for DGK ⁇ , and at least one siRNA molecule specific for EGR4. In some other embodiments, the disclosed re-programming agent may comprise at least one siRNA molecule specific for DGKa, and at least one siRNA molecule specific for DGK ⁇ , and at least one siRNA molecule specific for EGR2, and at least one siRNA molecule specific for EGR4.
  • the inhibiting compounds e.g., the disclosed siRNA molecules
  • the inhibiting compounds may provide in some embodiments a synergistic effect.
  • the re-programming agents of the present disclosure may be also referred to as synergistic re-programming agents, that synergistically functionalize and/or rewire, and/or reprogram, and/or activate dysfunctional NK cells.
  • synergistic re-programming agents that synergistically functionalize and/or rewire, and/or reprogram, and/or activate dysfunctional NK cells.
  • any of the combinations of siRNA molecules disclosed herein above is considered in some embodiments as a synergistic combination.
  • the combination of siRNA specific for EGR2 and an siRNA molecule specific for EGR4 that may be used herein as the re -programming agent that functionalize dysfunctional NK cells (e.g., exhausted or anergic NK cells).
  • a re-programming agent comprising a combination of siRNA specific for EGR2 and an siRNA molecule specific for DGKa, that may be used herein as the re -programming agent (or modulator and/or re -programmer) that functionalize dysfunctional NK cells (e.g., exhausted or anergic NK cells).
  • a synergistic combination as used herein in connection with the disclosed reprogramming agents refers to the interaction between two or more components, or the cumulative action of the two components (e.g., siRNA specific for EGR2 and an siRNA molecule specific for EGR4) that produce a combined effect greater than the sum of their individual effects.
  • the re-programming agents disclosed herein may comprise any of the inhibitory compounds (e.g., siRNA molecules) at any ratio, for example, 1:1, 1:1:1, 1 : 1 : 1 : 1 , 1:2 to 1: 10 6 , 2:2:1, 3:1:1, 4: 1 : 1 : 1 , or between 0.0001 to 10 6 : between 0.0001 to 10 6 : and between 0.0001 to 10 6 .
  • the effective amount of the NK re-programming agents e.g., siRNAs
  • the effective amount of the NK re-programming agents may be in some embodiments, the amount of the compounds sufficient to inhibit the expression, stability and/or activity of any one of the at least one EGR protein and/or of the at least one DGK protein as discussed above.
  • the active ingredient in the compositions of the disclosure may be provided in an amount effective for inhibiting the expression, stability and activity of at least one EGR protein and/or of the DGKs proteins. Percentage of such inhibitory effect is as indicated herein before in connection with the re-programming agents of the present disclosure. Such inhibitory effect is in extent for activating hematopoietic cells, specifically NK cells, more specifically, dysfunctional NK cells (e.g., anergic or exhausted NK cells).
  • the disclosed re-programming agent comprises an effective amount of at least one of: (i) at least one siRNA or esiRNA molecule specific for EGR2; (ii) at least one siRNA or esiRNA molecule specific for EGR4; (iii) at least one siRNA or esiRNA molecule specific for DGKa and (iv) at least one siRNA or esiRNA molecule specific for DGK ⁇ ; and/or any combinations of (i), (ii), (iii) and (iv).
  • siRNA sequences are only non-limiting embodiments and working examples for nucleic acid-based inhibitors (e.g., siRNA, or esiRNA, gRNAs, Oligonucleotides, etc.), of the expression of at least one of the EGR family and/or at least one of the DGK family. More specifically, it should be understood that any siRNA that target the nucleic acid sequence encoding the EGR2 molecule, specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 2, or any fragments or parts thereof, may be used herein as a suitable siRNA molecule.
  • nucleic acid-based inhibitors e.g., siRNA, or esiRNA, gRNAs, Oligonucleotides, etc.
  • any siRNA that target the nucleic acid sequence encoding the EGR4 molecule specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 4, or any fragments or parts thereof, may be used herein as a suitable siRNA molecule.
  • any siRNA that target the nucleic acid sequence encoding the DGKa molecule specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 6, or any fragments or parts thereof, may be used herein as a suitable siRNA molecule.
  • any siRNA that target the nucleic acid sequence encoding the DGKq molecule specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 8, or any fragments or parts thereof, may be used herein as a suitable siRNA molecule.
  • an siRNA molecule specific for EGR2 may be any siRNA molecule or a plurality of siRNA molecules, or specifically, an esiRNA stands, specific for the EGR2 molecule. More specifically, esiRNA for "endoribonuclease-prepared small interfering RNA", is a form of small interfering RNA (siRNA) generated through enzymatic cleavage of long double- stranded RNA (dsRNA) by endoribonucleases such as RNase III or Dicer, thereby randomly generating a pool of plurality of various siRNA and/or esiRNA molecules targeting various target sequences within the target member of the EGR family, at the coding and/or the non-coding regions thereof.
  • siRNA small interfering RNA
  • the present disclosure provides siRNA and/or esiRNA molecules targeting EGR 2 at exon 2, and/or at the 3' UTR , for example, from position 2242 of the cDNA sequence as denoted by SEQ ID NO: 2 (NM_000399).
  • an esiRNA molecule specific for EGR2 may comprise the nucleic acid sequence as denoted by SEQ ID NO: 9, or any complementary sequnce thereof.
  • the siRNA used herein for targeting EGR2 comprise any optional 21 to 25 mers of said esiRNA sequence and thus, at least one fragment of the nucleic acid sequence as denoted by SEQ ID NO: 9, or any complementary sequence thereof.
  • an esiRNA molecule useful in the present disclosure for specifically targeting EGR2 may be the esiEgr2 MISSION Sigma Aldrich Cat no. EHU124311, as denoted by SEQ ID NO: 9, or any complementary sequence thereof, in more specific embodiments, the siRNA used herein for targeting EGR2 comprise any fragment between about 21 to 25 mers of the nucleic acid sequence as denoted by SEQ ID NO: 9, or any complementary sequence thereof.
  • the siRNA used for targeting EGR2 may comprise for example bases 1-21, and/or bases 2-22, and/or bases 1-23, and/or bases 2-24, and/or bases 1- 25 and/or bases 2-26 and/or bases 30-34 and/or bases 60-81, and so on of the nucleic acid sequence as denoted by SEQ ID NO: 9.
  • an esiRNA molecule specific for EGR4 is disclosed in yet some further specific embodiments of the disclosed modulator and/or re-programmers.
  • the present disclosure provides siRNA and/or esiRNA molecules targeting EGR 4 at exon 2, and/or at the 3' UTR , for example, from position 1385 of the cDNA sequence as denoted by SEQ ID NO: 4 (NM_001965).
  • an esiRNA molecule specific for EGR4 may comprise the nucleic acid sequence as denoted by SEQ ID NO: 10.
  • the siRNA used herein for targeting EGR4 comprise at least one fragment (e.g., any optional 21 to 25 mers) of the nucleic acid sequence as denoted by SEQ ID NO: 10, or any complementary sequence thereof.
  • an esiRNA molecule useful in the present disclosure for specifically targeting EGR4 may be the esiEgr4 MISSION Sigma Aldrich Cat no. EHU135811, as denoted by SEQ ID NO: 10, or any complementary sequence thereof. It should be therefore appreciated that in some embodiments, the siRNA used herein for targeting EGR4 may comprise any fragment between about 21 to 25 mers of the nucleic acid sequence as denoted by SEQ ID NO: 10, or any complementary sequence thereof.
  • the siRNA used for targeting EGR4 may comprise for example bases 1-21, and/or bases 2-22, and/or bases 1-23, and/or bases 2-24, and/or bases 1- 25 and/or bases 2-26 and/or bases 30-34 and/or bases 60-81, and so on of the nucleic acid sequence as denoted by SEQ ID NO: 10.
  • the present disclosure further encompasses any siRNA molecule that targets the nucleic acid molecules that encode any one of EGR2, as denoted by SEQ ID NO: 2, or any variants or homologs thereof or of the EGR4 molecule as denoted by SEQ ID NO: 4, or any variants or homologs thereof.
  • an siRNA molecule specific for DGKa In yet some further specific embodiments of the disclosed modulator and/or re -programmers, an siRNA molecule specific for DGKa. Still further, in some embodiments, the present disclosure provides siRNA and/or esiRNA molecules targeting DGKa at exon 21, from position 2158 of the cDNA sequence as denoted by SEQ ID NO: 6. In yet some further specific embodiments of the disclosed re -programming agent, an siRNA molecule specific for DGKa may comprise the nucleic acid sequence as denoted by SEQ ID NO: 13, or any complementary sequnce thereof. In yet some further specific embodiments of the disclosed modulator and/or re -programmers, an siRNA molecule specific for DGK ⁇ .
  • the present disclosure provides siRNA and/or esiRNA molecules targeting DGK ⁇ at exon 15, from position 1624 of the cDNA sequence as denoted by SEQ ID NO: 8.
  • an siRNA molecule specific for DGK ⁇ may comprise the nucleic acid sequence as denoted by SEQ ID NO: 14, or any complementary sequnce thereof, or of any variants, homologs or derivatives thereof.
  • an siRNA molecule useful in the present disclosure may be the DGKq siRNA Sigma Aldrich SASI_HS02_00324291.
  • the present disclosure further encompasses any siRNA molecule that targets the nucleic acid molecules that encode any one of EGR2, as denoted by SEQ ID NO: 2, or any variants or homologs thereof or of the EGR4 molecule as denoted by SEQ ID NO: 4, or any variants or homologs thereof.
  • the present disclosure further encompasses any siRNA molecule that targets the nucleic acid molecules that encode any one of DGKa, as denoted by SEQ ID NO: 6, or any variants or homologs thereof or of the DGK ⁇ molecule as denoted by SEQ ID NO: 8, or any variants or homologs thereof.
  • the disclosed re -programming agent may comprise any combination of the siRNA and/or esiRNA nuclei acid molecules as denoted by SEQ ID NO: 9, any fragment between about 21 to 25 mers (nucleotides) of the nucleic acid sequence as denoted by SEQ ID NO: 9, SEQ ID NO: 10, any fragment between about 21 to 25 mers of the nucleic acid sequence as denoted by SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, or any combinations thereof, for example, SEQ ID NO: 9, and SEQ ID NO: 10; or SEQ ID NO: 9 and SEQ ID NO: 13; or SEQ ID NO: 9 and SEQ ID NO: 14; or SEQ ID NO: 10 and SEQ ID NO: 13; or SEQ ID NO: 10 and SEQ ID NO: 14; or SEQ ID NO: 13 and SEQ ID NO: 14; or SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13;
  • the siRNAs used herein for at least one of DGK-a and/or DGK- ⁇ as denoted by Catalog number Sigma Aldrich SASI_HSS01_00072301 (SEQ ID NO: 13) and Sigma Aldrich SASI_HS02_00324291 (SEQ ID NO: 14).
  • the disclosed reprogrammer of the present disclosure may comprise a nucleic acid-based inhibitor of at least one of the EGR family and/or of at least one of the DGK family, that may be at least one gRNA molecule, or any nucleic acid sequence encoding such gRNA molecule, to thereby applying in some embodiments the CRISPR/Cas system.
  • the disclosed re-programmer of the present disclosure may comprise the gRNA molecule that directs the CAS9 protein to a target sequence within the EGR2, or the EGR4, or the DGKa, or the DGK ⁇ , or any nucleic acid molecule encoding such gRNA.
  • the disclosed specification provides any of the disclosed siRNA and/or eiRNA molecules, as well as any complementary sequence of at least one of SEQ ID NO: 9, 10, 13 and 14, of any variants, homologs or derivatives thereof.
  • any gRNA that target the nucleic acid sequence encoding the EGR2 molecule specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 2, or any fragments or parts thereof, may be used herein as a suitable gRNA molecule.
  • any gRNA that target the nucleic acid sequence encoding the EGR4 molecule specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 4, or any fragments or parts thereof, may be used herein as a suitable gRNA molecule.
  • any gRNA that target the nucleic acid sequence of the DGKa molecule may be used herein as a suitable gRNA molecule.
  • any gRNA that target the nucleic acid sequence encoding the DGK ⁇ molecule specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 8, or any fragments or parts thereof, may be used herein as a suitable gRNA molecule.
  • the disclosed re -programming agent comprises at least one gRNA molecule specific for EGR2.
  • the gRNA molecule comprises the nucleic acid sequence as denoted by SEQ ID NO: 15 (sense), and/or the gRNA molecule comprising the nucleic acid sequence as denoted by SEQ ID NO: 16 (antisense).
  • the disclosed modulator or re-programmer comprises at least one gRNA molecule specific for DGKa.
  • the gRNA molecule comprises the nucleic acid sequence as denoted by SEQ ID NO: 17 (sense), and/or the gRNA molecule comprising the nucleic acid sequence as denoted by SEQ ID NO: 18 (antisense).
  • the disclosed reprogrammer or modulator may comprise any combination of the gRNAs of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
  • Variants of the polynucleotides of the present disclosure may have at least 80% sequence similarity to the entire sequence, often at least 85% sequence similarity, 90% sequence similarity, or at least 95%, 96%, 97%, 98%, or 99% sequence similarity or identity at the nucleic acid level, with the nucleic acid sequence of interest, such as the various polynucleotides of the disclosure.
  • the term "derivative" is used to define nucleic acid sequence variants, and covalent modifications of a polynucleotide made use of in the present disclosure, e.g. of a specified sequence.
  • the functional derivatives of any of the polynucleotides utilized according to the present disclosure e.g.
  • a specified sequence of any one of the polynucleotides of SEQ ID NOs: 9, 10, 13, 14, 15, 16, 17 and 18, or any complementary sequence thereof preferably have at least about 65%, more preferably at least about 75%, even more preferably at least about 85%, most preferably at least about 95% overall sequence homology with the nucleic acid sequence of the polynucleotide as structurally defined above, e.g.
  • the at least one nano- or micro-particle, micellar formulation, vehicle or matrix disclosed herein may comprise an effective amount of the disclosed nano- or micro-particle, micellar formulation, vehicle or matrix re-programming agents.
  • the amount of the re-programming agent in the disclosed nano- or micro-particle, micellar formulation, vehicle or matrix is the amount effective for activation of NK cells, specifically, activation of dysfunctional NK cells.
  • the re-programming agent comprised within the vehicle, matrix, nano- or micro-particle or micellar formulation of the disclosure may comprise at least one compound that comprise at least one nucleic acid molecule. More specifically, each of the nucleic acid molecules is specific for or specifically directed against one of at least one of: (i) at least one of EGR-2 and/or EGR-4; and/or (ii) at least one of DGK-a and/or DGK- ⁇ .
  • the re-programming agents comprised within the nano- or micro-particle or micellar formulation or vehicle or matrix of the disclosure may comprise at least one dsRNA molecules. More specifically, each of the dsRNA molecules may be directed against or specific for one of at least one of: (i) at least one of EGR-1 and EGR-2; and (ii) at least one of: (i) at least one of EGR-2 and/or EGR-4; and/or (ii) at least one of DGK-a and/or DGK- ⁇ . In yet more specific embodiments, such dsRNA molecules, may be at least one of siRNA, miRNA, shRNA and piRNAs.
  • the re-programming agents comprised within the nano- or micro-particle or micellar formulation or vehicle or matrix of the disclosure may comprise at least one siRNA molecule specifically directed against, or specific for EGR-2, and/or at least one siRNA molecule specifically directed against, or specific for DGK-a. It should be understood that any of the disclosed nano- or micro-particle or micellar formulation or vehicle or matrix, are applicable for any aspect of the present disclosure.
  • the at least one re-programming agent may be encapsulated within the intra-nanoparticle core and/or cavity of the nanoparticle of the disclosure.
  • the compounds of the modulator and/or re -programmers of the present disclosure specifically, the siRNAs of the disclosure may be surrounded, enveloped, encapsulated, entrapped and comprised within a nanoparticle, specifically, within the inner core and/or cavity of a nano-or microparticle.
  • at least one targeting moiety is connected and/or associated directly, or indirectly, with the outer nanoparticle surface of the nano-micro particle, vehicle, matrix, microparticle or micellar formulation of the disclosure.
  • the at least one targeting moiety of the nano- or micro-particle or micellar formulation or vehicle or matrix of the disclosure may be any affinity molecule, for example, at least one of an antibody, an aptamer, a ligand (e.g., for an activating receptor, or alternatively, a ligand for inhibitory receptor) or any combinations thereof, that specifically recognizes and binds at least one molecule expressed on the surface of at least one hematopoietic cell.
  • the targeting moiety used by the nano-particles of the disclosure may comprise an antibody or any antigen binding fragments thereof.
  • the antibody used as a targeting moiety for the vehicle, matrix, nano- or micro-particle or micellar formulation of the disclosure may be any one of: full length antibody, antibody fragment, single-chain variable fragment (scFv), bi-specific antibody, tri-specific antibody and variable new antigen receptor antibody (V-NAR).
  • antibody as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen.
  • CDR complementarity determining region
  • the term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM).
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CHI, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region comprises one domain (CL1).
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • antigen-binding domains that can be used in the context of the present disclosure include antibodies, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins comprising a ligand-binding portion of a receptor that specifically binds a particular antigen or antigen-binding scaffolds.
  • the antigen binding domains in accordance with the disclosure may recognize and bind a specific antigen or epitope.
  • binding specificity specifically binds to an antigen
  • binding reaction which is determinative of the presence of the epitope in a heterogeneous population of proteins and other biologies.
  • epitope is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody.
  • Epitopes or "antigenic determinants” usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • an "antigen-binding domain” can comprise or consist of an antibody or antigen-binding fragment of an antibody.
  • antigen-binding fragment of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.
  • the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)).
  • CDR complementarity determining region
  • Other engineered molecules such as domain-specific antibodies, single domain antibodies, domain- deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g.
  • an antigenbinding fragment of an antibody will typically comprise at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the VH and VL domains may be situated relative to one another in any suitable arrangement.
  • variable region may be dimeric and contain VH-VH (e.g., VHH nanobodies), VH-VL or VL-VL dimers.
  • VH-VH e.g., VHH nanobodies
  • VH-VL e.g., VHH nanobodies
  • VL-VL dimers e.g., VHH nanobodies
  • the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
  • the antibody suitable for the targeting moiety of the nanoparticle of the disclosure may also be a bi-specific antibody (such as Bi-specific T-cell engagers-BiTEs) or a tri-specific antibody.
  • the antibody suitable for the disclosure may also be a variable new antigen receptor antibody (V-NAR).
  • VNARs are a class of small, immunoglobulin-like molecules from the shark immune system. Humanized versions of VNARs could be used to bind protein epitopes that are difficult to access using traditional antibodies.
  • the targeting moiety of the nanoparticle of the disclosure may comprise aptamers that specifically recognize and bind at least one molecule expressed on the surface of at least one hematopoietic cell.
  • aptamer or “specific aptamers” denotes single-stranded nucleic acid (DNA or RNA) molecules which specifically recognizes and binds to a target molecule.
  • the aptamers according to the disclosure may fold into a defined tertiary structure and can bind a specific target molecule with high specificities and affinities. Aptamers are usually obtained by selection from a large random sequence library, using methods well known in the art, such as SELEX and/or Molinex.
  • aptamers may include single-stranded, partially single-stranded, partially double-stranded or doublestranded nucleic acid sequences; sequences comprising nucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified nucleotides and nucleotides comprising backbone modifications, branch points and non-nucleotide residues, groups or bridges; synthetic RNA, DNA and chimeric nucleotides, hybrids, duplexes, heteroduplexes; and any ribonucleotide, deoxyribonucleotide or chimeric counterpart thereof and/or corresponding complementary sequence.
  • aptamers used by the disclosure are composed of deoxyribonucleotides .
  • the aptamer that may be applicable herein may optionally comprise a spacer between the nucleic acid sequence and the reactive group.
  • the spacer may be an alkyl chain such as (CH2)e/i2, namely comprising six to twelve carbon atoms.
  • the at least one target moiety of the nano-particles of the disclosure specifically recognizes and binds and thereby targets at least one molecule expressed on the surface of at least one hematopoietic cell.
  • Hematopoietic cells are cellular blood components all derived from hematopoietic stem cells in the bone marrow. It should be appreciated that in certain embodiments, hematopoietic cells as used herein include cells of the myeloid and the lymphoid lineages of blood cells. More specifically, myeloid cells include monocytes, (macrophages and dendritic cells (DCs)), granulocytes (neutrophils), basophils, eosinophils, erythrocytes, and megakaryocytes or platelets. The Lymphoid cells include T cells, B cells, and natural killer (NK) cells.
  • NK natural killer
  • the cells targeted by the modulator and/or re-programmers of the present disclosure may be any hematopoietic cell described herein.
  • blood cells are divided into three lineages: red blood cells (erythroid cells) which are the oxygen carrying, white blood cells (leukocytes that are further subdivided into granulocytes, monocytes and lymphocytes) and platelets (thrombocytes).
  • red blood cells erythroid cells
  • white blood cells leukocytes that are further subdivided into granulocytes, monocytes and lymphocytes
  • platelets thrombocytes
  • the at least one target moiety of the nano-particles of the disclosure specifically recognizes and binds and thereby targets at least one molecule expressed on the surface of a lymphocyte.
  • Lymphocytes are mononuclear nonphagocytic leukocytes found in the blood, lymph, and lymphoid tissues. They comprise the body's immunologically competent cells and their precursors. They are divided on the basis of ontogeny and function into two classes, B and T lymphocytes, responsible for humoral and cellular immunity, respectively. Most are small lymphocytes 7-10 pm in diameter with a round or slightly indented heterochromatic nucleus that almost fills the entire cell and a thin rim of basophilic cytoplasm that contains few granules.
  • lymphocytes When “activated" by contact with antigen, small lymphocytes begin macromolecular synthesis, the cytoplasm enlarges until the cells are 10-30 pm in diameter, and the nucleus becomes less completely heterochromatic; they are then referred to as large lymphocytes or lymphoblasts. These cells then proliferate and differentiate into B and T memory cells and into the various effector cell types: B cells into plasma cells and T cells into helper, cytotoxic, and suppressor cells.
  • the hematopoietic cell recognized or targeted by the targeting moiety of the nano- or micro-particle or micellar formulation or vehicle or matrix of the disclosure may be a natural killer (NK) cell.
  • NK natural killer
  • the nanoparticle, vehicle, matrix, microparticle or micellar formulation of the disclosure may be associated directly or indirectly by the outer nanoparticle surface thereof with at least one antibody, at least one aptamer or any combinations thereof, that specifically recognize and bind at least one of at least one NK cell activating receptor and at least one NK cell inhibitory receptor or any combinations thereof.
  • Natural killer (NK) cells are a type of cytotoxic lymphocytes that are critical to the innate immune system in providing rapid responses to viral-infected cells and tumor formation.
  • NK cells do not express T cell antigen receptors (TCR) or pan T marker CD3 or surface immunoglobulins (Ig) B cell receptors, instead they express the surface markers CD16 (FcyRIII) and CD56 in humans (NK1.1 or NK1.2 in mice), up to 80% of human NK cells also express CD8. Further, NK cells are effectors of innate immunity in expressing activating and inhibitory NK receptors, which play an important function in self-tolerance and in sustaining NK activity.
  • TCR T cell antigen receptors
  • Ig surface immunoglobulins
  • NK cell inhibitory receptors are part of either the immunoglobulin-like (IgSF) superfamily or the C-type lectin-like receptor (CTLR) superfamily.
  • IgSF immunoglobulin-like
  • CLR C-type lectin-like receptor
  • KIR human killer cell immunoglobulin-like receptor
  • ITT Immunoglobulin-like transcripts
  • KIRs Killer-cell immunoglobulin-like receptors
  • KIRs are a family of type I transmembrane glycoproteins expressed on the plasma membrane of natural killer (NK) cells and a minority of T cells.
  • MHC major histocompatibility
  • KIR receptors can distinguish between major histocompatibility (MHC) class I allelic variants, which allows them to detect virally infected cells or transformed cells.
  • MHC major histocompatibility
  • Most KIRs are inhibitory, meaning that their recognition of MHC molecules suppresses the cytotoxic activity of their NK cell. Only a limited number of KIRs are activating, meaning that their recognition of MHC molecules activates the cytotoxic activity of their cell.
  • Activating receptors do not have the immunoreceptor tyrosine-base inhibition motif (ITIM) characteristic of inhibitory receptors, and instead contain a positively charged lysine or arginine residue in their transmembrane domain (with the exception of KIR2B4) that helps to bind DAP12, an adaptor molecule containing a negatively charged residue as well as immunoreceptor tyrosinebased activation motifs (IT AM).
  • Activating KIR receptors include KIR2DS, KIR2DL1, KIR3DS and CD244 (Cluster of Differentiation 244), also known as Natural Killer Cell Receptor 2B4.
  • the disclosure relates to the nano- or micro-particle or micellar formulation or vehicle or matrix of the disclosure targeting (by way of having a targeting moiety targeted at) any of the activating and/or inhibitory receptors of NK cells or T cells, and to any derivatives, splice, homologs and orthologs variants thereof or any combinations thereof.
  • the activating or inhibitory receptors of the NK cells targeted by the targeting moiety of the nano-particles of the disclosure may be any of the molecules described herein that may comprise 'equivalent amino acid residues'. This term refers to an amino acid residue capable of replacing another amino acid residue in a polypeptide without substantially altering the structure and/or functionality of the polypeptide.
  • Equivalent amino acids thus have similar properties such as bulkiness of the sidechain, side chain polarity (polar or non-polar), hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral or basic) and side chain organization of carbon molecules (aromatic/aliphatic). As such, equivalent amino acid residues can be regarded as conservative amino acid substitutions.
  • Amino acids having polar side chains (Asp, GIu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys); (ii) Amino acids having non-polar side chains (Gly, Ala, Vai, Leu, lie, Phe, Trp, Pro, and Met); (iii) Amino acids having aliphatic side chains (Gly, Ala Vai, Leu, ile); (iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro); (v) Amino acids having aromatic side chains (Phe, Tyr, Trp); (vi) Amino acids having acidic side chains (Asp, GIu); (vii) Amino acids having basic side chains (Lys, Arg, His); (viii) Amino acids having amide side chains (Asn, Gin); (ix) Amino acids having hydroxy side chains (Ser, Thr); (x) Amino acids having sulphide (
  • the activating or inhibitory NK receptors targeted by the targeting moiety of the nanoparticles of the disclosure may have secondary modifications, such as phosphorylation, acetylation, glycosylation, sulfhydryl bond formation, cleavage and the likes, as long as said modifications retain the functional properties of the original protein, specifically, act as inhibitory or activating receptors.
  • Secondary modifications are often referred to in terms of relative position to certain amino acid residues. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • the disclosure further encompasses any derivatives, enantiomers, analogues, variants or homologues of any of the NK activating and or inhibitory receptors disclosed herein.
  • derivative is used to define amino acid sequences (polypeptide), with any insertions, deletions, substitutions and modifications to the amino acid sequences (polypeptide) that do not alter the activity of the original polypeptides.
  • derivative it is also referred to homologues, variants and analogues thereof, as well as covalent modifications of a polypeptides made according to the present disclosure.
  • any splice variants of the indicated receptors are also encompassed by the present disclosure.
  • the nanoparticles of the disclosure may be connected and/or associated directly or indirectly in the outer nanoparticle surface thereof with at least one antibody directed against NKp46 or any derivative, splice variant, homolog, ortholog or valiant thereof.
  • the nanoparticles of the disclosure may be associated as a targeting moiety, with any of the anti-NKp46 antibodies disclosed by the Examples section.
  • the nanoparticle of the disclosure may be associated with a plurality of antibodies, aptamers or any combinations thereof. It should be noted that each of the plurality of antibodies or aptamers specifically recognizes and binds an NK cell inhibitory or activating receptor of a plurality of inhibitory or activating receptors expressed by NK cells of a subject suffering from an immune-related disorder.
  • the targeting moiety may be directed against a variety of molecules expressed on a target cell, specifically, any hematopoietic cell, more specifically, NK cell.
  • NK cells within the tumor microenvironment often display a decrease in expression of activating receptors, an increased expression of inhibitory receptors, and a decrease in cytokine secretion and cytotoxic ability.
  • the present nanoparticles of the disclosure may offer personalized therapy, by efficient targeting of the reprogramming agents of the present disclosure to the particular NK present in the diseased subject, for example, NK cells obtained from a tumor of a particular patient.
  • the targeting moiety may target receptors, specifically activating and/or inhibitory receptors expressed on an NK cell isolated or obtained from the treated subject or a subject suffering from an immune-related disorder.
  • the profiling of the specific molecules (e.g., activating and/or inhibitory receptors) in the patient's NK cells should be determined in order to design specific targeting moieties for the nanoparticles of the disclosure.
  • the nano-particles of the disclosure may be connected to any commercial antibody specific for any of the inhibitors and/or activating NK cell receptor. Examples for NKp46 antibody may be the LS-C662543 by LSBio.
  • the nano-particles of the disclosure may be connected to any commercially available antibody specific for any one of CD57, NKG2A, CD96, the natural cytotoxicity receptors (NCRs) NKp30, NKp44, NKp80, CD16, NKG2D, NKG2C, DNAX Accessory Molecule-1 (DNAM-1), and 2B4, or any of the receptors disclosed by the disclosure.
  • the NK cells obtained from the patient may be used for screening antibodies libraries or alternatively, aptamer libraries to design the most appropriate targeting moieties that may be used for a specific patient.
  • the targeting moieties may be aptamers.
  • Aptamers are produced by a combinatorial procedure named SELEX (Systematic Evolution of Ligands by Exponential enrichment), that are emerging as promising diagnostic and therapeutic tools.
  • SELEX Systematic Evolution of Ligands by Exponential enrichment
  • cell-SELEX procedures using living cells as complex targets
  • a major advantage of the cell-based procedures is that they may be employed for the targeting of a specific cell type, without any prior knowledge of the specific target, leading to identify multiple aptamers able to recognize specific cell phenotypes and discover new cell biomarkers.
  • aptamers have been developed for different cell types and other complex systems, especially for live cancer cells, and thus, may be also developed for NK cells of a subject suffering from an immune-related disorder.
  • the SELEX procedure involves repeated cycles of: 1. Incubation of the high complexity library with the targets (binding); 2. Removal of unbound sequences and recovery of the bound oligonucleotides (partitioning); 3. Amplification of the bound sequences by PCR (for DNA library) or RT-PCR and transcription (for RNA library).
  • a fundamental aspect is the inclusion of a counter-selection step, for example, using NK cells obtained from a healthy subject or a subject that is not suffering from the specific immune-related disorder, to avoid the parallel enrichment of aptamers for unwanted targets.
  • the negative selection step is introduced before the positive selection at each round, allowing filtering out sequences against those molecules commonly expressed on both the target and control cell lines.
  • an alternative cell-SELEX strategy (referred as “differential cell- SELEX”) has been developed to isolate aptamers able to recognize a specific cell phenotype, rather than a single specific target of interest. This strategy offers the possibility to select multiple ligands discriminating between even closely related cell types, without any prior knowledge of the target.
  • the procedure consists of the incubation of the starting library on a non-target cell line (with undesired phenotype, negative selection step) followed by the recovery of unbound oligonucleotides that are, then, incubated on cells with the desired phenotype (positive selection step).
  • Fluorescence-Activated Cell Sorting (FACS)-SELEX An extension of the cell-SELEX strategy is a Fluorescence-Activated Cell Sorting (FACS)-based protocol that allows to select aptamers targeting a specific subpopulation.
  • FACS Fluorescence-Activated Cell Sorting
  • a cell-sorting device is used to differentiate and separate the cell subpopulations that are bound or unbound to the aptamers. Bound aptamers are, then, eluted and amplified.
  • the protocol permits to eliminate dead cell population that, absorbing single-stranded nucleic acid molecules, may negatively influence the selection procedure.
  • such an approach has two additional advantages. First, it allows the reduction of experimental steps, incorporating in one round both positive and negative selection. Second, it allows to simultaneously monitor the selection process during the rounds without the need of additional binding assays.
  • aptamers are emerging as one of the most promising tools for the specific deliver to diseased cells of secondary reagents. Indeed, it has been shown that upon binding to their targets, aptamers can be rapidly internalized, allowing the tissue specific internalization of active therapeutic substances, including nanoparticles, anti-cancer therapeutics, small interfering RNAs (siRNAs), microRNAs, and anti-microRNAs. This permits the exposition to secondary reagents only of target cells, increasing the efficacy and reducing the toxicity of the therapy. Based on these considerations, modified cell-based selection approaches was developed to isolate internalizing aptamers, eliminating those sequences that do not, or very slowly, internalize.
  • the targeting moieties of the nanoparticles of the disclosure may be antibodies, each antibody recognizes and binds at least one of a plurality of antigens expressed on the surface of a particular immune-cell (e.g., NK cell) of a patient suffering from an immune-related disorder, or cancer.
  • a particular immune-cell e.g., NK cell
  • immune-cells of a patient may be used for screening antibody libraries, to select appropriate plurality of antibody targeting moieties for a specific relevant subject.
  • the disclosure further encompasses the use of any combination of antibodies and aptamers that recognize and target to the plurality of receptors expressed by the patient's immune cell (e.g., NK cell).
  • the targeting moiety may be connected, linked, conjugated, or associated either directly to the outer surface of the nano-particle of the disclosure, or any matrix or micellar formulation described herein, or indirectly, for example, via at list one linker.
  • the at least one targeting moiety is connected (conjugated) to the carrier surface via chemical or physical bonding as described herein below.
  • the association of the at least one targeting moiety with the carrier may be direct or may be via a linker.
  • the linker can be inert, or the linker can have biological activity.
  • the linker must be at minimum bivalent; however, in some embodiments, the linker can be bound to more than one active agent, in which case, the linker is polyvalent.
  • the linker can be composed of any assembly of atoms, including oligomeric and polymeric chains, which functions to connect one or more of the targeting moieties (e.g., antibodies, aptamers), to the nanoparticles.
  • the linker may be an oligomeric and polymeric chain, such as an oligo- or polyethylene glycol chain, or an oligo- or poly(amino acid) chain.
  • the linker is a non-polymeric organic functional group, such as an alkyl group or an alkylaryl group.
  • the linker may be hydrophilic to facilitate passage of the nanoparticles across biological membranes.
  • the linker is a linear chain; however, in some embodiments, the linker/s may contain one or more branch points. In the case of branched linker, the terminus of each branch point can be functionalized with the targeting moiety. Still further, it should be noted that the targeting moiety may be associated or linked to the nano-or microparticles of the disclosure either directly or indirectly, for example, via a linker or any adaptor molecule, for example, an adaptor molecule that is based on affinity interactions, for example, avidin-biotin, leucine zipper adaptor and the like.
  • adaptor molecules may enable the use of common or universal nanoparticles loaded with the inhibitory compounds of the re-programming agents of the present disclosure (e.g., the siRNAs of the disclosure), and adapted for personalized treatments by connecting to the affinity adaptor variety of antibodies that are specific for receptors expressed by infiltrating NK cells isolated from the particular patient and profiled by the present disclosure.
  • a linker that links the targeting moiety to the nano-particle of the disclosure may be used.
  • linker in the context of the disclosure concerns an amino acid sequence of from about 1 to about 10 or more amino acid residues positioned on the outer layer of the nanoparticles of the disclosure. The linker is covalently linked or joined to the amino acid residues in its vicinity.
  • a linker in accordance with the disclosure may be of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acid residues long.
  • Linkers are often composed of flexible amino acid residues, for example but not limited to glycine and serine so that the adjacent targeting moiety (e.g., an antibody or any other affinity molecule) are free to move relative to one another.
  • the targeting moieties of the nano- or micro-particle or micellar formulation, or vehicle or matrix of the disclosure recognize and bind NK cells of a subject suffering from an immune -related disorder, for example, at least one of a cancer, a proliferative disorder, an infectious disease, a graft versus host disease, an inflammatory disorder, an immunecell mediated disorder and an autoimmune disorder.
  • an immune -related disorder for example, at least one of a cancer, a proliferative disorder, an infectious disease, a graft versus host disease, an inflammatory disorder, an immunecell mediated disorder and an autoimmune disorder.
  • formulations of the NK reprogramming agent (or modulator and/or re-programmer) of the present disclosure are combinations encompassed within a nano- or micro-particles.
  • Nanoscale drug delivery systems using liposomes and nanoparticles are emerging technologies for the rational drug delivery, which offers improved pharmacokinetic properties, controlled and sustained release of drugs and, more importantly, lower systemic toxicity.
  • a particularly desired solution allows for externally triggered release of encapsulated compounds. Externally controlled release can be accomplished if drug delivery vehicles, such as liposomes or polyelectrolyte multilayer capsules, incorporate nanoparticle (NP) actuators.
  • NP nanoparticle
  • Controlled drug delivery systems have several advantages compared to the traditional forms of drugs.
  • a drug is transported to the place of action, hence, its influence on vital tissues and undesirable side effects can be minimized. Accumulation of therapeutic compounds in the target site increases and, consequently, the required doses of drugs are lower. This modern form of therapy is especially important when there is a discrepancy between the dose or the concentration of a drug and its therapeutic results or toxic effects.
  • Cell-specific targeting as described above, can be accomplished by attaching drugs to specially designed carriers.
  • Various nanostructures, including liposomes, polymers, dendrimers, silicon or carbon materials, and magnetic nanoparticles are applicable in the present disclosure. Polymeric nanoparticles are one technology being developed to enable clinically feasible oral delivery.
  • the carrier is an organized collection of lipids.
  • the structure forming lipids specifically, micellar formulations or liposomes, it is to be understood to mean any biocompatible lipid that can assemble into an organized collection of lipids (organized structure).
  • the lipid may be natural, semi-synthetic or fully synthetic lipid, as well as electrically neutral, negatively or positively charged lipid.
  • the lipid may be a naturally occurring phospholipid.
  • lipids forming glycerophospholipids include, without being limited thereto, glycerophospholipid.
  • phosphatidylglycerols including dimyristoyl phosphatidylglycerol (DMPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), l-palmitoyl-2- oleoylphosphatidyl choline (POPC), hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC); phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS).
  • PG phosphatidylglycerols
  • DMPG dimyristoyl phosphatidylglycerol
  • PC phosphatidylcholine
  • POPC l-palmitoyl
  • Examples of cationic lipids may include, for example, 1 ,2-dimyristoyl- 3 -trimethylammonium propane (DMTAP) l,2-dioleyloxy-3-(trimethylamino) propane (DOTAP); N-[l-(2,3,- ditetradecyloxy )propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide
  • DMTAP 1 ,2-dimyristoyl- 3 -trimethylammonium propane
  • DOTAP dioleyloxy-3-(trimethylamino) propane
  • DMRIE N-[l-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethyl- ammonium bromide
  • DORIE N-[l-(2,3-dioleyloxy) propyl]-N,N,N- trimethylammonium chloride
  • DOTMA 3p[N- (N',N'- dimethylaminoethane) carbamoly] cholesterol
  • DC-Chol dimethyl-dioctadecylammonium
  • DD AB dimethyl-dioctadecylammonium
  • the lipids may be combined with other lipid compatible substances, such as, sterols, lipopolymers etc.
  • a lipopolymer may be a lipid modified by inclusion in its polar headgroup a hydrophilic polymer.
  • the polymer headgroup of a lipopolymer may be preferably water-soluble.
  • the hydrophilic polymer has a molecular weight equal or above 750Da.
  • polymers which may be attached to lipids to form such lipopolymers, such as, without being limited thereto, polyethylene glycol (PEG), poly sialic acid, polylactic (also termed polylactide), poly glycolic acid (also termed poly glycolide), apolylactic-poly glycolic acid, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polyaspartamide, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyvinylmethylether, polyhydroxyethyl acrylate, derivatized celluloses such as hydroxymethylcellulose or hydroxy ethylcellulose.
  • PEG polyethylene glycol
  • poly sialic acid polylactic
  • polylactide poly glycolic acid
  • poly glycolide also termed poly glycolide
  • apolylactic-poly glycolic acid polyvinyl alcohol, poly
  • the polymers may be employed as homopolymers or as block or random copolymers.
  • the lipids derivatized into lipopolymers may be neutral, negatively charged, as well as positively charged.
  • the most commonly used and commercially available lipids derivatized into lipopolymers are those based on phosphatidyl ethanolamine (PE), usually, distearoylphosphatidylethanolamine (DSPE).
  • the structure forming lipids may be combined with other lipids, such as a sterol.
  • Sterols and in particular cholesterol are known to have an effect on the properties of the lipid's organized structure (lipid assembly), and may be used for stabilization, for affecting surface charge, membrane fluidity.
  • a sterol e.g. cholesterol is employed in order to control fluidity of the lipid structure.
  • the liposomes employed in the context of the present disclosure may be multilamellar vesicles (MLVs), multivesicular vesicles (MVVs), small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs) or large multivesicular vesicles (LMVV).
  • MLVs multilamellar vesicles
  • MVVs multivesicular vesicles
  • SUVs small unilamellar vesicles
  • LUVs large unilamellar vesicles
  • LMVV large multivesicular vesicles
  • the re -programming agents of the present disclosure may be encapsulated or associated with any of the nanostructures described above, specifically, any of the micellar formulations, liposomes, polymers, dendrimers, silicon or carbon materials, polymeric nanoparticles and nanoparticles disclosed herein above.
  • association may be used interchangeably with the term “entrapped” , “attachment” , “linked”, “embedded”, “absorbed” and the like, and contemplates any manner by which the compounds of the invention is held. This may include for example, physical or chemical attachment to the carrier. Chemical attachment may be via a linker, such as polyethylene glycol.
  • the association provides capturing of the at least one compounds of the disclosure by the nanostructure such that the release of the at least one compound used by the re-programming agents of the present disclosure may be controllable.
  • the nanostructure in accordance with the present disclosure may further comprise at least one targeting moiety on the surface.
  • targeting moiety may facilitate targeting the compound-nanostructures of the disclosure into a particular target cell, target tissue, target organ or particular cellular organelle target.
  • that targeting moiety targets the nano-particles to NK cells.
  • the transporting or targeting moiety may be attached directly or indirectly via any linker, and may comprise affinity molecules, for example, antibodies or aptamers or any other affinity molecule, as described herein above, that specifically recognize target antigen on specific hematopoietic cells.
  • the nanoparticles of the disclosure may be composed of phosphatidylcholine (PC), dipalmitoylphosphatidylethanolamine (DPPE), and cholesterol (Choi).
  • the nanoparticles of the disclosure may comprise PC, DPPE and Choi at molar ratios of 3:1:1 (PC:DPPE:Chol).
  • the nanoparticles of the disclosure may be prepared by a lipid-film method.
  • the nanoparticles of the disclosure are further surface-modified with high molecular weight glycosaminoglycan hyaluronic acid (HA), thereby obtaining HA-NPs.
  • the HA-NPs of the disclosure are coated with monoclonal anti-NKp46 antibody.
  • Natural killer cell p46-related protein NKp46
  • NCR1 Natural cytotoxicity triggering receptor 1
  • LY94 Lymphocyte antigen 94 homolog
  • CD335 Antigen cluster of differentiation 335, NKP46, NKp46, NK-p46, and CD33.
  • NKp46 is a member of the natural cytotoxicity receptor (NCR) family and was identified as an important regulator of NK cell function. Engagement of the CD335 receptor on NK cells results in increased cellular activation, manifesting as increased cytokine production and release of cytolytic granules.
  • NCR natural cytotoxicity receptor
  • NKp46 refers to any one of: the human NKp46 isoform b precursor as denoted by NP_001138929, the isoform c precursor as denoted by NP_001138930.2, isoform e precursor as denoted by NP_001229286, as well as other isoporms as denoted by any one of NP_001229285, NP_001229286 and NP_004820.
  • the nanoparticles of the disclosure may be conjugated or covered by at least one anti-NKp44 antibody.
  • Natural cytotoxicity triggering receptor 2 is a protein that in humans is encoded by the NCR2 gene is a transmembrane glycoprotein characterized by a single extracellular V-type Ig-like domain and a cytoplasmic tail containing an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) and no known activating signaling motifs.
  • the NKp44 refers to any one of: the human NKp44 as denoted by any one of NP_001186438, NP_001186439 and NP_004819.
  • the disclosed nanoparticles that encapsulate the modulators of the present disclosure are composed of Phosphatidylcholine(PC), cholesterol (Choi), and 1, 2-dihexadecanoyl-snglycero-3- phosphoethanolamine (DPPE), were prepared by a method previously described by the present inventors (Biber G, et al., 2021, EMBO Mol Med 14:el4073).
  • lipids were mixed at 6:2:1.9:0.1 molar ratios: (i) Phosphatidy lcholine(PC), (ii) cholesterol (Choi), (iii) 1, 2- dihexadecanoyl-snglycero-3-phosphoethanolamine (DPPE), and (iv) DPPE labeled with rhodamine red (DPPE-PE, excitation/emission: 560/583 nm;Avanti).
  • DPPE-PE DPPE labeled with rhodamine red
  • Liposomes were coated with 6 mg hyaluronic acid(HA; high molecular weight, R&D) and 40 mg of l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDAC; Sigma).
  • NKp46 antibodies For each 50 pg of liposomes, 400 mM of EDAC and 100 mM of NHydroxysuccinimide (NHS) were added (Sigma). The solution was incubated with 50 pg of NKp46 antibodies.
  • EDAC NHydroxysuccinimide
  • the present disclosure further encompasses the option of using any other vector or vehicle for delivery of the re -programming agents of the present disclosure, specifically any combination of inhibitory compounds that inhibit the expression, stability and/or activity of at least one EGR protein and at least one DGK proteins, that are based on nucleic acid molecules, for example, the siRNAs of the present disclosure, or any oligonucleotide or even any gene editing system as described herein before.
  • such vector may be any one of a viral vector, a non- viral vector and a naked DNA vector.
  • Vectors are nucleic acid molecules of particular sequence can be incorporated into a vector that is then introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art, including promoter elements that direct nucleic acid expression.
  • Many vectors e.g. plasmids, cosmids, minicircles, phage, viruses, etc., useful for transferring nucleic acids into target cells may be applicable in the present disclosure.
  • the vectors comprising the nucleic acid(s) may be maintained episomally, e.g.
  • plasmids as plasmids, minicircle DNAs, viruses such cytomegalovirus, adenovirus, etc., or they may be integrated into the target cell genome, through homologous recombination or random integration, e.g. retrovirus-derived vectors such as AAV, MMLV, HIV-1, ALV, etc.
  • Vectors may be provided directly to the subject cells.
  • the cells are contacted with vectors comprising the oligonucleotides of the disclosure such that the vectors are taken up by the cells.
  • Methods for contacting cells with nucleic acid vectors that are plasmids such as electroporation, calcium chloride transfection, and lipofection, are well known in the art.
  • DNA can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV).
  • viruses e.g., adenovirus, AAV
  • a vector useful in the preset disclosure may be a viral vector.
  • such viral vector may be any one of recombinant adeno associated vectors (rAAV), single stranded AAV (ssAAV), self-complementary rAAV (scAAV), Simian vacuolating virus 40 (SV40) vector, Adenovirus vector, helper-dependent Adenoviral vector, retroviral vector and lentiviral vector.
  • viral vectors may be applicable in the present disclosure.
  • the term "viral vector” refers to a replication competent or replication-deficient viral particle which are capable of transferring nucleic acid molecules into a host.
  • the vector may be a naked DNA vector. More specifically, such vector may be for example, a plasmid, minicircle or linear DNA.
  • Naked DNA alone may facilitate transfer of a gene (2-19 kb) into skin, thymus, cardiac muscle, and especially skeletal muscle and liver cells when directly injected. It enables also long-term expression.
  • naked DNA injection is a safe and simple method, its efficiency for gene delivery is quite low.
  • the present disclosure provides at least one cell comprising the re-programming agent (or modulator and/or re-programmers) of the present disclosure or any nano-particles comprising these re-programming agents, specifically as described by the disclosure, as well as any cell population comprising at least 10% or more of the cells of the disclosure.
  • the cell of the disclosure may comprise any of the re-programming agents disclosed by the disclosure, specifically, the cell in accordance with some embodiments of the disclosure may comprise any inhibitory nucleic acid molecules, SMCs, aptamers, peptide, or any combinations thereof, that specifically inhibit the expression and/or stability and/or activity of at least one of: (i) at least one of EGR-2 and/or EGR-4; and (ii) at least one of DGK-a and/or DGK
  • the re -programming agents comprised within the cell of the disclosure may comprise at least one compounds that comprise at least one nucleic acid molecule. More specifically, each of the nucleic acid molecules is specific for or specifically directed against one of at least one of: (i) at least one of EGR-2 and/or EGR-4; and/or (ii) at least one of DGK-a and/or DGK- ⁇ .
  • the nucleic acid molecule of the re-programming agent comprised within the cell of the present disclosure may be RNA molecules or any nucleic acid sequence encoding the RNA molecules.
  • such RNA molecules may be at least one of a dsRNA, an antisense RNA, a ssRNA, a gRNA and a Ribozyme.
  • the re-programming agents comprised within the cell of the present disclosure may comprise at least one dsRNA molecules. More specifically, each of the dsRNA molecules may be directed against or specific for one of at least one of: (i) at least one of EGR-2 and/or EGR-4; and/or (ii) at least one of DGK-a and/or DGK- ⁇ . In yet more specific embodiments, such dsRNA molecules, may be at least one of siRNA, miRNA, shRNA and piRNAs.
  • the re-programming agents comprised within and/or functionalizing and/or activating the cell of the present disclosure may comprise at least one of: at least one siRNA molecule specifically directed against, or specific for EGR-2, and/or at least one siRNA molecule specifically directed against DGK-a. In yet some further embodiments, the reprogramming agents comprised within and/or functionalizing and/or activating the cell of the present disclosure may comprise least one siRNA molecule specifically directed against, or specific for EGR-2, and/or at least one siRNA molecule specifically directed against EGR-4.
  • the cell of the present disclosure may comprise any of the nano- or micro-particle or micellar formulation or vehicle or matrix as disclosed by the disclosure.
  • the cell of the present disclosure may be any hematopoietic cell.
  • the cell is a lymphocyte of the T lineage.
  • the cell of the present disclosure may be an NK cell.
  • the cell of the present disclosure may be a cell obtained from a subject suffering from an immune related disorder, specifically from a proliferative disorder.
  • the cells or population of cells may be obtained from at least one allogeneic healthy subject or from a pool of at least two allogeneic subjects.
  • the present disclosure further encompasses any population of the cells disclosed herein.
  • population of cells may be an enriched population of cells comprising the re-programming agents of the present disclosure, or any nano-particles thereof, or cells activated and/or functionalized and/or rewired, and/or reprogramed by the re-programming agents of the present disclosure, i.e. a population of cells in which a high percentage of cells among the total number of cells in the population of cells comprise the re-programming agents of the present disclosure.
  • such a percentage of cells may be 10%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,
  • the cells of the population may be cells that were manipulated to comprise the re -programming agents of the present disclosure or any nano-particles or any vectors or vehicles thereof, and/or cells (NK cells) that were activated and/or functionalized and/or rewired, and/or reprogramed by the re -programming agents of the present disclosure.
  • NK cells cells that were activated and/or functionalized and/or rewired, and/or reprogramed by the re -programming agents of the present disclosure.
  • "activated by the re-programming agents of the present disclosure” is meant, changed from dysfunctional to functional cells.
  • the present disclosure further encompasses any composition or preparation of NK cells or cell population thereof activated by the disclosed re-programming agents (being functional).
  • Such composition contains a population of cells having an increased proportion of activated NK cells.
  • the disclosure provides a population of NK cells activated by the re-programming agents of the present disclosure, that display reduced proportion of dysfunctional NK cells.
  • the disclosed NK cell population is an improved cell population as it is composed of less than 20% of dysfunctional cells, specifically, less than 20% of the NK cells in the NK cell population of the present disclosure or of any composition thereof are anergic NK cells.
  • the disclosed NK cell population is an improved cell population as it is composed of less than 13% of dysfunctional cells, specifically, less than 13% of the NK cells in the NK cel population of the present disclosure or of any composition thereof are anergic NK cells. In yet some further embodiments, less than 13% of the NK cells in the cell population of the present disclosure or of any composition thereof, is exhausted NK cells. In some embodiments, the disclosed NK cell population contain 80% or more functional NK cells (cells that can be activated, and/or cells that are not exhausted cells, or cells that are not anergic cells).
  • less than 20% as used herein, and also in context with all other aspects of the present disclosure is meant, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, and also for the term “less than 20%, or “less than 13%), I is meant: 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.09% or less, 0.08% or less, 0.07% or less, 0.06% or less, 0.05% or less, 0.04% or less, 0.03% or less, 0.02% or less, 0.01% or less, 0.009% or less, 0.009% or less, 0.00
  • the present disclosure provides in some other aspects thereof, a method for improving a population of cells, specifically, hemopoietic cells, and more specifically a population of cells comprising NK cells, by contacting the cells in the population of cells (that can be of either an autologous source, or alternatively, of an allogeneic source), with an activating effective amount of any of the re -programming agents of the present disclosure or any nano-or micro-particles thereof, or any composition or preparation thereof, thereby providing a population of cells having less than 20% thereof or even less than 13% thereof, or less than 10% thereof dysfunctional cells, as defined above.
  • less than 20% of the NK cells in the cell population prepared by the disclosed methods are exhausted NK cells.
  • NK cells in the cell population prepared by the disclosed methods are anergic NK cells.
  • This method therefore provides an improved population of cells (of either an autologous source or an allogeneic source) that contain more functional NK cells, and/or less dysfunctional NK cells as specified above.
  • the improved cell population provided by be disclosed methods is rewired and reprogramed thereby functionated.
  • a further aspect of the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of at least one re -programming agent (or modulator and/or re -programmer) comprising at least one compound that specifically inhibit the expression, activity and/or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family; and/or any nano- or micro-particle, micellar formulation, vehicle, matrix comprising the re-programming agent.
  • the re-programming agent of the disclosed composition activates and/or functionalize and/or rewire, and/or reprogram dysfunctional NK cells.
  • composition further comprises at least one of pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
  • the disclosed composition comprises an effective amount of the disclosed reprogramming agent (or modulator and/or re-programmer) that is sufficient for and effectively activate and/or functionalize and/or rewire, and/or reprogram NK cells, specifically, dysfunctional NK cells.
  • the re-programming agent of the disclosed compositions is any of the reprogramming agents as defined by the present disclosure, defined above, and in connection with other aspects of the present disclosure.
  • the nano- or microparticle, micellar formulation, vehicle or matrix of the compositions of the present disclosure is any of the nano- or micro-particles as disclosed and defined herein by the present disclosure.
  • the effective amount of the re-programming agents of the present disclosure may range between 0.001 mg to 10,000mg each, specifically, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 8000, 9000, 10000 mg a day or more.
  • the effective amount of the re-programming agents of the present disclosure may range between about 0.1 to 100 microgram/kg, specifically, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 microgram/kg and more.
  • an effective amount for in vitro treatment may be about 1 to 10 micrograms, specifically, 6.6 micrograms. Still further, in some embodiments the effective amount for in vivo treatment may range between about 1 to 50 microgram/kg, specifically, about 20 microgram/kg.
  • the present siRNA and/or esiRNA molecules of the disclosed modulator or re-programmer were injected to mice at an amount of 1500pmole of each, specifically, an amount of about 0.0025 mg/per dose (mouse). Accordingly, in some specific embodiments, the effective amount of the siRNA and/or esiRNA molecules of the disclosed modulator or re-programmer may be about 0.125 mg per kilogram of body weight/dose.
  • the disclosed modulator or re-programmer is provided in nano-particles (e.g., the disclosed liposomes)
  • about 300 micrograms of liposomes were injected to each mouse (300 microgram per dose), in addition to the siRNA molecules as discussed above.
  • the effective amount of the liposomes of the present disclosure that encompass the disclosed modulator or re -programmer may be about 15 mg per kilogram of body weight/dose.
  • the subject is treated with 1 dose each day.
  • the subject is treated using one dose each 3 days.
  • the subject is treated using one dose every 3 days for 21 days period.
  • compositions of the disclosure can be administered and dosed by the methods of the disclosure, in accordance with good medical practice, systemically, for example by parenteral, e.g. intravenous, intraperitoneal or intramuscular injection.
  • the pharmaceutical composition can be introduced to a site by any suitable route including intravenous, subcutaneous, transcutaneous, topical, intramuscular, intraarticular, subconjunctival, or mucosal, e.g. oral, intranasal, or intraocular administration.
  • injectable compositions comprising any of the disclosed re-programming agents or any nano- or microparticles thereof (e.g., liposomes), an any formulation thereof.
  • Local administration to the area in need of treatment may be achieved by, for example, by local infusion during surgery, topical application, direct injection into the specific organ, etc.
  • the re -programming agents of the present disclosure or any nanoparticles or compositions thereof, described herein after may be adapted for administration by parenteral, intraperitoneal, transdermal, oral (including buccal or sublingual), rectal, topical (including buccal or sublingual), vaginal, intranasal and any other appropriate routes.
  • Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). It should be noted that any of the administration modes discussed herein, may be applicable for any of the methods of the disclosure as described in further aspects of the disclosure herein after.
  • compositions and formulations for oral administration may include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, solid solution, liposome, films, ovules, sprays or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or enemas.
  • Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
  • compositions used to treat subjects in need thereof according to the disclosure may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the compositions may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions of the present disclosure also include, but are not limited to, emulsions and liposome-containing formulations. It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
  • compositions of the disclosure may also be administered directly to the eye or ear, typically in the form of drops of a micronized suspension or solution in isotonic, pH-adjusted, sterile saline.
  • Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g. silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes.
  • a polymer such as crossed-linked polyacrylic acid, polyvinylalcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethylcellulose, hydroxyethylcellulose or methyl cellulose or a heteropolysaccharide polymer, for example, gelan gum, may be incorporated together with a preservative, such as benzalkonium chloride.
  • a preservative such as benzalkonium chloride.
  • Such formulations may also be delivered by iontophoresis.
  • Formulations for ocular and aural administration may be formulated to be immediate and/or modified release. Modified release includes delayed, sustained, pulsed, controlled, targeted, and programmed release.
  • the unit dosage formulations are those containing a daily dose or subdose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.
  • the disclosure relates to a method for activating, and/or functioning, and/or rewiring, and/or reprograming at least one hematopoietic cells, specifically, dysfunctional cells (e.g., anergic or exhausted).
  • the methods of the disclosure may be suitable for activating at least one of dysfunctional NK cells, dysfunctional T cells and dysfunctional B cells.
  • the methods of the disclosure may be particularly suitable for activating dysfunctional NK cells (e.g., anergic or exhausted).
  • the method may comprise the step of contacting the NK cell (specifically, the dysfunctional NK cell) with an activating effective amount of at least one re-programming agent (or modulator and/or re-programmer) comprising at least one compound that specifically inhibit at least one of, the expression, activity and stability of at least one transcription factor, specifically, at least one member of the EGR family and of at least one Kinase, specifically, at least one member of the DGK family, or any vehicle, matrix, nano- or micro-particle, micellar formulation or composition comprising the re-programming agents of the present disclosure.
  • at least one re-programming agent or modulator and/or re-programmer comprising at least one compound that specifically inhibit at least one of, the expression, activity and stability of at least one transcription factor, specifically, at least one member of the EGR family and of at least one Kinase, specifically, at least one member of the DGK family, or any vehicle, matrix, nano- or micro-particle, micellar formulation or composition comprising the
  • a further aspect of the present disclosure relates to a method for activating and/or functionalizing, and/or re-wiring, and/or reprograming at least one dysfunctional NK cell.
  • the method comprising the step of contacting the dysfunctional NK cell with an activating effective amount of at least one re-programming agent (or modulator and/or re-programmer) comprising at least one compound that specifically inhibit the expression, activity and/or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family; and/or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising the re-programming agent.
  • the re-programming agent activates and/or re- wires dysfunctional NK cells.
  • activating NK cells is meant, changing the cells from dysfunctional cells (either anergic or exhausted), to functional NK cells.
  • activating the NK cells in the context of the present disclosure is meant enhancing the functional properties of the cells, such that they are able to function and be activated.
  • NK cells or reprogramming NK cells refers to the deliberate modification of the signaling pathways, molecular networks, or genetic programs within natural killer (NK) cells to alter or enhance their functional properties. These processes aim to improve the cells’ ability to recognize, target, and eliminate abnormal cells, such as tumor cells or virally infected cells, or to modulate their interactions with the immune system.
  • the dysfunctional NK cells are anergic NK cells and/or exhausted NK cells.
  • the present disclosure provides methods for activating and/or rewiring dysfunctional NK cells that may be anergic NK cells and/or exhausted NK cells.
  • the present disclosure provide method for functionalizing, and/or rewiring and/or reprograming and/or activating anergic NK cells.
  • the present disclosure provide method for functionalizing, and/or rewiring and/or reprograming and/or activating exhausted NK cells.
  • the at least one compound that specifically inhibits the expression, activity and/or stability of at least one of: at least one member of the EGR family of transcription factors; and/or at least one member of the DGK family may comprise at least one nucleic acid molecule, at least one amino acid-based molecule, and/or at least one chemical inhibitor. Each of these molecules is specific for one of: (i) at least one member of the EGR family of transcription factors; and/or (ii) at least one member of the DGK family.
  • the nucleic acid molecule is a ribonucleic acid (RNA) molecule or any nucleic acid sequence encoding said RNA molecule, said RNA molecule is at least one of a double-stranded RNA (dsRNA), an antisense RNA, a single- stranded RNA (ssRNA), guide RNA (gRNA) and a Ribozyme. Still further, in some embodiment of the disclosed methods, dsRNA is at least one of siRNA, esiRNA, miRNA, shRNA and piRNAs. Accordingly, in some embodiments, the disclosed methods use at least one of these dsDNA molecules for activating and/or rewiring and/or functioning or functionalizing dysfunctional NK cells.
  • dsRNA double-stranded RNA
  • ssRNA single- stranded RNA
  • gRNA guide RNA
  • Ribozyme Ribozyme.
  • dsRNA is at least one of siRNA, esiRNA, miRNA
  • the compound of the re-programming agent used in the disclosed methods may comprise at least one siRNA and/or esiRNA molecule, each siRNA and/or esiRNA molecule is specific for one of: (i) one member of the EGR family of transcription factors; and/or (ii) one member of the DGK family.
  • the EGR family of transcription factors comprises at least one of EGR2, EGR4, EGR1 and EGR3.
  • the disclosed methods may use at least one siRNA molecule, each siRNA molecule is specific for at least one of EGR2, EGR4, EGR1 and EGR3.
  • the DGK family comprises at least one of DGKa, DGK ⁇ , DGK5, DGK0 and DGKy.
  • the disclosed methods may use at least one siRNA molecule, each siRNA molecule is specific for at least one of DGKa, DGK ⁇ DGK5, DGK0 and DGKy.
  • the at least one member of the EGR family of transcription factors comprises at least one of EGR2 and EGR4; and the at least one member of the DGK family comprises at least one of DGKa and DGK ⁇ .
  • the disclosed methods use at least one siRNA molecule, each siRNA molecule is specific for at least one of EGR2 and/or EGR4, and/or DGKa and DGK ⁇ . It should be understood that the disclosed methods may use any combination of the disclosed siRNA molecules as disclosed herein above in connection with other aspects of the present disclosure.
  • the re -programming agent of the disclosed methods comprises an effective amount of at least one of: (i) at least one siRNA molecule specific for EGR2; (ii) at least one siRNA molecule specific for EGR4; (iii) at least one siRNA molecule specific for DGKa and (iv) at least one siRNA molecule specific for DGK ⁇ ; and/or any combinations of (i), (ii), (iii) and (iv).
  • the esiRNA molecule specific for EGR2 that comprise the nucleic acid sequence as denoted by SEQ ID NO: 9, and any complementary sequence thereof, is randomly fragmented to generate a plurality of siRNA molecules targeting EGR2 and/or the esiRNA molecule specific for EGR4 comprising the nucleic acid sequence as denoted by SEQ ID NO: 10, and any complementary sequence thereof, is randomly fragmented to generate a plurality of siRNA molecules targeting EGR4, and/or the siRNA molecule specific for DGKa comprises the nucleic acid sequence as denoted by SEQ ID NO: 13, and/or the siRNA molecule specific for DGK ⁇ comprises the nucleic acid sequence as denoted by SEQ ID NO: 14, or of any variants, homologs or derivatives thereof.
  • the re-programming agent is comprised within at least one of a nano- or micro-particle, a micellar formulation, a vehicle, a matrix, or a composition.
  • the at least one targeting moiety of the nano-or micro particles used in the methods is at least one of an antibody, an aptamer, a ligand or any combinations thereof, that specifically recognizes and binds at least one molecule expressed on the surface of at least one NK cell.
  • the at least one antibody comprises antibody against at least one member of the NCR family.
  • the member of the NCR family comprises NKp46, and the targeting moiety comprises at least one antibody that specifically recognizes and binds NKp46.
  • the disclosed methods are particularly useful for activating and/or rewiring dysfunctional NK cell in a subject suffering from an immune-related disorder.
  • the immune-related disorder may be at least one of a cancer, a proliferative disorder, an infectious disease, a graft versus host disease, an inflammatory disorder, an immune-cell mediated disorder and an autoimmune disorder.
  • the disorder is cancer.
  • the cancer is at least one of AML and glioma.
  • the NK cells are Tumor infiltrating NK cells (TINK).
  • a lymphocyte cell that is a NK cell, specifically, a dysfunctional NK cell.
  • Natural killer cells or NK cells are a type of cytotoxic lymphocyte critical to the innate immune system. NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL). The role NK cells play is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to viral-infected cells, acting at around three days after infection, and respond to tumor formation.
  • NK cells typically detect major histocompatibility complex (MHC) presented on infected cell surfaces, triggering cytokine release, causing lysis or apoptosis.
  • MHC major histocompatibility complex
  • NK cells are unique, however, as they have the ability to recognize stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. They were named “natural killers” because of the initial notion that they do not require activation to kill cells that are missing "self” markers of MHC class 1.
  • immunological synapse (“NKIS”) denotes the dynamic interface formed between an NK cell and its target cell.
  • NKIS Formation of NKIS involves several distinct stages, beginning with the initiation of contact with a target cell and culminating in the directed delivery of lytic granule contents to lyse the target cell. Progression through the individual stages is methodical and underlies the precision with which NK cells select and kill susceptible target cells (including virally infected cells and cancerous cells) that they encounter during their routine surveillance of the body.
  • susceptible target cells including virally infected cells and cancerous cells
  • functions of NK cells comprise the formation of activating NK cell immunological synapse (IS), that is activating NKIS, or alternatively, the formation of inhibitory NK cell immunological synapse (IS), that is inhibitory NKIS.
  • IS activating NK cell immunological synapse
  • IS inhibitory NK cell immunological synapse
  • NKIS a mature and functional NKIS
  • the recognition and initiation stage the effector stage and the termination stage.
  • these processes enable the delivery of lytic granules to the synapse followed by their close association with the NK cell membrane to which they can fuse and release their contents onto the target cell.
  • lytic granules exist in resting NK cells before activation, each stage must be controlled to prevent accidental release of cytotoxic mediators and to enable rapid directed secretion at the appropriate moment.
  • molecules related to the above processes can be used as markers for evaluating activity and effectivity of the reprogramming agents of the present disclosure and any nano-particles thereof.
  • the initial stage is characterized by formation of a close association between the NK cell and a target cell, initial signaling and adherence of NK cell to its target cell.
  • This stage is facilitated by a number of molecules, including, although not limited to, members of the selectin family, the CD2 receptor, and receptors from the integrin family of adhesion molecules in particular such as the integrins lymphocyte function-associated antigen 1 (LFA1; CDl la/CD18) and MAC1 (CDl lb/CD18).
  • LFA1 lymphocyte function-associated antigen 1
  • CDl la/CD18 integrins lymphocyte function-associated antigen 1
  • MAC1 CDl lb/CD18
  • NK cell progresses to maturation and molecular reorganization at NKIS depends on the level of signals through inhibitory receptors (KIRs, Killer-cell Immunoglobulin-like Receptors), which can establish a so-called inhibitory synapse.
  • KIRs Killer-cell Immunoglobulin-like Receptors
  • Such regulation ensures that NK cells effectively carry out their surveillance function, by leaving most cells undisturbed, while being poised to destroy those that are diseased.
  • the inhibitory NKIS is especially elegant in that it directly interferes with the ability of the lytic synapse to progress past the initiation stage.
  • the effector stage is characterized by a number of processes, most prominent of which are (1) formation of a stable NK cell-target cell interface with a 'cleft' into which cytolytic molecules are secreted; (2) recruitment of lytic granules to the synapse; (3) clearance of a conduit in the NK cell cortex through which lytic granules could be directed to the cell membrane; and (4) fusion of the lytic-granule membrane with plasma membrane for release of lytic-granule contents.
  • Parallel events include receptor clustering, lipid-raft aggregation, further activation signaling and lytic- granule redistribution.
  • CDl la the most important for both, adhesion and triggering of cytotoxicity
  • CDl lb Another requirement for effector function is polarization of lytic granules to NKIS, or in other words movement of the granules along the microtubules to the microtubule-organizing centre (MTOC).
  • Signals required for MTOC polarization include ERK (extracellular-signal-regulated kinase) phosphorylation, VAV1 activation and PYK2 (protein tyrosine kinase 2) activities.
  • RAB27a also performs this function in docking lytic granules in CTLs.
  • Muncl3-4 putative vesicle priming factor
  • SNAREs N-ethylmaleimide-sensitive fusion protein attachment protein receptors
  • SHP-1 a SH2 domain containing tyrosine phosphatase
  • Termination stages of NKIS refer to those that occur after the lytic-granule contents have been secreted. Those include a period of inactivity and down modulation of the accumulated activating receptors followed by NK cell detachment from the target cell and recycling of cytolytic capacity. Once NK cell has carried out its cytolytic function, it can detach from the target cell and restore its ability to kill another susceptible cell. At the inhibitory synapse, detachment may result from reduced integrity of interactions between the F-actin cortex and the plasma membrane through dephosphorylation of ERM protein targets.
  • NK cytolytic capacity The signals initiating the process of recycling NK cytolytic capacity are largely unknown, apart from activation of the nuclear factor-KB (NF-KB) which has been shown to serve as a transcription factor for expression of the lytic granule component perforin.
  • NF-KB nuclear factor-KB
  • secretion of cytolytic granules in response to increased intracellular Ca 2+ flux is characteristic of 'Termination stage' of a lymphocyte responding to an activating stimulus at IS, a which is common to NK cells and CTLs, i.e. innate or adaptive immune response.
  • CTL or NK cells kill infected or cancerous cells they secrete cytolytic proteins (perforin and granzymes) into the target cell.
  • cytolytic granules are pre-stored in cytolytic granules within the CTL until an increase in the intracellular Ca2 + drives granule to exocytosis. Secretion of cytolytic granules and increased intracellular Ca 2+ flux are measurable and can serve as markers for evaluating the activity of the presently conceived compounds.
  • the lymphocyte cell modulated, specifically activated by the reprogramming agents of the present disclosure, or any nano-particles thereof may be at least one of an NK cell, a T cell and a B cell forming an inhibitory IS, specifically, NK cells.
  • a further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an immune-related disorder in a subject in need thereof.
  • the method comprising the step of administering to the subject a therapeutically effective amount of at least one re-programming agent (or modulator and/or re-programmer) comprising at least one compound that specifically inhibit the expression, activity and/or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family; and/or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising the re-programming agent, wherein said reprogramming agent activates dysfunctional NK cells.
  • the disclosed methods may comprise an ex vivo step of functionalizing and/or activating and/or rewiring the NK cells ex vivo or in vitro, by contacting the cells with the effective amount of the re -programming agents of the preset disclosure.
  • the cells are then transferred back to the subject, for example, by adoptive transfer.
  • the cells may be of an autologous or of allogeneic source.
  • the subject is administered with at least one NK cell, or a population of NK cells or any composition or preparation thereof, that were functionalized, activated and/or rewired.
  • the NK cells may be in some embodiments of an autologous source.
  • the cells may be of an allogeneic source (either of a single donor or a pool of two or more donors).
  • the disclosed therapeutic methods may use any of the re-programming agents disclosed by the present disclosure, and any of the nano- or micro-particle, micellar formulation, vehicle, matrix as defined by the present disclosure.
  • the subject treated by the disclose therapeutic methods is suffering from an immune-related disorder.
  • the immune-related disorder applicable for the disclosed methods is at least one of a cancer, a proliferative disorder, primary or secondary immunodeficiency, a graft versus host disease, an inflammatory disorder, an immune-cell mediated disorder, an autoimmune disorder and a viral infection.
  • the subject is further treated with at least one immunomodulatory therapeutic agent prior to, after or simultaneously with the at least one re-programming agent of the present disclosure.
  • the at least one immunomodulatory therapeutic agent comprises at least one of: (i) at least one checkpoint inhibitor; (ii) at least one cytokine; and (iii) chimeric antigen receptor (CAR) T cells.
  • a further aspect of the present disclosure relates to at least one re-programming agent (or modulator and/or re-programmer) or any nano- or micro-particle, micellar formulation, vehicle, matrix, cell or composition comprising said re-programming agent, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an immune- related disorder in a subject in need thereof.
  • the re-programming agent used herein comprises at least one compound that specifically inhibit the expression, activity and/or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and/or (ii) at least one member of the DGK family; and/or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising the re-programming agent.
  • the re -programming agent activates and/or functionalize and/or rewire, and/or reprogram dysfunctional NK cells.
  • the compound used herein is any of the compounds as defined by the present disclosure, and the nano- or microparticle, micellar formulation, vehicle or matrix is as defined by the present disclosure, and the compositions used herein are any of the compositions disclosed y the present disclosure.
  • the methods and uses of the disclosure may be relevant for treating any immune- related disorder, for example, an infectious disease, specifically, a viral infection, cancer or any other proliferative disorder, a graft versus host disease, an inflammatory disorder, an immune-cell mediated disorder and an autoimmune disorder.
  • An "Immune-related disorder” or “Immune- mediated disorder”, as used herein encompasses any condition that is associated with the immune system of a subject, more specifically through inhibition of the immune system, or that can be treated, prevented or ameliorated by reducing degradation of a certain component of the immune response in a subject, such as the adaptive or innate immune response.
  • an 'immune-related disorder' encompasses a range of dysfunctions of the innate and adaptive immune systems.
  • immune-related disorder can be characterized, for example, (1) by the component(s) of the immune system; (2) by whether the immune system is overactive or underactive; (3) by whether the condition is congenital or acquired, as will be specified herein after.
  • the methods of the disclosure may be used for treating cancer or any other proliferative disorders.
  • proliferative disorder “cancer”, “tumor” and “malignancy” all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include nonsolid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors.
  • the methods of the present disclosure may be applicable for treatment of a patient suffering from any one of non-solid and solid tumors.
  • Malignancy may be any one of carcinomas, melanomas, lymphomas, leukemias, myeloma and sarcomas.
  • Carcinoma refers to an invasive malignant tumor consisting of transformed epithelial cells. Alternatively, it refers to a malignant tumor composed of transformed cells of unknown histogenesis, but which possess specific molecular or histological characteristics that are associated with epithelial cells, such as the production of cytokeratins or intercellular bridges.
  • Melanoma as used herein, is a malignant tumor of melanocytes.
  • Melanocytes are cells that produce the dark pigment, melanin, which is responsible for the color of skin. They predominantly occur in skin, but are also found in other parts of the body, including the bowel and the eye. Melanoma can occur in any part of the body that contains melanocytes.
  • Leukemia refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood-leukemic or aleukemic (subleukemic).
  • Sarcoma is a cancer that arises from transformed connective tissue cells. These cells originate from embryonic mesoderm, or middle layer, which forms the bone, cartilage, and fat tissues. This is in contrast to carcinomas, which originate in the epithelium. The epithelium lines the surface of structures throughout the body, and is the origin of cancers in the breast, colon, and pancreas. Myeloma as mentioned herein is a cancer of plasma cells, a type of white blood cell normally responsible for the production of antibodies. Collections of abnormal cells accumulate in bones, where they cause bone lesions, and in the bone marrow where they interfere with the production of normal blood cells.
  • lymphoma is a cancer in the lymphatic cells of the immune system. Typically, lymphomas present as a solid tumor of lymphoid cells. These malignant cells often originate in lymph nodes, presenting as an enlargement of the node (a tumor). It can also affect other organs in which case it is referred to as extranodal lymphoma.
  • lymphoma include Hodgkin's disease, non-Hodgkin's lymphomas and Burkitt's lymphoma.
  • the disclosed modulator or re-programmer, methods, compositions and kits of the present disclosure are applicable for any type and/or stage and/or grade of any metastasis, metastatic cancer or status of any of the cancerous conditions disclosed herein.
  • metastatic cancer or “metastatic status” refers to a cancer that has spread from the place where it first started (primary cancer) to another place in the body.
  • malignancies that may find utility in the present invention can comprise but are not limited to hematological malignancies (including lymphoma, leukemia, myeloproliferative disorders, Acute lymphoblastic leukemia; Acute myeloid leukemia), hypoplastic and aplastic anemia (both virally induced and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune mediated and idiopathic) and solid tumors (including GI tract, colon, lung, liver, breast, prostate, pancreas and Kaposi's sarcoma.
  • hematological malignancies including lymphoma, leukemia, myeloproliferative disorders, Acute lymphoblastic leukemia; Acute myeloid leukemia), hypoplastic and aplastic anemia (both virally induced and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune mediated and idiopathic) and solid tumors
  • the invention may be applicable as well for the treatment or inhibition of solid tumors such as tumors in lip and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbladder, extrahepatic bile ducts, ampulla of vater, exocrine pancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma, carcinoma and malignant melanoma of the skin, breast, vulva, vagina, cervix uteri, corpus uteri, ovary, fallopian tube, gestational trophoblastic tumors, penis, prostate, testis, kidney, renal pelvis, ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma of the conjunctiva, malignant melanoma of the conjunctiva, malignant
  • AML Acute Myeloid Leukemia
  • AML is an aggressive form of blood and bone marrow cancer characterized by the rapid proliferation of abnormal myeloid cells, which interfere with the production of healthy blood cells. It originates in the bone marrow but often spreads quickly to the blood and, in advanced stages, to other parts of the body such as the lymph nodes, liver, and spleen.
  • AML leads to symptoms like fatigue, frequent infections, easy bruising or bleeding, and anemia due to the overcrowding of abnormal cells in the bone marrow. It is most common in adults but can occur in children as well.
  • Diagnosis typically involves blood tests, bone marrow biopsies, and genetic studies to classify the subtype, which is crucial for treatment planning. Management of AML often includes chemotherapy, targeted therapy, and in some cases, stem cell transplantation. The prognosis varies depending on factors such as age, genetic mutations, and the patient’s overall health
  • Glioblastoma is an aggressive and malignant form of brain cancer that originates in the glial cells, which support and protect neurons. It is the most common and lethal primary brain tumor in adults, often arising in the cerebral hemispheres but capable of occurring in other brain regions or the spinal cord. Glioblastoma is classified as a Grade IV astrocytoma by the World Health Organization due to its rapid growth, infiltration into surrounding brain tissue, and resistance to treatment. Symptoms vary depending on the tumor's location but commonly include headaches, seizures, cognitive impairment, and neurological deficits such as weakness or speech difficulties. Diagnosis is typically confirmed through imaging techniques like MRI and a biopsy.
  • Treatment involves a combination of surgery, radiation therapy, and chemotherapy, although complete surgical removal is usually not possible due to the tumor's invasive nature. Despite aggressive treatment, glioblastoma has a poor prognosis, with a median survival of 12-18 months after diagnosis. Research into novel therapies, including immunotherapy and targeted treatments, is ongoing.
  • the methods of the disclosure may be used to treat a proliferative disorder, cancer, tumor and malignancy by activating/ enhancing antitumor immunity.
  • antitumor immunity refers to innate and adaptive immune responses which may lead to tumor control.
  • the immune system can be activated by tumor antigens and, once primed, can elicit an antitumor response.
  • Natural Killer (NK) cells are a front-line defense against drug-resistant tumors and can provide tumoricidal activity to enhance tumor immune surveillance. Cytokines like IFN-y or TNF play a crucial role in creating an immunogenic microenvironment and therefore are key players in the fight against metastatic cancer. Critical aspects in the tumor-immune system interface include the processing and presentation of released antigens by antigen-presenting cells (APCs), interaction with T lymphocytes, subsequent immune/T-cell activation, trafficking of antigen-specific effector cells, and, ultimately, the engagement of the target tumor cell by the activated effector T cell.
  • APCs antigen-presenting cells
  • NK and T cells a phenomena known as exhaustion.
  • the methods, as well as the re -programming agents, nanoparticles, compositions and kits of the disclosure may be also applicable for treating a subject suffering from an infectious disease. More specifically, such infectious disease may be any one of viral diseases, protozoan diseases, bacterial diseases, parasitic diseases, fungal diseases and mycoplasma diseases.
  • infectious disease as used herein also encompasses any infectious disease caused by a pathogenic agent.
  • Pathogenic agents include viruses, prokaryotic microorganisms, lower eukaryotic microorganisms, complex eukaryotic organisms, fungi, prions, parasites, yeasts, toxins and venoms. Of particular relevance are infectious diseases caused by a bacterial pathogen.
  • a prokaryotic microorganism includes bacteria such as Gram positive, Gram negative and Gram variable bacteria and intracellular bacteria.
  • bacteria contemplated herein include the species of the genera Treponema sp., Borrelia sp., Neisseria sp., Legionella sp., Bordetella sp., Escherichia sp., Salmonella sp., Shigella sp., Klebsiella sp., Pseudomonas sp., Yersinia sp., Vibrio sp., Hemophilus sp., Rickettsia sp., Chlamydia sp., Mycoplasma sp., Staphylococcus sp., Streptococcus sp., Bacillus sp., Clostridium sp., Corynebacterium sp., Proprionibacterium sp., Mycobacterium sp., Ureaplasma sp. and Listeria sp.
  • a lower eukaryotic organism includes a yeast or fungus such as but not limited to Pneumocystis carinii, Candida albicans, Aspergillus, Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans, Trichophyton and Microsporum.
  • yeast or fungus such as but not limited to Pneumocystis carinii, Candida albicans, Aspergillus, Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans, Trichophyton and Microsporum.
  • a complex eukaryotic organism includes worms, insects, arachnids, nematodes, aemobe, Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, Trypanosoma brucei gambiense, Trypanosoma cruzi, Balantidium coli, Toxoplasma gondii, Cryptosporidium or Leishmania.
  • viruses are infectious diseases caused by a viral pathogen.
  • viruses is used in its broadest sense to include viruses of the families adenoviruses, papovaviruses, herpesviruses: simplex, varicella-zoster, Epstein-Barr, CMV, pox viruses: smallpox, vaccinia, hepatitis B, rhinoviruses, coronaviruses, retroviruses, zika virus, ebola virus, hepatitis A, poliovirus, rubella virus, hepatitis C, arboviruses, rabies virus, influenza viruses A and B, measles virus, mumps virus, HIV, HTLV I and II.
  • fungi includes for example, fungi that cause diseases such as ringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichosis, coccidioidomycosis, paracoccidio-idoiny cosis, and candidiasis.
  • parasite includes, but not limited to, infections caused by somatic tapeworms, blood flukes, tissue roundworms, ameba, and Plasmodium, Trypanosoma, Leishmania, and Toxoplasma species.
  • the methods, as well as the re-programming agent (or modulator and/or re-programmers), nanoparticles, compositions and kits and compositions of the disclosure may be applicable for treating disorders associated with immunodeficiency.
  • the immune -related disorder or condition may be a primary or a secondary immunodeficiency.
  • any of the immune-related disorders described herein after in connection with other aspects of the disclosure is also applicable or the present aspect as well.
  • 'Immunodeficiency' primary or secondary, meaning inherited or acquired, respectively.
  • the term 'immunodeficiency' is intended to convey a state of an organism, wherein the immune system's ability for immuno-surveillance of infectious disease or cancer is compromised or entirely absent.
  • PIDs primary immunodeficiency diseases
  • Secondary immuno-deficiencies are caused by various conditions, aging or agents such as viruses or immune suppressing drugs.
  • SCID Severe combined immunodeficiency
  • DiGeorge syndrome DiGeorge syndrome
  • Hyperimmunoglobulin E syndrome also known as Job’s Syndrome
  • CVID Common variable immunodeficiency
  • CTD Chronic granulomatous disease
  • NADPH NADPH oxidase enzyme
  • Classical recurrent infection from catalase positive bacteria and fungi Wiskott-Aldrich syndrome (WAS); autoimmune lymphoproliferative syndrome (ALPS); Hyper IgM syndrome: X- linked disorder that causes a deficiency in the production of CD40 ligand on activated T-cells. This increases the production and release of IgM into circulation.
  • B-cell and T-cell numbers are within normal limits. Increased susceptibility to extracellular bacteria and opportunistic infections.
  • LAD Leukocyte adhesion deficiency
  • NEMO Essential Modifier
  • Selective immunoglobulin A deficiency the most common defect of the humoral immunity, characterized by a deficiency of IgA. Produces repeating sino-pulmonary and gastrointestinal infections.
  • X-linked agammaglobulinemia XLA; also known as Bruton type agammaglobulinemia
  • XLA X-linked agammaglobulinemia
  • XLP X-linked lymphoproliferative disease
  • Ataxia-telangiectasia X-linked lymphoproliferative disease
  • secondary immunodeficiencies those can be manifested in both the young and the elderly. Under normal conditions immune responses are beginning to decline at around 50 years of age, what is called immunosenescence.
  • the term 'immunosenescence' refers to the gradual deterioration of the immune system brought on by natural age advancement. It involves both the host’s capacity to respond to infections and the development of long-term immune memory. Additional common causes of secondary immunodeficiency include severe burns, malnutrition, certain types of cancer, and chemotherapy in cancer patients.
  • a cellular immunodeficiency refers to a deficiency the count or function of T lymphocytes, which are the main type of cells responsible for the cellular adaptive immune response in attacking viruses, cancer cells and other parasites.
  • T lymphocytes which are the main type of cells responsible for the cellular adaptive immune response in attacking viruses, cancer cells and other parasites.
  • AIDS Acquired Immunodeficiency Syndrome
  • HIV as a direct cause of cellular immunodeficiency, particularly the deficiency of the CD4+ T helper lymphocyte population, has been well established.
  • Additional examples of viral- or pathogen- induced immunodeficiencies include, although not limited to chickenpox, cytomegalovirus, German measles, measles, tuberculosis, infectious mononucleosis (Epstein-Barr virus), chronic hepatitis, lupus, and bacterial and fungal infections.
  • SARS virus- induced Severe Acute Respiratory Syndrome
  • disorders related to cellular immunodeficiency may include Aplastic anemia, Leukemia, Multiple myeloma, Sickle cell disease, chromosomal disorders such as Down syndrome, infectious diseases caused by pathogens such as Cytomegalovirus, Epstein-Barr virus, Human immunodeficiency virus (HIV), Measles and certain bacterial infections.
  • Aplastic anemia Leukemia, Multiple myeloma
  • Sickle cell disease chromosomal disorders
  • infectious diseases caused by pathogens such as Cytomegalovirus, Epstein-Barr virus, Human immunodeficiency virus (HIV), Measles and certain bacterial infections.
  • Chronic kidney disease Nephrotic syndrome, Hepatitis, Liver failure and other conditions caused by Malnutrition, alcoholism and burns.
  • patients' populations diagnosed with one of the secondary immunodeficiencies, and particularly one of the cellular immunodeficiencies as above, can particularly benefit from methods, as well as the re -programming agents, nanoparticles, cells, compositions and kits of the present disclosure. Differential diagnosis of such immunodeficient patients is routinely performed in various clinical settings.
  • Hematopoietic stem cell transplantation is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. It may be autologous (the patient's own stem cells are used), allogeneic (the stem cells come from a donor) or syngeneic (from an identical twin). Performance of this medical procedure usually requires the destruction of the recipient's immune system using radiation or chemotherapy before the transplantation.
  • the donor should preferably have the same human leukocyte antigens (HLA) as the recipient.
  • HLA human leukocyte antigens
  • the HSC are removed from a large bone of the donor, typically the pelvis, through a large needle that reaches the center of the bone.
  • Peripheral blood stem cells are now the most common source of stem cells for HSCT. They are collected from the blood through a process known as apheresis. The donor's blood is withdrawn through a sterile needle in one arm and passed through a machine that removes white blood cells. The red blood cells are returned to the donor.
  • the peripheral stem cell yield is boosted with daily subcutaneous injections of Granulocyte-colony stimulating factor (G-CSF), serving to mobilize stem cells from the donor's bone marrow into the peripheral circulation.
  • G-CSF Granulocyte-colony stimulating factor
  • amniotic fluid as well as umbilical cord blood may be also used as a source of stem cells for HSCT.
  • Hypersensitivities are divided into four classes (Type I- IV) based on the mechanisms involved and the time course of the hypersensitive reaction.
  • Type I is an immediate or anaphylactic reaction, often associated with allergy; it is mediated by IgE antibodies that trigger degranulation of mast cells and basophils.
  • Type II also called antibody-dependent or cytotoxic
  • IgG and IgM antibodies are also called antibody-dependent or cytotoxic antibodies.
  • Type III and Type IV are mediated by T cells, monocytes, and macrophages; Type IV reactions are involved in many autoimmune and infectious diseases.
  • a partial list including the most common allergies includes but not limited to Seasonal allergy, Mastocytosis, Perennial allergy, Anaphylaxis, Food allergy, Allergic rhinitis and Atopic dermatitis.
  • Splenomegaly enlargement of spleen
  • hypersplenism a condition in which abnormal red blood cells being destroyed in the spleen
  • Splenomegaly of between 11-20 cm greater than 20 cm in the size of spleen has been associated with hemolytic anemias, and other diseases involving abnormal red blood cells being destroyed in the spleen, as well as with other disorders, including congestion due to portal hypertension, and infiltration by leukemias and lymphomas.
  • compositions and methods of the present disclosure can be applied to prevent an immunodeficiency and/or GvHD in immunocomprimised cancer patients, being it a result of cancer itself (as mentioned above) or an adverse effect of high doses of chemotherapy or radiotherapy (which may induce burns).
  • NK cell deficiency and dysfunctionality A number of human diseases were specifically related to NK cell deficiency and dysfunctionality. Those include certain PIDs characterized by genetic aberrations that impair NK cells function. Several of these diseases induce a specific blockade in the stages leading to the formation of a functional lytic synapse. Most of these diseases can result in haemophagocytic lymphohistiocytosis (HLH), i.e. an inappropriately robust immune response to infection (typically with herpesviruses), which results in a persistent systemic inflammatory syndrome. This leads to the physiological symptoms of septic shock, but is also associated with the pathological finding of haematophagocytosis (the ingestion of red blood cells by phagocytes).
  • the NK cells are most relevant to the HLH phenotype, given their localization to marginal zones in lymphoid organs after viral infection, their innate function early in the course of infection and their inherent ability to eliminate hyperactivated macrophages.
  • the re-programming agents of the present disclosure or any nanoparticles or compositions or methods thereof are particularly applicable to patients diagnosed with one of the disorders related to NK cell or NKIS deficiency, or abnormal NK lytic granule trafficking. Notable examples of disorders belonging to this group are detailed below.
  • LAD-I Leukocyte adhesion deficiency type I results from a defect in the CD 18 (P-integrin) component of leukocyte integrin heterodimers.
  • P-integrin CD 18
  • LAD-I leukocytes do not appropriately adhere to inflamed or activated cells and cannot localize effectively to tissues and sites of inflammation. This leads to increased numbers of leukocytes in the blood and susceptibility to infectious diseases. Because early steps in NK-cell synapse formation - adhesion and activation signaling depend on integrins, NK cells from patients with LAD-I do not adhere to their target cells, resulting in defective cytotoxicity. LAD-I is also distinguished from other diseases discussed here because it does not lead to HLH.
  • Wiskott-Aldrich syndrome results from a hematopoietic- cell-specific defect in actin reorganization and cell signaling due to WASP deficiency.
  • Patients lacking WASP expression or expressing abnormal WASP have NK cells with decreased cytolytic capacity.
  • patients with WAS are susceptible to herpesviruse and can develop HLH, thereby demonstrating the functional relevance of WASP deficiency for the NK-cell lytic synapse. Formation of the lytic synapse is abnormal in NK cells from WAS patients and includes decreased F-actin accumulation and adhesion-receptor clustering at the synapse.
  • Chediak-Higashi syndrome CHS
  • Hermansky-Pudlak syndrome type II HPS2
  • albinism which is caused by aberrant functioning of melanocytes, which pigment skin via secretion of melanosomes (an equivalent of lytic granules).
  • CHS and HPS2 are similar in that they represent a failure in generation of the NK-cell lytic synapse at the end of the effector stages, as the abnormal lytic granules will not migrate along the microtubules to the MTOC.
  • Familial erythrophagocytic lymphohistiocytosis (FHL) types 3 and 4 are similar to CHS and HPS2, but are not associated with albinism demonstrating that the affected genes are not essential in melanocytes.
  • FHL3 is caused by mutation in the UNC13D gene, which encodes MUNC13-4.
  • FHL4 is caused by mutations in the STX11 gene, which encodes syntaxin- 11.
  • autoimmune disorders also referred to as disorders of immune tolerance
  • T cells lymphocytes and the NK cells in particular, play a pivotal role in the control of immune tolerance under normal conditions, and in T- and B-cell mediated human autoimmune disorders.
  • the NK cells have been further implicated in rheumatoid arthritis, systemic lupus erythematosus, and in multiple sclerosis.
  • the methods of the disclosure, as well as any reprogramming agents, nanoparticles, cells, compositions and kits of the disclosure may be used for the treatment of a patient suffering from any autoimmune disorder.
  • the methods as well as any re-programming agents, nanoparticles, cells, cell populations, compositions and kits of the disclosure may be used for treating an autoimmune disease such as for example, but not limited to, inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, fatty liver disease, Lymphocytic colitis, Ischaemic colitis, Diversion colitis, Behcet's syndrome, Indeterminate colitis, rheumatoid arthritis, systemic lupus erythematosus (SLE), Eaton-Lambert syndrome, Goodpasture's syndrome, Greave's disease, Guillain-Barr syndrome, autoimmune hemolytic anemia (AIHA), Idiopathic thrombocytopenic purpura (ITP),
  • IBD inflammatory bowel disease
  • GvHD Graft versus Host Disease
  • aGvHD acute GvHD
  • cGvHD chronic GvHD
  • late acute GVHD and overlap syndrome with features of both aGvHD and cGvHD.
  • aGvHD has been tightly linked to the activity and maturation of the donor T cells and NK cells that are transferred along with the marrow graft, i.e. cells that are directly responsible for recognition of antigenic differences on antigen-presenting cells of the host. Once activated, donor anti-host-specific T cells can mediate tissue destruction. GvHD continues to be a major life-threatening complication after allogeneic bone marrow transplantation.
  • GvHD a GvHD is characterized by selective damage to the liver, skin (rash), mucosa, and the gastrointestinal tract.
  • Other types of GvHD may further involve the hematopoietic system, e.g., the bone marrow and thymus, and the lungs in the form of immune-mediated pneumonitis.
  • Differential diagnosis of GvHD is further based on specific biomarkers.
  • the compounds as well as any re-programming agents, nanoparticles, compositions and kits according to the present disclosure are applicable to patients that are at risk of developing GvHD.
  • recipients who have received peripheral blood stem cells/bone marrow from an HLA mismatched related donor (or from an HLA matched unrelated donor) have an increased risk of developing a GvHD.
  • compositions and methods of the present disclosure can be applied to prevent the development of aGvHD.
  • the re-programming agents of the present disclosure or any nanoparticles, formulations, cells or compositions thereof may be applicable in boosting the immune response of a subject suffering from a secondary immunosuppression caused by chemotherapy, specifically, treatment with a chemotherapeutic agent.
  • chemotherapeutic agent or “chemotherapeutic drug” (also termed chemotherapy) as used herein refers to a drug treatment intended for eliminating or destructing (killing) cancer cells or cells of any other proliferative disorder.
  • the mechanism underlying the activity of some chemotherapeutic drugs is based on destructing rapidly dividing cells, as many cancer cells grow and multiply more rapidly than normal cells. As a result of their mode of activity, chemotherapeutic agents also harm cells that rapidly divide under normal circumstances, for example bone marrow cells, digestive tract cells, and hair follicles.
  • chemotherapeutic drugs are available.
  • a chemotherapeutic drug may be used alone or in combination with another chemotherapeutic drug or with other forms of cancer therapy, such as a biological drug, radiation therapy or surgery.
  • Certain chemotherapy agents have also been used in the treatment of conditions other than cancer, including ankylosing spondylitis, multiple sclerosis, hemangiomas, Crohn’s disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, lupus and scleroderma.
  • Chemotherapeutic drugs affect cell division or DNA synthesis and function and can be generally classified into groups, based on their structure or biological function.
  • the present disclosure generally pertains to chemotherapeutic agents that are classified as alkylating agents, anti- metabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other anti-tumor agents such as DNA-alkylating agents, anti-tumor antibiotic agents, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonist agents, protein kinase inhibitors, HMG-CoA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple-helix DNAs, nucleic acids aptamers, and molecularly-modified viral, bacterial or exotoxic agents.
  • chemotherapeutic drugs may be classified as relating to more than a single group. It is noteworthy that some agents, including monoclonal antibodies and tyrosine kinase inhibitors, which are sometimes referred to as “chemotherapy”, do not directly interfere with DNA synthesis or cell division but rather function by targeting specific components that differ between cancer cells and normal cells and are generally referred to as “targeted therapies”, “biological therapy " or “immunotherapeutic agent” as detailed below.
  • alkylating agents function by alkylating many nucleophilic functional groups under conditions present in cells.
  • chemotherapeutic agents that are considered as alkylating agents are cisplatin and carboplatin, as well as oxaliplatin.
  • Alkylating agents impair cell function by forming covalent bonds with amino, carboxyl, sulfhydryl, and phosphate groups in various biologically-significant molecules.
  • agents which function by chemically modifying DNA are mechlorethamine, cyclophosphamide, chlorambucil and ifosfamide.
  • An additional agent acting as a cell cycle non-specific alkylating antineoplastic agent is the alkyl sulfonate agent busulfan (also known as Busulfex).
  • the immune-suppressive condition may be caused by treatment with oxaliplatin.
  • Oxaliplatin is a platinum-based chemotherapy drug in the same family as cisplatin and carboplatin. It is typically administered in combination with fluorouracil and leucovorin in a combination known as Folfox for the treatment of colorectal cancer. Compared to cisplatin the two amine groups are replaced by cyclohexyldiamine for improved antitumour activity. The chlorine ligands are replaced by the oxalato bidentate derived from oxalic acid in order to improve water solubility.
  • Oxaliplatin is marketed by Sanofi- Aventis under the trademark Eloxatin®.
  • anti-metabolites also termed purine and pyrimidine analogues
  • purines or pyrimidines mimic the structure of purines or pyrimidines which are the building blocks of DNA and may thus be incorporated into DNA.
  • the incorporation of anti-metabolites into DNA interferes with DNA syntheses, leading to abnormal cell development and division.
  • Anti-metabolites also affect RNA synthesis.
  • anti-metabolites include 5 -fluorouracil (5-FU), azathioprine and mercaptopurine, fludarabine, cladribine (2-chlorodeoxyadenosine, 2-CdA), pentostatin (2'-deoxycoformycin, 2'-DCF), nelarabine, Floxuridine (FUDR), gemcitabine (Gemzar, a synthetic pyrimidine nucleoside) and Cytosine arabinoside (Cytarabine).
  • 5-FU 5 -fluorouracil
  • azathioprine and mercaptopurine fludarabine
  • cladribine (2-chlorodeoxyadenosine, 2-CdA
  • pentostatin (2'-deoxycoformycin
  • 2'-DCF pentostatin
  • nelarabine Floxuridine (FUDR)
  • gemcitabine Gemcitabine
  • Gamzar a synthetic pyrimidine nucleoside
  • the re-programming agents, nanoparticles, compositions and kits of the present disclosure may be applicable for boosting an immune-response by activating and/or functionalizing and/or rewiring, and/or reprograming NK cells, specifically, dysfunctional NK cells in a subject treated with a chemotherapeutic agent that may be at least one Plant alkaloid and terpenoid.
  • Plant alkaloids and terpenoids are agents derived from plants that block cell division by preventing microtubule function, thereby inhibiting the process of cell division (also known as “mitotic inhibitors” or “anti-mitotic agents”).
  • Examples of alkaloids include the vinca alkaloids (e.g.
  • vincristine, vinblastine, vinorelbine and vindesine include, for example, taxanes (e.g. paclitaxel and docetaxel). Taxanes act by enhancing the stability of microtubules, preventing the separation of chromosomes during anaphase.
  • taxanes e.g. paclitaxel and docetaxel. Taxanes act by enhancing the stability of microtubules, preventing the separation of chromosomes during anaphase.
  • the re-programming agents, nanoparticles, compositions and kits used by the present disclosure may be applicable for boosting an immune-response by activating and/or functionalizing and/or rewiring, and/or reprograming NK cells, specifically, dysfunctional NK cells, in a subject treated with chemotherapeutic agent that may be at least one Topoisomerase inhibitor.
  • Topoisomerases are essential enzymes that maintain DNA topology (i.e. the overall three dimensional structure of DNA). Inhibition of type I or type II topoisomerases interferes with both transcription and replication of DNA by inhibiting proper DNA supercoiling.
  • Type I topoisomerase inhibitors include camptothecins [e.g.
  • irinotecan and topotecan examples include amsacrine, etoposide, etoposide phosphate, and teniposide.
  • Anthracyclines or anthracycline antibiotics are a class of drugs used in cancer chemotherapy that are derived from the streptomyces bacterium. These compounds are used to treat many cancers, including leukemias, lymphomas, breast, uterine, ovarian, and lung cancers. These agents include, inter alia, the drugs daunorubicin (also known as Daunomycin), and doxorubicin and many other related agents (e.g., Valrubicin and Idarubicin).
  • the anthracycline agent Idarubicin acts by interfering with the enzyme topoisomerase II.
  • the compounds, as well as any re-programming agents, nanoparticles, cells, vectors, compositions and kits used by the present disclosure may be applicable for boosting an immune-response by activating and/or functionalizing and/or rewiring, and/or reprograming NK cells, specifically, dysfunctional NK cells in a subject treated with Doxorubicin.
  • the chemotherapeutic agent Doxorubicin also known by the trade name Adriamycin and by the name hydroxydaunorubicin
  • the compounds, as well as any re-programming agents, nanoparticles, cells, vectors, vehicles, compositions and kits used by the present disclosure may be applicable for boosting an immune-response by activating and/or functionalizing and/or rewiring, and/or reprograming NK cells, specifically, dysfunctional NK cells, in a subject treated with chemotherapeutic agent that may comprise at least one Cytotoxic antibiotics.
  • chemotherapeutic agent that may comprise at least one Cytotoxic antibiotics.
  • the anthracyclines agents described above are also classified as “cytotoxic antibiotics”.
  • Cytotoxic antibiotics also include the agent Actinomycin D (also known generically as Actinomycin or Dactinomycin), which is the most significant member of the actinomycines class of polypeptide antibiotics (that were also isolated from streptomyces). Actinomycin D is shown to have the ability to inhibit transcription by binding DNA at the transcription initiation complex and preventing elongation of RNA chain by RNA polymerase. Other cytotoxic antibiotics include bleomycin, epirubicin and mitomycin.
  • the re-programming agents as well as any nanoparticles, cells, vectors, vehicles, compositions and kits of the present disclosure or any nanoparticles or compositions thereof may be applicable for subjects suffering from immune-deficiency caused by immune-therapy or a biological therapy.
  • cancer vaccines, antibody treatments, and other “immunotherapies” are potentially more specific and effective and less toxic than the current approaches of cancer treatment and are generally termed “immunotherapy”, and therefore, an agent used for immunotherapy, is defined herein as an immuno-therapeutic agent.
  • immunotherapy as herein defined is a treatment that uses certain components of the immune system to fight diseases (e.g. cancer), by, inter alia, stimulating the immune system to become more efficient in attacking cancer cells (e.g., by administering vaccines) or by administering components of the immune system (e.g., by administering cytokines, antibodies, etc.).
  • immunotherapy has become an important part of treating several types of cancer with the main types of immunotherapy used being monoclonal antibodies (either naked or conjugated), cancer vaccines (i.e. that induce the immune system to mount an attack against cancer cells in the body) and non-specific immunotherapies.
  • Antibody-mediated therapy refers to the use of antibodies that are specific to a cancer cell or to any protein derived there-from for the treatment of cancer. As a non-limiting example, such antibodies may be monoclonal or polyclonal which may be naked or conjugated to another molecule.
  • Antibodies used for the treatment of cancer may be conjugated to a cytotoxic moiety or radioactive isotope, to selectively eliminate cancer cells.
  • biological treatment refers to any biological material that affects different cellular pathways.
  • agent may include antibodies, for example, antibodies directed to cell surface receptors participating in signaling, that may either activate or inhibit the target receptor.
  • biological agent may also include any soluble receptor, cytokine, peptides or ligands.
  • monoclonal antibodies that are used for the treatment of cancer include bevacizumab (also known as Avastin), rituximab (anti CD20 antibody), cetuximab (also known as Erbitux), anti-CTLA4 antibody and panitumumab (also known as Vectibix) and anti Grl antibodies.
  • the anti-cancer agent is epidermal growth factor receptor (EGFR) inhibitor.
  • the EGFR inhibitor is selected from the group consisting of: Cetuximab (Erbitux®), Panitumumab (Vectibix®), and necitumumab (Portrazza®).
  • the EGFR inhibitor is Cetuximab (Erbitux®).
  • cancer vaccines as referred to herein are vaccines that induce the immune system to mount an attack against cancer cells in the body.
  • a cancer treatment vaccine uses cancer cells, parts of cells, or pure antigens to increase the immune response against cancer cells that are already in the body. These cancer vaccines are often combined with other substances or adjuvants that enhance the immune response.
  • Non-specific immunotherapies as referred to herein do not target a certain cell or antigen, but rather stimulate the immune system in a general way, which may still result in an enhanced activity of the immune system against cancer cells.
  • a non-limiting example of non-specific immunotherapies includes cytokines (e.g. interleukins, interferons).
  • cytokines e.g. interleukins, interferons.
  • the compounds, as well as any re-programming agents, nanoparticles, compositions and kits used by the disclosure may be used as a combined supportive treatment for patients suffering from immune suppression. This supportive treatment may be combined with other supportive therapies as discussed herein.
  • the re-programming agents of the present disclosure or any nanoparticles, cells, vectors, vehicles, kits, methods or compositions thereof may be applicable for subjects undergoing at least one of adoptive cell transfer, a cancer vaccine, antibody-based therapy, a hormone, a cytokine or any combination thereof.
  • the compounds, as well as any re -programming agents, nanoparticles, cells, vectors, vehicles, compositions and kits of the disclosure may be used for boosting the immune response in subjects undergoing radiotherapy.
  • Radiation therapy or radiotherapy often abbreviated RT, RTx, or XRT, is therapy using ionizing radiation, generally as part of cancer treatment to control or kill malignant cells and normally delivered by a linear accelerator.
  • Radiation therapy may be curative in a number of types of cancer if they are localized to one area of the body. It may also be used as part of adjuvant therapy, to prevent tumor recurrence after surgery to remove a primary malignant tumor (for example, early stages of breast cancer).
  • the radiation is ionizing radiation, which may be any one of X-rays, gamma rays and charged particles.
  • the radiation may be employed in the course of total body irradiation, brachytherapy, radioisotope therapy, external beam radiotherapy, stereotactic radio surgery (SRS), stereotactic body radiation therapy, particle or proton therapy, or body imaging using the ionizing radiation.
  • the compounds, as well as any re -programming agents, nanoparticles, cells, vectors, vehicles, compositions, methods and kits used by the disclosure may be used for boosting the immune response in subjects undergoing gene therapy.
  • Gene therapy is the therapeutic delivery of nucleic acid polymers into a patient's cells as a drug to treat disease.
  • the most common form uses DNA, optionally packed in a vector that encodes a functional, therapeutic gene to replace a mutated gene.
  • the compounds, as well as any re -programming agents, nanoparticles, cells, vectors, vehicles, compositions, methods and kits used by the disclosure may be used for boosting the immune response in subjects undergoing immunotherapy using checkpoint inhibitors.
  • the phrase "boosting the immune response" when referred to herein relates to an action caused by activating and/or functionalizing and/or rewiring, and/or reprograming NK cells, specifically, dysfunctional NK cells by the re-programming agents of the present disclosure and any methods using the reprogramming agent.
  • immune checkpoint molecules are co-stimulatory and co- inhibitory molecules that act in coordination to modulate the immune response of autoreactive T cells.
  • Immune checkpoint molecules like CTLA-4, TIM-3, PD-1, are negative regulators of immune responses to avoid immune injury.
  • Recent studies have identified several new immune checkpoint targets, like lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin and mucin-domain containing-3 (TIM-3), T cell immunoglobulin and ITIM domain (TIGIT), V- domain Ig suppressor of T cell activation (VISTA), and so on.
  • a checkpoint inhibitor applicable in the method of the disclosure may be an antibody against an immune checkpoint molecule selected from the group consisting of human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), lymphocyte activation gene 3 (LAG3), CD137, 0X40 (also referred to as CD 134), killer cell immunoglobulin-like receptors (KIR), TIGIT, VISTA and any combination thereof.
  • PD-1 human programmed cell death protein 1
  • CEACAM1 carcinoembryonic antigen-related cell adhesion molecule 1
  • LAG3 lymphocyte activation gene 3
  • CD137 also referred to as CD 134
  • KIR killer cell immunoglobulin-like receptors
  • TIGIT TIGIT
  • VISTA any combination thereof.
  • immune check point inhibitors applicable in the present disclosure include but are not limited to Yervoy (ipilimumab) that targets CTLA-4, Keytruda (pembrolizumab) that targets PD-1 ligands, Opdivo (nivolumab) that targets PD-1, Tecentriq (atezolizumab) from Genentech, targets PD-L1, Bavencio (avelumab) blocks PD-L1, Imfinzi (durvalumab) targets PD- Ll, Libtayo (cemiplimab-rwlc) that targets the PD-1 cellular pathway, and any combinations thereof.
  • the therapeutic methods disclosed by the disclosure may use any of the administration modes described herein before, in connection with the compositions of the disclosure, for example, administration by parenteral, intraperitoneal, transdermal, oral (including buccal or sublingual), rectal, topical (including buccal or sublingual), vaginal, intranasal and any other appropriate routes.
  • the therapeutic methods of the present disclosure are applicable for various disorders that include disorders caused by or exprobrated and/or worsened by subjecting the treated subject to therapy (e.g., chemotherapy, immunotherapy, cell therapy) as discussed above.
  • therapy e.g., chemotherapy, immunotherapy, cell therapy
  • the present methods further offer a combined therapy solution using any of the above-indicated treatment regimen while in parallel functionalizing the immune system, by functionalizing exhausted or anergic dysfunctional NK cells, together with the additional therapy given.
  • the disclosed methods provide combined therapy using the present re-programming agents or any nano-particles or compositions thereof, with an additional therapeutic agent.
  • an additional therapeutic agent for example, any of the agents discussed herein above, in particular, the agents or treatment regimens that cause a secondary immunodeficiency. - I l l -
  • the methods of the disclosure provides in vivo activation and/or functionalization and/or rewiring, and/or reprograming of NK cells, specifically, dysfunctional NK cells (e.g., anergic NK cells or exhausted NK cells) by administering to the treated subject a therapeutically effective amount of the re-programming agents of the present disclosure, specifically the combined inhibitors of at least one EGR proteins and at least one DGK protein as disclosed by the disclosure, or any nano-particles, micro-particles, micellar formulations, vectors, vehicles and matrix thereof, or any combinations thereof with other therapeutic compounds.
  • the disclosure therefore provides in vivo treatment of the subject that suffers from any of the described disorders.
  • the re -programming agents of the present disclosure and specifically, the nano-particles provided by the disclosure target hematopoietic cell/s, specifically, NK cells in the treated subject and reduce the expression, stability and/or activity of at least one EGR protein and/or of at least one DGK protein in the targeted NK cells, thereby leading to activation of these NK cells in inhibitory immunological synapses in the treated subject.
  • This targeted activation enhances the immune response against any of the described disorders, specifically, cancer in the subject and therefore provide treatment thereof.
  • the disclosure further encompasses and provides therapeutic methods involving ex vivo or in vitro steps for activating immune cells of the subject, or of a suitable donor, and introducing the activated cells, for example, NK cells, to the treated subject.
  • such methods may involve obtaining hematopoietic cells of a subject suffering from any of the described disorders or of a suitable donor (specifically, allogeneic donor), contacting in vitro/ ex vivo the hematopoietic cell/s (e.g., T cell, B cells and/or NK cells) with an activating effective amount of the re-programming agents of the present disclosure or any nanoparticle, vehicles, vectors, micellar formulations, composition or kits thereof, and re-introducing the activated hematopoietic cells (that were activated in accordance with the methods of the disclosure) to the subject (adoptive transfer).
  • hematopoietic cells of allogeneic or syngeneic subjects are also applicable in the methods of the disclosure.
  • the cells used by the methods of the disclosure may be cells, specifically, cells of an autologous source.
  • autologous when relating to the source of cells, refers to cells derived or transferred from the same subject that is to be treated by the method of the disclosure.
  • the cells used by the methods of the disclosure may be cells of an allogeneic source, or even of a syngeneic source.
  • allogeneic when relating to the source of cells, refers to cells derived or transferred from a different subject, referred to herein as a donor, of the same species.
  • sergeneic when relating to the source of cells, refers to cells derived or transferred from a genetically identical, or sufficiently identical and immunologically compatible subject (e.g., an identical twin).
  • these cells may be than administered to the subject by adoptive transfer.
  • adaptive cell transfer is the transfer of cells into a patient.
  • the cells may have originated from the patient or from another individual.
  • the cells are most commonly derived from the immune system, with the goal of improving immune functionality and characteristics.
  • T cells are extracted from the patient, genetically modified and cultured in vitro and returned to the same patient.
  • the term “adoptive transfer” as used herein defined applies to all the therapies that consist of the transfer of components of the immune system, specifically cells such as NK cells that are already capable of mounting a specific immune response.
  • the activation of the NK cells by inhibiting the expression, stability and/or activity of at least one EGR protein and/or of at least one DGK protein by the reprogramming agents of the present disclosure is performed in cells of an autologous or allogeneic source, that are then administered to the subject, specifically, by adoptive transfer.
  • the disclosure further encompasses the in vivo or ex vivo therapeutic methods described herein performed in a subject suffering from any of the disorders disclosed herein before, that is further treated with an additional therapeutic agent, for example, an immune check point inhibitor.
  • the methods of the disclosure comprise the step of administering to a subject treated with at least one immune checkpoint inhibitor, an effective amount of the re-programming agents of the present disclosure or any nanoparticles, cells, vectors, vehicles, compositions and kits thereof.
  • the immune checkpoint inhibitor is any of the inhibitors specified herein before.
  • the at least one re-programming agent for use according to the disclosure refers to any of the re -programming agents, cells, vectors, vehicles, compositions and kits thereof, as described by the present disclosure herein above.
  • any of the nanoparticle disclosed herein above are also applicable in this aspect as well.
  • the present disclosure provides any of the reprogramming agent (or modulator and/or re-programmers), nanoparticles and compositions disclosed by the disclosure for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an immune-related disorder in a subject in need thereof.
  • the present disclosure provides any of the reprogramming agents, nanoparticles and compositions disclosed by the disclosure for use in a method for activating NK cells, specifically, dysfunctional NK cells (e.g., anergic NK cells or exhausted NK cells) more specifically, in a subject in need thereof.
  • dysfunctional NK cells e.g., anergic NK cells or exhausted NK cells
  • an immune-related disorder is any of the disorders specified herein before in connection with other aspects of the disclosure.
  • the at least one re -programming agent for use according to this aspect of the disclosure may be used in a method of treating a subject that is already treated with at least one additional therapeutic agent, for example, an immune checkpoint inhibitor, or any of the other therapeutic agents disclosed by the disclosure in connection with other aspects.
  • the terms “effective amount” or “sufficient amount” used by the methods of the disclosure mean an amount necessary to achieve a selected result.
  • the “effective treatment amount” is determined by the severity of the disease in conjunction with the preventive or therapeutic objectives, the route of administration and the patient's general condition (age, sex, weight and other considerations known to the attending physician).
  • the terms “treat, treating, treatment” as used herein and in the claims mean ameliorating one or more clinical indicia of disease activity by administering a pharmaceutical composition of the disclosure in a patient having a pathologic disorder.
  • percentage values such as, for example, 10%, 50%, 100%, 120%, 500%, etc., are interchangeable with "fold change” values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively.
  • the present disclosure further provides combined therapy involving the use of at least one re-programming agent, that may comprise at least one compound, that specifically inhibit at least one of, the expression, activity and stability of at least one member of the EGR family, and/or compounds that specifically inhibit at least one of, the expression, activity and stability of at least one member of the DGK family and optionally, at least one additional therapeutic agent, specifically, checkpoint inhibitor that may be administered either together in a pharmaceutical composition, or in separate compositions through different routes, dosages and combinations.
  • at least one re-programming agent may comprise at least one compound, that specifically inhibit at least one of, the expression, activity and stability of at least one member of the EGR family, and/or compounds that specifically inhibit at least one of, the expression, activity and stability of at least one member of the DGK family and optionally, at least one additional therapeutic agent, specifically, checkpoint inhibitor that may be administered either together in a pharmaceutical composition, or in separate compositions through different routes, dosages and combinations.
  • the re-programming agent is any of the reprogramming agents as defined by the present disclosure.
  • the disclosed kit is applicable for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an immune-related disorder in a subject in need thereof.
  • At least one immunomodulatory therapeutic agent comprises at least one of: (said at least one immunomodulatory therapeutic agent comprises at least one of: (i) at least one checkpoint inhibitor; (ii) at least one cytokine; and (iii) chimeric antigen receptor (CAR) T cells.
  • an additional therapeutic agent in the context of the present aspect further encompasses any of the therapeutic agents discussed above, in connection with agents that may cause, or lead to, or worsen immunodeficiency, in particular, secondary immunodeficiency.
  • any of the chemotherapeutic agents, immunomodulatory agents and cell therapy agents disclosed by the present disclosure are examples of the chemotherapeutic agents, immunomodulatory agents and cell therapy agents disclosed by the present disclosure.
  • kits of the disclosure may comprise any of the components and compounds described above in connection with other aspects of the disclosure.
  • the disclosure provides the kits as described above for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an immune -related disorder in a subject in need thereof.
  • the disclosure provides the kits as described above for use in a method for activating NK cells, specifically, dysfunctional NK cells (e.g., anergic NK cells or exhausted NK cells), more specifically, in a subject in need thereof.
  • the re-programming agent (or modulator and/or re-programmers), as well as any nanoparticles, compositions and kits of the present disclosure may be administered in a form of combination therapy, i.e. in combination with one or more additional therapeutic agents.
  • Combination therapy may include administration of a single pharmaceutical dosage formulation comprising at least one composition of the disclosure and additional therapeutics agent(s); as well as administration of at least one composition of the disclosure and one or more additional agent(s) in its own separate pharmaceutical dosage formulation.
  • compositions of the disclosure and one or more additional agents can be administered concurrently or at separately staggered times, i.e. sequentially. Still further, the concurrent or separate administrations may be carried out by the same or different administration routes.
  • the other therapeutic agent may involve the administration or inclusion of at least one additional factor that may in some specific embodiments be any checkpoint inhibitor, for example, any antibody against an immune checkpoint molecule selected from the group consisting of human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), lymphocyte activation gene 3 (LAG3), CD137, 0X40 (also referred to as CD134), killer cell immunoglobulin-like receptors (KIR), TIGIT, VISTA and any combination thereof.
  • PD-1 human programmed cell death protein 1
  • CEACAM1 carcinoembryonic antigen-related cell adhesion molecule 1
  • LAG3 lymphocyte activation gene 3
  • CD137 also referred to as CD134
  • KIR killer cell immunoglobulin-like receptors
  • TIGIT TIGIT
  • VISTA any combination thereof.
  • immune check point inhibitors applicable in the present disclosure include but are not limited to Yervoy (ipilimumab) that targets CTLA-4, Keytruda (pembrolizumab) that targets PD-1 ligands, Opdivo (nivolumab) that targets PD-1, Tecentriq (atezolizumab) from Genentech, targets PD-L1, Bavencio (avelumab) blocks PD-L1, Imfinzi (durvalumab) targets PD- LI, Libtayo (cemiplimab-rwlc) that targets the PD-1 cellular pathway, and any combinations thereof.
  • the other therapeutic agent may involve the administration or inclusion of at least one additional factor that may in some specific embodiments be selected from among EPO, G-CSF, M-GDF, EGF, SCF, GM-CSF, M-CSF, CSF- 1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, or other various interleukins, IGF-1, LIF, interferon (such as a, beta, gamma or consensus), neurotrophic factors (such as BDNF, NT-3, CTNF or noggin), other multi-potent growth factors (such as, to the extent these are demonstrated to be such multi-potent growth factors, flt-3/flk-2 ligand, stem cell proliferation factor, and totipotent stem cell factor), fibroblast growth factors (such as FGF), and analogs, fusion molecules, or other derivatives of EPO, G-CSF, M-GDF
  • treatment with the re-programming agents of the present disclosure or any nanoparticles or compositions thereof may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the other therapeutic agent and the compounds are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the other agent and the compounds would still be able to exert an advantageously combined effect.
  • the re-programming agents of the present disclosure or any nanoparticles or compositions thereof may be applicable in combined treatment with G-CSF.
  • G-CSF Granulocyte-colony stimulating factor
  • CSF 3 colony-stimulating factor 3
  • G-CSF is a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream. Functionally, it is a cytokine and hormone, a type of colony- stimulating factor, and is produced by a number of different tissues.
  • the pharmaceutical analogs of naturally occurring G-CSF are called filgrastim and lenograstim.
  • G- CSF also stimulates the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils.
  • G-CSF In oncology and hematology, a recombinant form of G-CSF is used with certain cancer patients to accelerate recovery and reduce mortality from neutropenia after chemotherapy, allowing higher- intensity treatment regimens. G-CSF is also used to increase the number of hematopoietic stem cells in the blood of the donor before collection by leukapheresis for use in hematopoietic stem cell transplantation. G-CSF may also be given to the receiver in hematopoietic stem cell transplantation, to compensate for conditioning regimens.
  • the present disclosure establishes the application of member of the EGR family, specifically, ERG2 and/or ERG4, and members of the DGK family, specifically, DGKa and/or DGK ⁇ and any combination thereof, as diagnostic biomarkers, or specifically, as a diagnostic signature for dysfunctional cells, specifically, anergic cells and/or exhausted cells, more specifically, anergic NK cells, and/or exhausted NK cells.
  • the diagnostic potential of such signature may be applicable for monitoring patients and for determining and personalizing treatment regimens given to patients suffering from immune-related disorders.
  • a further aspect of the present disclosure relates to a method for determining a personalized treatment regimen for a subject suffering from an immune-related disorder, and optionally for monitoring the effectiveness of a treatment regimen in the subject.
  • the method comprising the steps of: In step (a), determining the levels of at least one of ERG2 and DGKa in at least one NK cell of at least one biological sample of the subject, to obtain the level value of the at least one of ERG2 and DGKa.
  • step (b) classifying said subject as: (i), a subject displaying a decreased NK cell functioning, (or having dysfunctional NK cells), if the level value determined in (a), for the at least one of: ERG2 and/or DGKa in the at least one sample is higher as compared to a predetermined standard value.
  • the subject is classified as (ii), a subject displaying functioning NK cells, if the level value determined in (a) for the at least one of ERG2 and DGKa in the at least one sample is equal or lower as compared to a predetermined standard value.
  • the next step (c) involves selecting for a subject displaying a decreased NK cell functioning (or dysfunctional NK cells), an activating treatment regimen that decreases the levels of at least one of ERG2 and DGKa in at least one NK cell of the subject.
  • the activating treatment regimen selected in (c), comprises at least one reprogramming agent as defined by the present disclosure, specifically, any of the re-programming agents disclosed herein in connection with other aspects of the present disclosure.
  • the personalized therapeutic method is applicable for a subject is a subject treated with at least one immunomodulatory therapeutic compound or treatment regimen. In some embodiments, any of the additional therapeutic compounds disclosed by the present disclosure herein before.
  • the at least one immunomodulatory therapeutic agent comprises at least one of: (i), at least one checkpoint inhibitor; (ii), at least one cytokine; and (iii), chimeric antigen receptor (CAR) T cells, at least one PROTAC molecule, at least one of Pomalidomide, Lenalidomide, Thalidomide, and/or immunomodulatory drug (IMiD).
  • CAR chimeric antigen receptor
  • the step of selecting for a subject displaying a decreased NK cell functioning (or dysfunctional NK cells), an activating and/or functionalizing treatment regimen comprises one of: (a), ceasing a treatment regimen with the at least one immunomodulatory therapeutic agent, and/or replacing the treatment with a treatment regimen comprising the re -programming agents as defined by the present disclosure.
  • (b) administering re-programming agent as defined by the present disclosure, prior to, after or concomitantly with the immunomodulatory therapeutic agent as disclosed by the present disclosure.
  • the present disclosure further provides a personalized therapeutic method that involves a diagnostic step that facilitates determination and/or adjustment of the treatment regimen, as well as monitoring the subject. More specifically, the present disclosure relates to a method of treating a subject suffering from an immune-related disorder, by providing a personalized treatment regimen for the subject and/or by monitoring the effectiveness of a treatment regimen in the subject. The method comprising the steps of: In step (a), determining the levels of at least one of ERG2 and DGKa in at least one NK cell of at least one biological sample of said subject, to obtain the level value of said at least one of ERG2 and DGKa.
  • step (b) classifying said subject as: (i), a subject displaying a decreased NK cell functioning, (or having dysfunctional NK cells), if the level value determined in (a), for the at least one of: ERG2 and/or DGKa in the at least one sample is higher as compared to a predetermined standard value.
  • the subject is classified as (ii), a subject displaying functioning NK cells, if the level value determined in (a) for the at least one of ERG2 and DGKa in the at least one sample is equal or lower as compared to a predetermined standard value.
  • the next step (c) involves selecting for a subject displaying a decreased NK cell functioning (or dysfunctional NK cells), an activating treatment regimen that decreases the levels of at least one of ERG2 and DGKa in at least one NK cell of said subject.
  • the methods of the present disclosure that comprise a diagnostic step, further provide a prognostic tool for providing a prognosis for a subject, for monitoring a treatment regimen and/or the disease progression, where samples are obtained from the subject in various time points (also referred to herein as temporally separated samples).
  • a sample may be obtained prior to initiating a treatment regimen, and/or in various time points after the initiation of a treatment regimen.
  • a treatment regimen with at least one immunomodulatory therapeutic agent for example, a treatment regimen with at least one immunomodulatory therapeutic agent.
  • the disclosed methods may be useful for determining the likelihood for a successful treatment, for monitoring a patient and determining the optimal timing and/or amount of a treatment regimen, for evaluating responsiveness of a subject, for predicting relapse of a disease, and for evaluating the survival of a subject.
  • the expression level of at least one of the EGR family member and/or of at least one DGK family proteins described herein is being determined.
  • level of expression or “expression leve ' are used interchangeably and generally refer to a numerical representation of the amount (quantity) of an amino acid product or polypeptide or protein in a biological sample.
  • level of expression or “expression level” refers to the numerical representation of the amount (quantity) of polynucleotide which may be gene in a biological sample.
  • “Expression” generally refers to the process by which gene-encoded information is converted into the structures present and operating in the cell.
  • gene expression values may be measured in the protein level, for example by MS methods or alternatively by immunological methods.
  • the expression may be measured in the nucleic acid level, for example using Real-Time Polymerase Chain Reaction, sometimes also referred to as RT-PCR or quantitative PCR (qPCR).
  • RT-PCR Real-Time Polymerase Chain Reaction
  • qPCR quantitative PCR
  • expression of a gene may refer to transcription into a polynucleotide and translation into a polypeptide. Fragments of the transcribed polynucleotide, the translated protein, or the post- translationally modified protein shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the protein, e.g., by proteolysis. Methods for determining the level of expression of the ERG and/or DGK proteins are known in the art.
  • the methods of the disclosure refer to the level of the biomarker protein/s in the sample.
  • the "expression level” as referred to herein simply refers to the levels, amount, quantity of the biomarker protein/s in the sample, specifically where the "expression level” is determined at the protein level. The determined level and/or amount reflects not only the level of expression but may also reflect the stability of the biomarker protein.
  • the expression level of the ERG and/or DGK proteins of the invention is determined to obtain an expression value.
  • level value refers to the result of a calculation, that uses as an input the “level of expression” or “expression level” obtained experimentally. It should be appreciated that in some optional embodiments, determination of the expression value may further involves normalizing the “level of expression” or “expression level” by at least one normalization step as detailed herein, where the resulting calculated value termed herein "expression value” is obtained. More specifically, as used herein, "normalized values” in some embodiments, are the quotient of raw expression values of marker proteins, divided by the expression value of a control reference protein from the same sample.
  • the levels of the at least one EGR family member and/or of at least one DGK family member are compared with predetermined standard levels.
  • “Standard” or a “predetermined standard” as used herein denotes either a single standard value or a plurality of standards with which the p The levels of the at least one EGR family member and/or of at least one DGK family member value determined for the tested sample is compared.
  • the standards may be provided, for example, in the form of discrete numeric values or in the form of a chart for different values of the levels of these proteins, or alternatively, in the form of a comparative curve prepared on the basis of such standards (standard curve).
  • the prognostic methods of the present disclosure may optionally further involve the use of a calibration curve created by detecting and quantitating the levels of the at least one EGR family member and/or of at least one DGK family member in cells of known populations of responders and non-responders to the indicated treatment. Obtaining such a calibration curve may be indicative to provide standard values. It should be noted that for determining the expression value/s of at least one of the ERGs and/or DGKs proteins disclosed herein, the methods of the invention may further comprise the step of providing at least one detecting molecule specific for determining the expression of at least one of the ERGs and/or DGKs proteins of the invention.
  • such detecting molecules may be provided as a mixture, as a composition or as a kit.
  • the at least one detecting molecule may be provided as a mixture of detecting molecules, wherein each detecting molecule is specific for one ERGs and/or DGKs protein. It should be appreciated however, that for each ERG and/or DGK protein, one or several specific detecting molecules may be used and provided.
  • the detecting molecules may be provided separately for each ERGs and/or DGKs protein, e.g., in specific tube, containers, slots, spots, wells, and the like. It further alternative embodiments, the detecting molecules may be attached or immobilized to a solid support, specifically, in recorded location.
  • the present disclosure further provides at least one functional NK cell, or a population of cells comprising at least one of the functional NK cells.
  • the functional NK cells disclosed herein were obtained by in vivo or ex vivo manipulation thereof, such that they display inhibited or reduced expression, activity and/or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family.
  • the functional NK cells were contacted with or comprise at least one compound that specifically inhibits the expression, activity and/or stability of at least one of at least one member of the EGR family of transcription factors; and/or at least one member of the DGK family comprises at least one nucleic acid molecule.
  • the compound may be at least one amino acid-based molecule, and/or at least one chemical inhibitor, each specific for one of: (i) at least one member of the EGR family of transcription factors; and/or (ii) at least one member of the DGK family.
  • the cell may be genetically modified NK cells that was modified for reduced levels of at least one member of the EGR family of transcription factors; and/or at least one member of the DGK family.
  • the cells may be obtained from an autologous subject, manipulated ex vivo and transferred back (e.g., adoptive transfer) to the subject.
  • the functional NK cells may be of an allogeneic source, that were manipulated ex vivo or in vivo, and than administered to the allogeneic patient in need.
  • the manipulation of the NK cells is performed in vivo, using any suitable vehicle, for example, a nano-particle vehicle.
  • the present disclosure provides a method for the preparation of improved population of cells comprising lymphocytes, specifically, lymphocytes of the T lineage, more specifically, a population of cells comprising NK cells.
  • a population of cells comprising T cells comprise the spec of contacting a population of cells with an effective amount of the disclosed re-programming agent, such that the dysfunctional cells in the cell population are activated and/or functionalized and/or reactivated and/or rewired.
  • the disclosed method results in an improved population of cells, specifically, a population of cells having an increase portion of functional cells, specifically, functional NK cells.
  • the disclosed method results in an improved population of cells, specifically, a population of cells having a decreased portion of dysfunctional cells, specifically, dysfunctional NK cells.
  • the resulting improved population display less than 20% anergic cells, specifically, specifically, less than 19% anergic cells, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, and less than 0.001% of anergic cells, specifically, anergic NK cells.
  • the disclosed improved cell population comprise a reduced portion of exhausted cells, for example, 20% exhausted cells, specifically, specifically, less than 19% anergic cells, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, and less than 0.001% of exhausted cells, specifically, anergic NK cells.
  • 20% exhausted cells specifically, specifically, less than 19% anergic cells, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less
  • references to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
  • composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.
  • a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases "ranging/ranges between" a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number "to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.
  • 721.221 cell lines (RRID:CVCL_6263), and K562 (ATCC- CCL-243) CML cell lines were generously provided by Prof. Ofer Mandelboim (Department of Microbiology and Immunology, Faculty of Medicine, Hebrew University, Israel).
  • 721.221 HLA negative and 721.221 expressing HLA-Cw7 cells were cultured in RPMI 1640 (Sigma- Aldrich) supplemented with 10% FBS, 2mM L-glutamine, penicillin (50 pg/ml), streptomycin (50 pg/ml), 1% non- essential amino acids, and 1% sodium pyruvate.
  • Stable 721.221 HLA Cw7 cell lines expressing mCherry were previously established in the inventors' laboratory (Ben-Shmuel et al, 2022, eLife l l:e73282; Biber et al, 2021, EMBO Mol Med 14:el4073) and were used for Incucyte assay.
  • the human pancreatic ductal carcinoma, PANC-1 cell line was used (ATCC - CRL1469) for the in- vivo tumor experiments.
  • Human K562 CML cell lines stably expressing CFP were used for the OTS system (Ben-Shmuel et al, 2022, eLife 11 :e73282). The cells were maintained in culture at a density of 0.3-0.5xl0 6 cells/mL.
  • Human primary PBMCs were isolated from whole blood of healthy donors or patients, as previously described (Barda-Saad et al, 2005, Nat Immunol 6:80-89; Ben-Shmuel et al, 2022, eLife l l:e73282; Biber et al, 2021, EMBO Mol Med 14:el4073). Blood samples from healthy donors were randomly collected and provided by Magen David Adorn (MDA) (Israeli National Blood Bank, no. 12-0016). Informed consent was obtained from all donors. The donor’s identification information remained anonymous. The research was performed with the approval of and according to the guidelines of the Bar- Ilan University Ethics Committee.
  • PBMCs peripheral blood mononuclear cells
  • pNK cells Primary NK (pNK) cells were isolated from PBMCs of healthy donors using the EasySepTM human NK cell enrichment kit (STEMCELL Technologies) with a purity of CD3 CD56 + NK cells >95%. The efficiency of the magnetic separation was checked after each separation via flow cytometry. Subsequently, pNK cells were stained with pan anti KIR2D, anti KIR3DL1 and anti-NKG2 antibodies (Miltenyi biotec and BioLegend) and then subjected to cell sorting using Aria Cell Sorter II (Fig. 1). PBMCs were used as a negative control during sorting. NK cell isolation and sorting was 95% accurate and the resulting cells were then used for functional assays.
  • Sorting was conducted for pNK obtained from a healthy individual donor in every experiment involving anergic and responsive NK cells.
  • Antibodies and their sources were as follows:
  • FACS surface staining
  • PE-anti-Human KIR2D PE-anti- Human NKG2A (Miltenyi Biotech); PE-anti- Human KIR3DL1, Alexa 647- anti- Human Tim-3, Alexa 647-anti- Human PD-1, Alexa 647- anti- Human TIGIT, FITC anti- Human CD107a, mouse antiHuman CD16, FITC-anti Human CD56 (Bio Legend); mouse anti- Human NKp46 (Merck Millipore), FITC-anti Human CD45, PE-Cy5 anti Human CD3 (BD Biosciences).
  • pNK cells were transfected with a Lonza NucleofectorTM 2b Device using the manufacturer’s protocol U-001, with either non-specific (NS) siRNA, DGKa, siRNA or Egr2 siRNA. Transfections were performed 48 hours before biochemical and functional assays.
  • Cytotoxicity assays were performed as described (Ben-Shmuel et al, 2022, eLife 11 :e73282; Biber et al, 2021, EMBO Mol Med 14:el4073).
  • the degranulation assay based on staining for CD 107a was performed as described (Ben-Shmuel et al, 2022, eLife l l:e73282; Biber et al, 2021, EMBO Mol Med 14:el4073).
  • Flow cytometric analysis i) Surface receptor staining: 5xl0 5 pNK cells were incubated for 30 min on ice with 1:25 diluted anti-PD-1- Alexa 647 or anti-Tim-3- Alexa 647 or anti-TIGIT- Alexa 647 conjugated antibodies. The cells were then washed twice with Phosphate Buffer Saline (PBS) and analyzed by flow cytometry.
  • PBS Phosphate Buffer Saline
  • pNK cells were stained with anti-NKG2A, pan KIR2D, KIR3DL1, CD3, CD56, PD1, TIGIT, TIM-3, CD16, NKp46 for 10 min in 4°C or 30 min on ice according to the manufacturer's protocols, and then acquired using the ARIAIII or Fortessa FACS, and analyzed by Flow Jo Software vl0.8.1.
  • Intracellular staining NK cells (anergic and responsive) (0.5-lxl0 6 cells per sample) and 721.221 target cells were incubated separately on ice for 10 min, at a ratio of 1:2. The cells were mixed, centrifuged and incubated on ice for 15 min.
  • the cell mixture was then transferred to 37°C for the indicated period of time, and subsequently fixed with 0.4 ml 3.7% paraformaldehyde for 20 min at room temperature (RT). After two additional washes, cells were permeabilized with 0.1% Triton X-100 in PBS for 4 min at RT. Cells were then blocked for 45 min with 2% goat serum, followed by 1 hour incubation with relevant antibody [pERK (Y204) (on ice)] . The samples were washed twice, and then incubated with l:2500-diluted Alexa Fluor 647-conjugated goat antiRabbit IgG antibody (Invitrogen), for 30 min on ice. Fluorescence was analyzed by flow cytometry on a BD Fortessa system using FACSDiva Software. For all experiments involving FACS, the gating strategy is shown in the extended view figures.
  • Intracellular calcium was determined as described, using Indo-1 AM staining (Sabag et al, 2022, Cancers 14:3756; Ben-Shmuel et al, 2022, eLife l l:e73282; Biber et al, 2021, EMBO Mol Med 14:el4073).
  • HLA-Cw7 cells (l*10 4 ) expressing mCherry were seeded in 100, u L Opti-MEM in Corning 96- well flat bottom plates (Cat no. TCP011096). Effector cells, consisting of anergic or responsive cells, treated with Egr2 siRNA or N.S. siRNA were seeded along with Cw7 cherry cells at an E:T ratio of 10:1. Cw7 cherry cells alone were used as target-only control for normalization of fluorescence values. The plates were maintained in the Incucyte SX5 - Live cell analysis and analyzed using Incucyte Spectral analysis software - basic analyzer for 14 hours.
  • phase confluences were divided to orange confluence, and mean intensity was calculated and normalized to time point 0:00 hrs.
  • NP based siRNA delivery particles were prepared as previously described (Biber et al, 2021, EMBO Mol Med 14:el4073). The NP’s were tagged with anti-NKp46 and labelled by Rhodamine to enable detection. Egr2 siRNA or NS siRNA were encapsulated by protamine entrapment as previously established (Biber et al, 2021, EMBO Mol Med 14:el4073).
  • K562 CFP (l*10 5 ) (using stable lines previously established in the lab (Ben-Shmuel et al, 2022, eLife ll:e73282)) were mixed with Matrigel (Phenol Red free. Cat no#) at a 1:1 ratio (v/v - 25uL:25uL) in Corning 24 well flat bottom plates.
  • the cells were cultured using OTS media, RPMI media supplemented with 0.1 ug/mL fibroblast growth factor (FGF), 0.18 ug/mL epidermal growth factor (EGF) and 20 ng/mL transforming growth factor (TGFP), and allowed to form domes for 48 hours.
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • TGFP transforming growth factor
  • pNK cells were obtained from PBMCs of healthy donors and subjected to sorting to obtain anergic and responsive populations. They were then seeded on the OTS at an E: T ratio of 5:1 in Opti-MEM for imaging.
  • Egr2 siRNA or NS siRNA were encapsulated in anti-NKp46-tagged liposomal nanoparticles using a previously established protocol in the inventors' lab (Biber et al, 2021, EMBO Mol Med 14:el4073) and delivered to the OTS post 6 hours.
  • Cultures were then subjected to the incucyte killing assay by measuring the decrease in fluorescence from the target cells (CFP) for 48 hours; K562 CFP cells alone were taken as OTS-only control for normalization of values.
  • the images were obtained using Standard Analyzer with a 20X objective. Whole well images were obtained using 4X objective at 48h.
  • the plates were maintained in the Sartorius SX5 Incucyte and analyzed using Incucyte Spectral analysis software, basic analyzer and adherent cell by cell analysis, for 48 hours. The phase confluences were divided to green confluence, and mean intensity was calculated and normalized to the measurement at the initiation of the experiment.
  • RNA interference siRNAs specific for human EGR2, EGR4, DGKa, DGK ⁇ and non-specific controls were purchased from Sigma-Aldrich.
  • specific oligonucleotides encoding siRNAs were as follows: esiEgr2 MISSION Sigma Aldrich Cat no. EHU124311 as denoted by SEQ ID NO: 9, esiEgr4 MISSION Sigma Aldrich Cat no. EHU135811 as denoted by SEQ ID NO: 10, DGKa siRNA Sigma Aldrich Cat no. SASI_HSS01_00072301 as denoted by SEQ ID NO: 13, DGK ⁇ siRNA Sigma Aldrich Cat no. SASI_HS02_00324291 as denoted by SEQ ID NO: 14.
  • Non-targeting (non-specific), negative control siRNA duplex sense 5'- UAGCGACUAAACACAUCAA-3', as denoted by SEQ ID NO: 11, anti-sense 5'- UUGAUGUGUUUAGUCGCUA-3' as denoted by SEQ ID NO: 12.
  • RNA isolation was performed based on the Quick-RNA Microprep (Cat no. R1050 & R1051; ZymoResearch) according to the manufacturers’ protocol.
  • the libraries were sequenced in multiplex as single-end, non-strand specific 81 -bp reads on an Illumina NextSeq 550 at the Bar-Ilan University Sequencing Unit, resulting in an average 30-50 million reads per sample.
  • the RNA sequencing data was trimmed using Trim_galore (version 0.5.0) and aligned using STAR (v2.7.3a) (Dobin et al., 2013) with default parameters.
  • the ENSEMBL human hg38 build and its corresponding GTF were used to build the STAR reference database.
  • the number of reads per gene was quantified using HTSeq-Count (v0.6.1pl) (Putri et al, 2022, Bioinformatics 38:2943-2945; Anders et al, 2015, Bioinformatics 31:166-169).
  • the R (v4.1.1) Bioconductor package, DESeq2 (vl.32.0) (Love et al., 2014, Genome Biol 15:550) was used to classify genes as differentially expressed (Benjamini-Hochberg adjusted P value ⁇ 0.05).
  • NOD/SCID IL-2Ry nu11 mice grafted with aggressive, NK-resistant pancreatic cancer cells (PANC-1 - ATCC CRL1469) were used to examine NK cell dysfunction. All experiments were performed in accordance with Bar-Ilan University Ethics rules, under protocols approved by Bar- Ilan University. Mice were maintained under specific-pathogen-free conditions, with a 12-h night/daylight cycle, and at stable ambient temperature with 40-70% relative humidity. Mice were monitored daily and were euthanized upon manifestation of hunched posture, impaired mobility, rough coat, or paralysis. Details on the in vivo conditions and study designs have been included in Table 1. In all the in vivo experiments, a total of three mice were euthanized prior to the endpoint of the experiment and were not included in the results.
  • the average tumor growth rate was calculated according to the formula:
  • Survival Genie open source software was used to perform survival analysis on AML and LGG datasets obtained from TCGA (Dwivedi et al, 2022, Sci Rep 12:1-10).
  • the gene set was defined according to the NK cell signature established by Bottcher et al, - NCRI, NCR3, KLRB1, CD160, and PRF1 (Bottcher et al, 2018, Cell 172:1022-1037.el4) along with the genes - DGKA-EGR2 low and high quartiles.
  • NK cells To characterize the “anergic” NK cell population, the inventors first obtained NK cells from the peripheral blood of healthy individuals and sorted, via flow cytometry, the anergic NK cell subset, lacking self-MHC-I-specific inhibitory receptors (pan KIR and NKG2A) and the responsive NK cell subset, which expresses the complete inhibitory receptor repertoire (Anfossi et al, 2006, Immunity 25:331-342; Fernandez et al, 2005, Blood 105:4416 ⁇ -423) (Fig. 1A). The inventors next confirmed that the isolated NK populations are each functionally anergic or responsive by performing degranulation and tumor lysis assays (Fig. IB, 1C).
  • TRRUST analysis using the Metascape tool was performed on the RNAseq dataset (Fig. IE), revealing the enrichment of several immune effector-related TFs within the anergic subset.
  • NFkB and RELA p65 subunit of NFkB
  • JUN responsible for AP-1 complex formation
  • SATB1 a TF showing functional relevance in immune regulation and tumorigenesis
  • a volcano plot was further generated to help identify significantly upregulated genes with high fold change in the anergic subset.
  • One such gene is CD27 which serves as a maturation marker for the NK cell lineage, with the CD27high NK cell subset exhibiting a distinct tissue distribution and responsiveness to chemokines and interacting productively with dendritic cells (Hayakawa and Smyth, 2006, J Immunol 176:1517-1524).
  • the increased transcript level (TL) of CCR7 in anergic cells was intriguing, as a previous study reported that CCR7 expression in NK cells improves migration toward lymphomas and helps in tumor control (Schomer et al, 2022, Cytotherapy 24:827-834).
  • the obtained results indicate the potential of the anergic NK cell subset in immune regulation (Fig. 2C).
  • the upregulation of KLRC1 (NKG2A) in the responsive NK population supports the positive role of NKG2A in acquiring functional competence, reflecting its significant role in NK cell education and functional responses (Kim et al, 2005, Nature 436:709-713).
  • its expression in responsive cells not only validates the sorting procedure and its efficiency but also confirms that the anergic population is devoid of NKG2A expression.
  • genes associated with NK cell function such as FCGR3A (encoding CD16), NCAM1 (encoding CD56), GZMB (encoding Granzyme B), CD226 (encoding DNAM-1), PLCy (encoding PLC gamma 1/2), B3GAT1 (encoding CD57), and CD 160 (encoding CD 160) in responsive cells indicates the compromised functionality of the anergic NK cells.
  • FCGR3A encoding CD16
  • NCAM1 encoding CD56
  • GZMB encoding Granzyme B
  • CD226 encoding DNAM-1
  • PLCy encoding PLC gamma 1/2
  • B3GAT1 encoding CD57
  • CD 160 encoding CD 160
  • the “anergic” population was previously shown to exhibit diminished effector functions (Ardolino et al, 2014, J Clin Investig 124:4781-4794) with the underlying signaling pathways unknown.
  • the inventors focused on examining the transcript levels (also referred to herein as TLs) of key genes known to contribute to effector functions and key genes playing a major role in the signaling cascades mediating NK cell cytotoxicity (Fig. 2D, 2E).
  • NK cells The dysfunction of the anergic NK population was reflected by the reduced TL of Eomes and Tbx21, which are transcription factors (TFs) known to play a role in the maturation of NK cells (Kiekens et al, 2021, Front Immunol 12:3741), suggesting compromised maturation and reduced ability of the anergic population to control tumor growth or infections.
  • Analysis of the TL of crucial signaling molecules associated with NK cell activation was conducted in anergic and responsive subsets (Fig. 2E); notably, the inventors observed significant reductions in PLCy2 and MAPK TL in anergic cells, collectively implying compromised functionality within the anergic population.
  • Naturally induced “anergic” NK cells display a transcriptional program analogous to that of “exhausted” NK/T cells, exhibiting an overlap of crucial regulators
  • FCGR3A encoding CD 16
  • NCR1 encoding NKp46
  • TL and surface expression of CD 160 an important biomarker for cytokine production in NK cells and early control of tumor growth (Tu et al, 2015, J Exp Med 212:415-429) was found to be reduced in anergic cells along with the TL and surface expression of DNAM-1 (Du et al, 2018, Proc Natl Acad Sci USA 115:E11731-E11740) and 2B4 (Meazza et al, 2017, Eur J Immunol 47:1051-1061), which are vital receptors responsible for NK cell responses (Fuchs and Colonna, 2006, Semin Cancer Biol 16:359-366; Enqvist et al, 2015, J Immunol 194:4518-4527) (Fig.
  • NK cell anergy is characterized by downregulation of key activating surface receptors. Furthermore, it is noteworthy that these anergic NK cells do not express any inhibitory surface markers such as NKG2A or classical KIR’s.
  • TL of PDCD1 the gene encoding PD-1 was found to be significantly elevated within the anergic subset, and TLs of HAVCR2 (encoding Tim-3) and TIGIT (encoding TIGIT) were lower.
  • the inventors determined and confirmed these at the protein level via flow cytometry (Fig. 3C). The data thus suggest that PD-1 could potentially serve as a marker for identifying anergic NK cells.
  • the combined transcriptional and protein data reveal a unique signature of signaling molecules playing a role in NK cell anergy.
  • GSEA gene set enrichment analysis
  • NK cell anergy exhibited enrichment towards the top of the ranked list, including genes obtained from individuals with HBV infections, notably DGKA. This observation elucidates a pool of genes shared between NK cell anergy and NK cells sourced from viral infection (Fig. 3D).
  • the inventors sought to understand whether anergic NK cells share a similar signature with T cells during viral infection and cancer with a focus on identifying key highly enriched genes. To address this, the inventor's dataset was compared to datasets from (West et al, 2011. Immunity 35:285) and revealed transcriptional commonalities of the anergic NK cells with virally exhausted T cells (Fig. 3E), highlighting noteworthy genes including DGKA, EGR2, and NR4A2.
  • anergic NK gene profile overlapped with the canonical “CD8+ T-cell exhaustion” program by obtaining gene sets from Martinez et al (NF AT signature) (Martinez et al, 2015, Immunity 42:265-278) and Scott et al (TOX signature) (Scott et al, 2019, Nature 571:270-274) (Fig. 3F, 3G).
  • anergic NK cells demonstrated a significant upregulation of key genes associated with the T-cell exhaustion program, exhibiting substantial enrichment in prominent genes such as PDCD1, NR4A1, NR4A3, EGR2, NFATcl, and TOX2, aligning with both the NF AT- and TOX-signatures.
  • NFAT2 NFATcl
  • TOX TOX2
  • TOX2 NFATc2
  • DGKA NK and T-cell viral infection
  • EGR2 T-cell viral infection and CD8 T-cell exhaustion
  • TOX2 TOX2 in driving NK cell anergy
  • Egr2 is a zinc finger TF known to have a role in regulating immune cell functions (Wagle et al, 2021, Nat Commun 12:2782; Dai et al, 2020, BMC Immunol 21:1-14).
  • DGKa and DGK ⁇ are enzymes playing a crucial role in lipid signaling and regulation of immune cell function via facilitating the conversion of Diacylglycerol (DAG) to phosphatidic acid (PA) (Zhong et al, 2008, Immunol Rev 224:249- 264).
  • DAG Diacylglycerol
  • PA phosphatidic acid
  • NK cells isolated from tumors exhibit a functional and phenotypic dysfunctional signature similar to naturally existing anergic NK cells.
  • a xenogeneic tumor-grafted mouse model of pancreatic cancer was established.
  • Cells from an aggressive human pancreatic ductal carcinoma (PANC-1) were introduced sub-cutaneously (s.c.) into NOD/SCID IL-2Rynull (NRG) mice.
  • Primary human NK cells obtained from healthy donors were injected intra-tumorally (i.t.) (Fig. 5A).
  • NK cells were retrieved from the tumors when the decrease in tumor size reached a plateau at day 18, signifying the inability of the NK cells to further control tumor growth (Fig. SB).
  • TINK tumor-infiltrating NK cells
  • Fig. 6A, 6B obtained from excised tumors on day 18 exhibited significantly reduced CD107a levels (Figs. SC and 6B, 6C), coupled with drastically upregulated PD-1 expression relative to the naive NK, indicating an “exhausted” phenotype (Fig. 5D).
  • Egr2 and DGKa levels were significantly elevated in the TINK cells relative to the naive NK cells (Figs.
  • Egr2 gene silencing resulted in the restoration of calcium levels in anergic NK cells to levels almost comparable to those in responsive cells (Fig. 7F).
  • PD-1 was significantly elevated in anergic cells. Moreover, effector functions negatively correlate with surface inhibitory receptor expression. Thus, to investigate the impact of EGR2 silencing on the expression of inhibitory checkpoint receptors, the inventors performed EGR2 gene silencing in anergic vs responsive cells. The inventors specifically observed a significant reduction in the expression levels of PD-1 (Fig. 7G) but no differences were observed in Tim-3 and TIGIT expression levels (Figs. 7H, 71 and 8F).
  • NK cell infiltration and function are subjects of key interest in various cancer types including primary glioma, therapy -resistant glioblastoma (Breznik et al, 2022, Commun Biol 5:1-15; Shaim et al, 2021, J Clin Investig 131:el42116; Sedgwick et al, 2020, Front Immunol 11:1549), and myeloid leukemia (Kaweme and Zhou, 2021, Front Immunol 12:2348; Carlsten and Jaras, 2019, Front Immunol 10:2357).
  • NK cell-associated genes NCR 1 , NCR3 , CD 160, PRF1, KLRB1 (hereby referred to as the NK signature) in these cancer datasets (Bottcher et al, 2018, Cell 172:1022-1037.el4; Ni J et al, 2020, Immunity 52:1075-1087.e8).
  • the NK cell signature was established along with DGKA-EGR2high and DGKA-EGR21ow profile of samples in these cancer datasets (Fig. 9A, 9B).
  • the NK-DGKA-EGR2high signature correlated with significantly reduced survival of both leukemia and glioma.
  • the NK cells were highly enriched in the immune cell infiltration profile, strengthening the contention that this signature belongs to the infiltrating NK cells.
  • NK cell activation by targeting EGR2 To determine the potential of NK cell activation by targeting EGR2, the inventors analyzed the cytolysis of tumor cells by anergic NK cells gene silenced for EGR2. Anergic cells (transfected with Egr2 siRNA or NS siRNA) or responsive cells (transfected with NS siRNA) were tested for lysis of 721.221.HLA-Cw7 target cells expressing mCherry using the Incucyte assay. Indeed, the inventors observed poor tumor cell lysis by the anergic cells treated with NS siRNA compared to the responsive population (Fig. 10A) reflected by the decrease in fluorescence intensity from the mCherry target cells.
  • cytotoxicity of the anergic cells was strongly enhanced, almost to the level of cytolysis displayed by the responsive cells (Figs. 9C and 10B). Moreover, at 48 h, tumor lysis was similar in anergic cells treated with Egr2 siRNA encapsulated NP and responsive cells treated with NS siRNA-NP (Fig. 10B). This provides preliminary proof of concept for use of a liposomal drug delivery platform to reprogram anergic and exhausted NK cells in situ to activate their anti-tumor activity.
  • the inventors' current study demonstrates that the naturally ‘anergic’ NK cell population shares similar phenotypic, transcriptional, and functional characteristics with the “exhausted” NK cells. Therefore, the inventors examined the ability of the exhausted NK cells to control tumor growth upon EGR2 gene silencing utilizing the NRG xenograft PDAC model (Fig. 5). Mice bearing PANC-1 tumors were injected i.t. with pNK when the tumors reached 250-300 mm3. Previously, the inventors observed upregulation of DGKa, Egr2, and PD-1 and reduced degranulation by day 5 following pNK administration (Fig. 5B-5F).
  • the results disclosed herein show that elevated expression of DGKa and EGR2 correlates with reduced patient survival.
  • the inventors further demonstrate that a liposomal nanoparticle-based siRNA delivery system effectively reprogrammed anergic NK cells within a 3D organotypic spheroid leukemia model.
  • EGR2 gene silencing enhanced the anti-tumor potency of exhausted NK cells, leading to augmented tumor control, reduced tumor growth, decreased PD-1 expression, and the restoration of the cytotoxic potential of exhausted NK cells.
  • the present disclosure unravels the potential molecular circuitry of anergic, and exhausted NK cells and demonstrates Egr2-DGKa-SHP-l- PLCyl/2-MAPK as a critical signaling axis in NK cell dysfunction (as illustrated in Fig. 9J).

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  • Animal Husbandry (AREA)
  • Virology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
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Abstract

La présente divulgation concerne un modulateur et/ou un reprogrammateur pour activer des cellules tueuses naturelles (NK) dysfonctionnelles, et des méthodes thérapeutiques personnalisées et des utilisations associées. L'agent de reprogrammation décrit comprend un composé qui inhibe spécifiquement l'expression, l'activité et/ou la stabilité d'au moins un élément de la famille EGR et/ou DGK.
PCT/IL2024/051199 2023-12-18 2024-12-18 Cellules tueuses naturelles dysfonctionnelles reprogrammées et utilisations associées Pending WO2025134119A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018030874A1 (fr) * 2016-08-12 2018-02-15 주식회사 툴젠 Élément immunorégulateur manipulé et immunité ainsi modifiée

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018030874A1 (fr) * 2016-08-12 2018-02-15 주식회사 툴젠 Élément immunorégulateur manipulé et immunité ainsi modifiée

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIBER GUY, SABAG BATEL, RAIFF ANAT, BEN€SHMUEL AVIAD, PUTHENVEETIL ABHISHEK, BENICHOU JENNIFER I C, JUBANY TAMMIR, LEVY MORIA, K: "Modulation of intrinsic inhibitory checkpoints using nano‐carriers to unleash NK cell activity", EMBO MOLECULAR MEDICINE, WILEY-BLACKWELL, US, vol. 14, no. 1, 11 January 2022 (2022-01-11), US , XP093325992, ISSN: 1757-4676, DOI: 10.15252/emmm.202114073 *
YANG ENJUN, SINGH BRENAL K, PAUSTIAN AMANDA M SCHMIDT, KAMBAYASHI TAKU: "Diacylglycerol Kinase ζ Is a Target To Enhance NK Cell Function", THE JOURNAL OF IMMUNOLOGY, AMERICAN ASSOCIATION OF IMMUNOLOGISTS, vol. 197, no. 3, 1 August 2016 (2016-08-01), pages 934 - 941, XP093325987, ISSN: 0022-1767, DOI: 10.4049/jimmunol.1600581 *

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