Dendritic cells-targeted nanoparticles for t cell activation and cancer immunotherapy

Abstract

The present disclosure generally relates to the preparation of biocompatible nanoparticles aimed to activate dendritic cells, in particular dendritic cells associated with tumors, and render them more prone to effective recognition by the immune system, by inhibition of immune checkpoint inhibitors. The present disclosure further relates to methods for preparing such nanoparticles and to use thereof in the treatment of diseases, in particular cancer.

Description

[0001] DENDRITIC CELLS-TARGETED NANOPARTICLES FOR T CELL ACTIVATION AND CANCER IMMUNOTHERAPY

[0002] FIELD OF THE INVENTION

[0003] The present disclosure generally relates to the preparation of biocompatible nanoparticles-based delivery systems that activate the immune cell function, in particular against cancer cells.

[0004] BACKGROUND OF THE INVENTION

[0005] Programmed death-ligand 1 / programmed cell death 1 (PD-L1 / PD-1) checkpoint inhibition is a promising strategy for cancer immunotherapy. PD-L1 was found to be upregulated on tumor cells (for example, melanoma, ovarian and lung cancers) and immune cells (such as dendritic cells, DCs) and its engagement with PD-1 on T cells results in reduction of T cell activity and immune surveillance escape. Antibodies directed against both PD-1 and PD-L1 have already been approved for clinical use, for example neutralizing antibodies that bind to PD-L1 and prevent the interaction between PD-1 and PD-L1 in order to restore antitumor T-cell functions (Bejarano, L. el al., Cancer Discovery 2021, 11 (4), 933-959). However, this line of cancer immunotherapy is associated with various limitations, such as induction of destructive immune-related adverse effects and insufficient penetration to the tumor site.

[0006] DCs are considered the most potent antigen presenting cells, capable of effectively stimulating a robust immune response against cancer. DCs capture a tumor antigen, process it, and present it to T cells to start adaptive immunity processes. However, it was previously shown that during antigen presentation, tumor-infiltrating DCs exhibit high levels of PD-L1, which inhibits T cell activity and dampens the immunological response.

[0007] It was previously reported that PD-L1 binds two receptors, PD-1 and CD80 (B7.1). Studies have shown that in cancer patients, PD-L1 is expressed more abundantly than CD80 on tumor associated DCs. It was also reported that when PD-L1 binds to CD80 (B7.1) on the same cell (in cis), it hampers the CD80 / CD28 co- stimulatory interaction and T cell priming. Furthermore, a recent study in cancer patients discovered that the anti- PD-L1 antibody atezolizumab, which inhibits both PD-1 / PD-L1 and PD-L1:CD80 interactions, improves DC function and triggers a potent antitumor T cell immunity (Mayoux, M. et al., Science Translational Medicine, 2020, 12 (546), 1-12). Blocking of the PD-1 / PD-L1 interaction was also shown by short D-peptide antagonists, e.g., an all D peptide having the amino acid sequence of NYSKPTDRQYHF (Chang, H. N. et al., Angew. Chem. Int. Ed. 2015, 54 (40), 11760 -11764).

[0008] In addition, a polymer functionalized with peptide antagonists of PD-L1 was designed, and was tested in combination with polymer-epirubicin conjugates which elicit immunogenic cell death (Li, L. et al., Adv. Funct. Mater. 2020, 1908961). Recently, clotrimazole was reported to promote DC-mediated antigen presentation and enhance T cell response (Wang, Z. et al., Journal for Immunotherapy of Cancer 2021, 9 (5)). Wang, Z. et al. has also shown that clotrimazole induced intra-tumor immune infiltration and inhibited tumor growth depending on both DCs and CD8+ T cells and potentiated the antitumor efficacy of anti-PDl antibody in a mice model.

[0009] The combination of immune check point blockade and immunogenic cell death (ICD) induction has been reported, among others, by Jia S. et al. (CCS Chem. 2022, 4, 501-514) relating to an aggregation-induced emission (AIE) sonosensitizer-based nano- system that produces reactive oxygen species under an external trigger to evoke ICD of tumor cells with surface modification of anti-PD-L1 peptide. Specifically, the peptide disclosed is an all D short PD-L1 peptide antagonist (also termed PD-L1 peptide antagonist,DPPA, having the amino acid sequence CNYSKPTDRQYHF) that can effectively block the PD-1 / PD-L1 pathway similar to the peptide mentioned above. Further nanoparticle carriers combining PD-L1 blockade with immunogenic cell death were reported by Moon, Y. et al. (Theranostics 2022, Vol. 12, Issue 5, 1999-2014) relating to nanoparticles comprising an anti-PD-L1 peptide having the amino acid sequence of CVRARTR and cathepsin B-specific cleavable peptide conjugated to doxorubicin.

[0010] SUMMARY OF THE INVENTION

[0011] The present disclosure is based on the design and preparation of biocompatible mesoporous silica nanoparticles (MSNPs) aimed to activate dendritic cells, in particular dendritic cells associated with tumors or in the tumor microenvironment (TME), and render them more prone to effective recognition by the immune system, specifically, T cells.

[0012] The nanoparticles of the present disclosure combine three functions, collectively targeting and activating DCs: they are decorated with mannose on their exterior surface in order to specifically target mannose receptors (also termed CD206) on DCs and for reducing off-target adverse effects. In addition, they are loaded with clotrimazole (CLT) that is embedded within the MSNPs and released upon internalization into the DCs or in the vicinity thereof and is intended for enhancing antigen presentation of DCs by upregulating major histocompatibility complexes (MHC) class II molecules. Furthermore, the nanoparticles are surface-functionalized with a PD-L1 binding peptide in order to block PD-L1 -mediated interactions, specifically, PDL1 / CD80 cis interactions on DCs as well as PD-L1 / PD-1, thereby enhancing CD80 / CD28 interactions between DCs and T cells. PDL1bp was conjugated to the mannosylated nanoparticles (MSNPs) via glutathione (GSH)- and hydrogen peroxide (H2O2)-cleavable linkers allowing the release of the peptide at the TME.

[0013] As shown in the Examples below, the functionalized MSNPs boosted DC-T cell interaction and promoted CD28-mediated T cell co-stimulation, as well as CLT-mediated antigen presentation. In vivo investigation revealed that administration of the CLT-loaded and PD-L1bp-functionalized MSNPs remarkably inhibited subcutaneous B16-F10 melanoma tumor growth when compared to the effect of an anti-PD-L1 (antibody) therapy.

[0014] In other words, the present disclosure provides a strategy for activating DCs and subsequently T cells at the TME with DC-targeted MSNPs, carrying at least one dendritic cell activator (e.g., CLT). Furthermore, when decorated with at least one immune checkpoint inhibitor (e.g., at least one peptide that binds to PD-L1, also termed herein PD-L1 binding peptide or PDL1bp), the MSNPs inhibited PD-L1 / CD80 cis interactions on DCs and released CD80 to co-stimulate T cell responses. As exemplified below, PDL1bp was cleaved from the surface of the MSNPs under conditions simulating the TME and the unmasked mannosylated MSNPs were taken-up rapidly by DCs and boosted their antigen presentation to T cells. Without wishing to be bound by theory, the functionalized delivery system simultaneously modulates three signaling pathways, namely, PD-1 / PD-L1, CD80 / CD28 and MHC II / T cell receptor, as a results of employing both PDL1bp- and CLT-mediated DC activation and increases T cell stimulation. The peptide decorated and CLT-loaded MSNPs promoted DC activity and enhanced antitumor efficacy when compared to anti- PD-L1 (i.e., antibody) therapy in mice with melanoma tumors. The above combination of features forms a basis for a therapy based on T cell stimulation (e.g., for treatment of melanoma), offering some solutions for overcoming the limitations of insufficient immune activation by conventional immunotherapies.

[0015] Furthermore, using nanoparticulate system (at the size of -250 nm) for targeting DCs is advantageous, since the nanoparticle size affects the ability of nanoparticles to drain to the lymph nodes and induce an adaptive immune response as shown by others. Due to their size the NPs (e.g., a diameter in the order of approximately 200-300 nm) can enhance cellular uptake by lymph-node-resident DCs and promote antigen presentation within hours of injection, while ensuring that the CLT payload reaches the target DC and reduce the bio distribution of the drug to undesired organs and tissues. All of these characteristics can potentially lead to faster T cell activation by DCs.

[0016] Therefore, by a first aspect thereof the present disclosure provides a nanoparticle- based delivery system comprising at least one dendritic cell activator, at least one immune checkpoint inhibitor and at least one ligand of a dendritic cell receptor.

[0017] The dendritic cell activator as herein defined may be embedded within the nanoparticle or coupled to the nanoparticle by at least one cleavable linker.

[0018] In some embodiments, the nanoparticle as herein defined is a hollow or porous nanoparticle, and the dendritic cell activator is embedded within the nanoparticle.

[0019] In other embodiments, the at least one dendritic cell activator is attached to the nanoparticles as herein defined by a cleavable linker sensitive to cleavage in the tumor microenvironment. In certain embodiments, the exterior surface of the nanoparticle is coated with at least one moiety modulating the release of the at least one dendritic cell activator. The moiety modulating the release of the dendritic cell activator may be coupled to the exterior surface of the nanoparticle as herein defined via at least one cleavable linker sensitive to cleavage in the tumor microenvironment, preferably an acid-labile linker, an enzyme-cleavable linker, a reduction sensitive linker or a reactive oxygen spices (ROS)- sensitive linker. In some embodiments, the moiety modulating the release of said at least one dendritic cell activator is a polypeptide (e.g., albumin, such as bovine serum albumin (BSA) or human serum albumin (HSA)), or a copolymer, preferably a pH sensitive copolymer. For example,

[0020] In certain further embodiments, the at least one dendritic cell activator suitable for the present disclosure is an agent enriching lysosomal pathway-associated genes, an inflammatory cytokine, a chemokine or an oligonucleotide. For example, the dendritic cell activator may be clotrimazole.

[0021] The immune checkpoint inhibitor suitable for the present disclosure is directed for example against any one of PD-1 / PD-L1, TIM-3 / Ceacam-l / Galectin-9, LAG-3 / Galectin- 3 / LSECtin / MHC-II, CTLA-4 / CD80(B7.1) / CD86(B7.2) or DIGIT / CD 155 / CD 112. In specific embodiments, the at least one immune checkpoint inhibitor as herein defined is directed against PD-L1 or PD-1, and in further specific embodiments, against PD-L1.

[0022] The present disclosure encompasses an immune checkpoint inhibitor which comprises a peptide, a polypeptide (in particular, a polypeptide which is not an antibody), an oligonucleotide, a polynucleotide, or a small molecule inhibitor. In specific embodiments, the nanoparticle-based delivery system according to the present disclosure comprises at least one immune checkpoint inhibitor which is not an antibody.

[0023] In some embodiments, the at least one immune checkpoint inhibitor as herein defined comprises a PD-L1 binding peptide, for example, a PD-L1 binding peptide which comprises an amino acid sequence which is at least 80% identical to the amino acid sequence Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe, denoted by SEQ ID NO. 1 or to the amino acid sequence Cys-Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His- Phe, denoted by SEQ ID NO. 2. In other embodiments, the PD-L1 binding peptide comprises or consists of the amino acid sequence Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg- Gln-Tyr-His-Phe, denoted by SEQ ID NO. 1 or the sequence Cys-Asn-Tyr-Ser-Lys-Pro- Thr-Asp-Arg-Gln-Tyr-His-Phe, denoted by SEQ ID NO. 2. Still further, the PD-L1 binding peptide suitable for the present disclosure comprises or consists of an amino acid sequence which is an all D amino acid configuration of the amino acid sequence Asn- Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe denoted by SEQ ID NO. 1, or an amino acid sequence which is an all D amino acid configuration of the amino acid sequence Cys-Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe, denoted by SEQ ID NO. 2.

[0024] The at least one immune checkpoint inhibitor as herein defined may be coupled to the exterior surface of said functionalized nanoparticle, optionally by a cleavable linker.

[0025] In further embodiments, the at least one ligand of a dendritic cell receptor is coupled to the exterior surface of the nanoparticle delivery system as herein defined, optionally by a cleavable linker. In other embodiments, the immune checkpoint inhibitor as herein defined is coupled to the exterior surface of the nanoparticle delivery system indirectly, via a cleavable linker attached to the at least one ligand of a dendritic cell receptor that is coupled to the exterior surface of said nanoparticle delivery system.

[0026] By way of example, the dendritic cell receptor suitable for the present disclosure is a mannose receptor or CD11c, in particular a mannose receptor.

[0027] A suitable ligand of a dendritic cell receptor is a mannose moiety or a peptide ligand of the DC receptor CD 11c, the peptide ligand having the amino acid sequence FYPSYHSTPQRP denoted by SEQ ID NO. 11. In particular, a suitable ligand of a dendritic cell receptor is a mannose moiety.

[0028] In certain embodiments, nanoparticles for preparing the delivery system as herein defined are selected from metal nanoparticles, silica nanoparticles (e.g., mesoporous silica nanoparticles), polymer-based nanoparticles (such as, polylactic-co-glycolic acid (PLGA) or polycaprolactone (PCL), polymeric micelles (such as PEGylated polymers), polymer conjugates (such as polymer-drug conjugates, polymer-ligand conjugate) or lipid-based nanoparticles (such as liposomes, stealth liposomes, vesicles or solid lipid nanoparticles), or a combination thereof. In particular, the nanoparticle is a silica nanoparticle, for example, a mesoporous silica nanoparticle (MSNP).

[0029] The present disclosure encompasses nanoparticles which have a mean particle size of between about 20 nm to about 1000, between about 20 to 800 nm in diameter, preferably between about 50 to 400 nm in diameter, more preferably wherein the nanoparticles have a mean particle size of about 80 to 300 nm in diameter.

[0030] By way of example, the present invention provides a nanoparticle-based delivery system as herein defined, wherein the nanoparticles are porous or hollow nanoparticles, said at least one immune checkpoint inhibitor is a PD-L1 binding peptide (e.g., comprising the amino acid sequence denoted by SEQ ID NO. 1 or SEQ ID NO. 2 or an amino acid sequence which is an all D amino acid configuration of the amino acid sequence denoted by SEQ ID NO. 1 or SEQ ID NO. 2), coupled to the exterior surface of said nanoparticles via a cleavable linker, wherein said at least one dendritic cell activator is clotrimazole, embedded within said nanoparticles, and wherein said exterior surface of said nanoparticles is decorated with a mannose moiety.

[0031] Clotrimazole may be loaded in an amount of about 2% to 20% by weight of the weight of the nanoparticles.

[0032] In some embodiment, the nanoparticle-based delivery system of the present disclosure further comprises an imaging group.

[0033] The present disclosure further provides a pharmaceutical composition comprising the nanoparticle-based delivery system as herein defined and at least one carrier and / or excipient.

[0034] By a still further aspect thereof, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of the nanoparticle-based delivery system or the pharmaceutical composition according as herein defined. For example, the disease or disorder encompassed by the present invention is a proliferative disease or disorder or an immune -related disease or disorder. In specific embodiments, the present disclosure provides means and methods suitable for use in treating cancer, e.g., melanoma, lung cancer (both non-small cell lung cancer (NSCLC) and small cell lung cancer), kidney cancer, bladder cancer or head and neck squamous cell carcinoma (HNSCC).

[0035] The present disclosure further provides a method of immunomodulation in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of the nanoparticle-based delivery system or the pharmaceutical composition as herein defined. In specific embodiments, the method of immunomodulation of the present disclosure is for at least one of dendritic cell activation and T cell activation.

[0036] By a further aspect thereof the present invention provides the nanoparticle -based delivery system or the pharmaceutical composition as herein defined for use in a method of treating a disease or disorder in a subject in need thereof. By still a further aspect thereof the present invention provides the nanoparticle-based delivery system or the pharmaceutical composition as herein defined for use in a method of immunomodulation in a subject in need thereof.

[0037] The present invention further provides a method for preparing the nanoparticle- based delivery system of the invention, said method comprising at least one of the following steps: a) providing organic or inorganic nanoparticles, b) encapsulating within the nanoparticles or attaching thereto at least one dendritic cell activator optionally using at least one cleavable linker, c) optionally coating the exterior surface of the nanoparticles with at least one moiety modulating the release of said at least one dendritic cell activator, d) attaching to said nanoparticles at least one immune checkpoint inhibitor, and at least one ligand of a dendritic cell receptor to the exterior surface of the nanoparticles, optionally using at least one cleavable linker. In certain embodiments of the method of preparation as herein defined, the nanoparticles are porous or hollow nanoparticles and the at least one dendritic cell activator is encapsulated therein, and the method further comprises coating the exterior surface of said nanoparticles with at least one moiety modulating the release of said at least one dendritic cell activator. In other specific embodiments of the method of preparation as herein defined, the immune checkpoint inhibitor, dendritic cell activator, and ligand of a dendritic cell receptor are attached to the surface of the nanoparticle through a cleavable linker.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0040] Fig. 1A - Fig. 1H: Physicochemical characteristics of functionalized MSNPs. Fig. 1A: TEM image shows nearly spherical morphology of MSNPs with mean average size of 50-80nm. Fig. IB: FTIR analysis of MSNPs functionalized with amine (N-H groups at 1520cm-1), amide (C-N bond stretching at 1365cm-1), carbonyl group (C-O- bond at 1427cm-1), and mercapto groups (S-H at 2625cm-1). Fig. 1C: UV absorption spectra of CLT-loaded MSNPs showing a peak of CLT absorbance at 254nm. Fig. ID: BSA conjugation to MSNP-NH2 reduces the BET calculated surface area from 463.37m2 / g to 58.467m2 / g. Fig. IE: MSNP-NH2 had a mean pore size of 3.2nm, as determined by BJH, while BSA-capped MSNPs had no detectable pore size. Fig. IF: TGA analysis confirms the surface modifications of MSNPs, weight loss between 200-400°C indicates the presence of peptide bonds, and weight loss between 450-550°C indicates the degradation of carbohydrates. Fig. 1G: Particle sizes measured by DLS and Fig. 1H: Zeta Potential analysis confirm successful functionalization of MSNPs after each step of modification.

[0041] Fig. 2: FTIR spectrum of the MSNP-MaN-BA peak around 2635 cm-1representing -SH group.

[0042] Fig. 3A - Fig. 3C: Schematic presentation of the synthesis of functionalized MSNPs and predicted mechanism of action. Fig. 3A: schematic presentation of the functionalization of MSNPs to create the CLT loaded, mannosylated and BSA-capped MSNPs, and further decorated with PDL1bp. The indicated step (1) is surface grafting of amine groups on silica NPs, followed by the indicated step (2) of CLT loading and the indicated step (3) of BSA capping. Conjugation of a mannose moiety (MaN) is performed at the indicated step (4) to BSA by amidation to give MSNP-BSA-MaN / CLT, which further caged with 4-mercaptophenyl boronic acid (4-MBA) at the indicated step (5) and linked to the peptide PDL1bp via disulfide bonds formation at the indicated step (6) to give the final functionalized nanoparticle MSNP-MaN-PDL1bp / CLT. Fig. 3B: schematic presentation of the proposed mechanism for PDL1bp cleavage at the tumor microenvironment (TME). Fig. 3C) schematic presentation of a suggested mechanism to improve adaptive immunity by MSNP-MaN-PDL1bp / CLT, combining both co-stimulatory signals to stimulate T cells.

[0043] Fig. 4A - Fig. 4B: In vitro release profiles of PDL1bp and CLT from functionalized MSNPs. Fig. 4A: PDL1bp release profile in PBS at the presence or absence of 10 mMH2O2or 10 mM DTT, as determined by Fluorescamine assay. Fig. 4B: CLT release profile with or without Trypsin (2.5%), as determined by HPLC.

[0044] Fig. 5 : A bar diagram of an XTT proliferation assay when DC2.4 cells (1x104) were incubated with formulations of various functionalized MSNPs at various concentrations for 24 hours.

[0045] Fig. 6A - Fig. 61: FITC fluorescent images when the DC2.4 cells (1X105) were incubated with 20 pg of formulations of various functionalized nanoparticles in presence (indicated as "+") or absence (indicated as "-") of LPS / IFNy (250 ng / mL). Fig. 6A: Control. Fig. 6B: MSNP-BSA / FITC (-LPS / IFNγ). Fig. 6C: MSNP-MaN / FITC (-LPS / IFNy). Fig. 6D: MSNP-BSA / FITC (+LPS / IFNy). Fig. 6E: MSNP-MaN / FITC (+LPS / IFNy). Fig. 6F: MSNP-MaN-PDL1bp / FITC (-LPS / IFNy). Fig. 6G: MSNP-MaN-PDL1bp / FITC (+LPS / IFNy). Fig. 6H: median intensity plot for the various formulations. Fig 61: graph representing proportion of the MSNP within the DC2.4 cells. Fig. 7A - Fig. 7B: Represents immature DC2.4 cells (Fig. 7A) and mature DC2.4 cells (Fig. 7B) when activated using IFNy & LPS.

[0046] Fig. 8A - Fig. 8B: PDL1 (Fig. 8A) and CD80 (Fig. 8B) expression levels on iDC2.4 and mDC2.4 cells.

[0047] Fig. 9A - Fig. 9F: Co-localization of PD-L1 and CD80 on DCs and MSNP -binding to PD-L1. Confocal microscopy images of iDC2.4 (Fig. 9A) and mDC2.4 (Fig. 9B) respectively, stained for PD-L1 (green) and CD80 (red), scale bar representing 10 pm. Intensity graphs show the fluorescence intensity of PD-L1 and CD80 over a specified distance marked in the image. Fig. 9C and Fig. 9D show the binding affinity of MSNPs with and without PDL1bp decoration to iDC2.4 (Fig. 9C) and mDC2.4 (Fig. 9D). Fig. 9E and Fig. 9F show mean fluorescence intensity (MFI) of labeled cells is quantified for iDC2.4 (Fig. 9E) and mDC2.4 (Fig. 9F). (n=3) ***p<0.001.

[0048] Fig. 10A - Fig. 10B: Fluorescent images (Fig. 10A) and Pearson correlation coefficient (Fig. 10B) for iDC2.4 and mDC2.4, scale bar represents 5pm.

[0049] Fig. 11A - Fig. 11C: PDL1bp-decorated MSNPs disrupt PD-L1 / CD80 interactions and enhance CD28 recognition. Fig. 11A is a schematic presentation of the suggested mechanism for CD80 release and recombinant CD28 (rCD28) binding by MSNPs. Fig. 11B and Fig. 11C show binding of fluorescently labeled recombinant CD28 (rCD28) to iDC2.4 cells and mDC2.4 cells that were pre-treated with free PDL1bp (25 μg / mL) or peptide-equivalent MSNP formulations, where Fig. 11B shows flow cytometry dot plots and Fig. 11C shows MFI of cells after incubation with 1 μg / mL fluorescently labeled rCD28., (n=3) *p<0.05, **p<0.01, ***p<0.001, ns not significant.

[0050] Fig. 12: shows the molecular interaction of rCD28 with the CD80 pattern with and without activated DC2.4 treated with anti-PDLl Ab. Upper panels: SSC pattern of DC2.4 cell suspension. Numbers indicate percentage of the total population, as determined by flow cytometry. Lower panel : mean fluorescence intensity (MFI) of unstained and stained cells. Fig. 13A - Fig. 13B: shows fluorescently labeled recombinant CD28 (rCD28) binding to iDC2.4 and mDC2.4 cells treated with either free PD-L1 or MSNP formulations. Dot plots for flow cytometry (Fig. 13A) and MFI for the cells post treatment (Fig. 13B).

[0051] Fig. 14A - Fig. 14B: FACS staining experiments showing CD28 expression level on unstained cells (Fig. 14A) and on approximately 92% cells of the EL4 T cells, as confirmed by anti-CD28 staining (Fig. 14B).

[0052] Fig. 15A - Fig. 15E: PDL1bp-decorated MSNPs promote DC and T cell interactions via the CD80 / CD28 co- stimulatory pathway and stimulate T cell activation. Fig. 15A shows a scheme of the suggested cellular interactions following treatment with MSNP-BSA- MaN-PDL1bp. Fig. 15B shows confocal images of molecular interactions between mDC2.4 cells (CD80+, red) and EL4 cells (CD28+, green) after indicated treatments with 5μg / mL free PDL1bp or peptide-equivalent MSNP doses. Fig. 15C shows fluorescent images captured using Zeiss Celldiscoverer7 to visualize the molecular interactions between mDC2.4 cells (CD80+, orange) and EL4 cells (CD28+, green) after indicated treatments; each image contains approximately 45 frames that were merged into one large scale image of a cell culture well. Fig. 15D is a graph showing quantification of the number of contacts between DC2.4 and EL4 within the merged images for the indicated treatments (n=3) ***p<0.001. Fig. 15E is a bar graph showing IL-2 secretion level of EL4 cells following co-culture with pre-treated mDC2.4 (n=3) ***p<0.001.

[0053] Fig. 16A - Fig. 16F : CLT-loaded MSNPs enhance DC activation, antigen presentation and subsequent T cell priming. Fig. 16A is a scheme for the proposed mechanism of CLT- mediated HEL antigen presentation on DC and subsequent T cell activation. Fig. 16B - Fig. 16D are bar graphs showing MFI after antibody staining of iDC2.4 for MHC II (Fig. 16B), CDl lc (Fig. 16C) and CD80 (Fig. 16D) following treatments with MSNP- MaN / CLT, MSNP-MaN and free CLT at drug-equivalent doses of 27.5μM CLT. Fig. 16E shows confocal images of fluorescently labelled HEL antigen (5(6)- carboxyfluorescein, green) uptake into iDC2.4 cells (LysoTracker™ Red DND-99, red) after control, MSNP-MaN, Free CLT or MSNP- MaN / CLT treatment. Fig. 16F is a bar graph showing IL-2 secretion by 3A9 cells, post co-culture with DC2.4 that were pre- treated with CLT or MSNP formulations at drug-equivalent doses, and HEL antigen. (n=3) *p<0.05, **p<0.01, ***p<0.001 ns not significant.

[0054] Fig. 17A - Fig. 17C: represent the expression level of MHCII (Fig. 17A), CDl lc (Fig. 17B) and CD80 (Fig. 17C) on DC2.4 cells following treatments with MSN-MaN / CLT, MSNP-MaN and free CTL.

[0055] Fig. 18: Manders colocalization coefficient (MCC) for DC2.4 cells which were pre- treated with MSNP formulations then incubated with labeled HEL for 2 hours.

[0056] Fig. 19: Clotrimazole induces the DC activation through increasing the lysosomal concentration. DC2.4 cells were treated with free CTL, MSNP-MaN / CTL, MSNP-MaN or control for 8 hours, then LysoTracker™ Red DND-99 was added into the medium for another 30 minutes. Confocal microscopy was used to detect the fluorescence signal of Lysosome.

[0057] Fig. 20: a bar graph showing IL-2 secretion by 3 A9 cells, after co-culture with pre-treated DC2.4 cells with free CLT, free PDL1bp or MSNP formulations at drug- and peptide- equivalent doses in the presence of HEL antigen. (n=3) **p<0.01, ***p<0.001 *p<0.05, ns not significant.

[0058] Fig. 21A - Fig. 21F: CLT-loaded, PDL1bp-decorated MSNPs inhibit B16-F10 melanoma growth and increase T activity at the TME. Fig. 21A shows a scheme of experimental setup of s.c. injection of B16-F10 cells and treatment with MSNPs at CLT- and peptide-equivalent doses or control anti-PD-L1 treatment. Fig. 21B shows a graphical presentation of the change in tumor volume of subcutaneous B 16-F10 tumors after MSNP or control treatment (n=8-9). Fig. 21C is a bar graph of tumor weight of excised subcutaneous tumors on day 16 after cancer cell inoculation (n=8-9). Fig. 21D - Fig. 21F are a bar graph of mRNA levels of TNFa (Fig. 21D), IFNγ (Fig. 21E) and Granzyme B (Fig. 21F) in the tissue of tumors excised on day 16 after cancer cell inoculation, as determined by qRT-PCR, relative to saline-treated control (n=6-7). *p<0.05, **p<0.01,

[0059] ***p<0.001. Fig. 22: graphic presentation of the changes in the average body weight (g) of tumor- bearing mice in saline control and different treated groups (n=8-9).

[0060] Fig. 23A - Fig. 23B: Scheme of synthesis of (Fig. 23A) PEG-b-PCL-IR806, (Fig. 23B) PCL-b-PEG-PD-L1 bp di-block copolymers, used for the preparation of polymeric micelles (PM).

[0061] Fig. 24A - Fig. 24D: Graphs showing particle size of the different weight ratio mixed polymeric micelle as measured by dynamic light scattering (Fig. 24A), a bar graph showing zeta potential values of PD-L1bp and micelles (Fig. 24B), TEM images of PEG- b-PCL-IR806:PCL-b-PEG-PDL1bp (70:30) micelle (Fig. 24C) and TEM images of PEG-b-PCL:PCL-b-PEG-PD-L1bp (70:30) (without IR806) (Fig. 24D).

[0062] Fig. 25: A bar graph shows the molecular interaction of rCD28 with CD80 on DC2.4 following peptide (PDL1bp) and micelles treatment (PEG-b-PCL:PCL-b-PEG-PD-L1bp (70:30)), with or without LPS+IFNy activation. Each value represents the mean ± S.E. of three experiments, performed in duplicate. ***p<0.001, ****p<0.0001, ns-not significant.

[0063] Fig. 26A - Fig. 26D: Confocal images of molecular interactions between stimulated DC2.4 cells (CD80, red) and EL4 cells (CD28, green) of untreated (Fig. 26A), polymeric micelles -treated (Fig. 26B), PD-L1bp-treated (Fig. 26C) and PD-L1bp-PM (Fig. 26D).

[0064] Fig. 27A - Fig. 27C: Bar graphs showing PD-L1bp and PM binding to DC and cancer cells. The binding of PD-L1bp and PD-L1bp-PM to activated DC2.4 cells (Fig. 27A), B16-F10 melanoma cells (Fig. 27B) and a bar graph showing rCD28 binding to DC2.4 cells following peptide or PM treatment (Fig. 27C). The MFI of DC2.4 or B16-F10 cells was significantly increased following PD-L1 -decorated PM (70:30) (peptide micelles) treatment relative to non-stimulated cells or blank micelles (without PD-L1bp) (a&b). PD-llbp and PM-treatment promotes rCD28 binding to DCs. Each value represents the mean ± S.E. of three experiments, performed in duplicate. *p<0.05, **p<0.01, ***p<0.001, ns-not significant. DETAILED DESCRIPTION OF THE INVENTION

[0065] The present disclosure relates to a delivery system, specifically, a nanoparticle- based delivery system that targets dendritic cells (DCs) and is capable of enhancing the activity thereof by carrying at least one dendritic cell activator and at least one immune checkpoint inhibitor. By a first aspect thereof the present disclosure provides a nanoparticle-based delivery system comprising at least one dendritic cell activator, at least one immune checkpoint inhibitor and at least one ligand of a dendritic cell receptor.

[0066] As known in the art, dendritic cells (DCs) are specialized antigen-presenting cells (APCs). Upon antigen uptake, activated DCs process and present self and non-self antigens to naive T lymphocytes, priming antigen-specific immune responses and regulating both tolerance and immunity. DCs are considered central components of the tumor microenvironment (TME) and can promote antitumor T-cell responses, e.g., by promoting the presentation of tumor-associated antigens.

[0067] DCs may be activated directly by conserved pathogen molecules and indirectly by inflammatory mediators produced by other cell types that recognize such molecules. By the term “dendritic cell activator” as used herein it is referred to any agent, drug or small molecule known in the art that induces DC activation, or a combination thereof. By way of example, dendritic cell activators encompassed by the present disclosure act by enriching lysosomal pathway-associated genes, which have been previously identified as an essential route for antigen processing and MHC II presentation in DCs. Other suitable dendritic cell activators are inflammatory cytokines (e.g., tumor necrosis factor (TNF)), chemokines (CCL2) or oligonucleotides (e.g., DNA, RNA). Therefore, in specific embodiments, the at least one dendritic cell activator is an agent enriching lysosomal pathway-associated genes, an inflammatory cytokine, a chemokine or an oligonucleotide.

[0068] As shown in the Examples, the antifungal agent clotrimazole (CLT) was loaded onto the nanoparticle delivery system to stimulate DCs. Without wishing to be bound by theory, clotrimazole acts by enriching lysosomal pathway-associated genes. Thus in specific embodiments, the at least one dendritic cell activator of the present disclosure acts by enriching lysosomal pathway-associated genes. Specifically, the at least one dendritic cell activator according to the present disclosure may be clotrimazole (CAS No. 23593-75-1, C22H17CIN2) which is an imidazole antifungal agent, and is commercially available.

[0069] The at least one dendritic cell activator may be carried by the nanoparticle-based delivery system by any means known in the art, for example, non-covalently, e.g., by being embedded or encapsulated within the nanoparticles (such as within pores or hollow spaces therein) or by a cleavable linker, allowing the release of the dendritic cell activator within DCs or in the vicinity thereof. In other words, the at least one dendritic cell activator of the present disclosure is embedded (or encapsulated) within the nanoparticle or coupled to said nanoparticle by a cleavable linker, e.g., a cleavable linker which is a linker sensitive to cleavage in the TME.

[0070] The exemplified drug delivery system was based on biocompatible mesoporous silica nanoparticles (MSNPs) and clotrimazole was encapsulated in the pores thereof. Therefore, in some cases, the nanoparticle-based delivery system of the present disclosure is wherein the nanoparticle is a hollow or porous nanoparticle (e.g., a mesoporous nanoparticle, for example MSNP, a micelle, a vesicle or a liposome) and wherein said at least one dendritic cell activator is embedded within the nanoparticle (i.e., non- covalently).

[0071] Alternatively, the at least one dendritic cell activator as herein defined may be coupled (i.e., chemically attached or conjugated) for example to the exterior surface of the nanoparticles as herein defined by a cleavable linker, preferably a linker that detaches (is cleaved) under conditions typical to the TME.

[0072] During preparation of the delivery system exemplified herein, after loading CLT into the pores in the mesoporous nanoparticles, the pores were capped (coated) with bovine serum albumin (BSA) using carbodiimide chemistry, thereby protecting CLT from being released from the pores. Thus, optionally, the exterior surface of the nanoparticle-based delivery system may be coated with at least one moiety modulating the release of said at least one dendritic cell activator, namely a moiety preventing or at least slowing-down the rate of release of the dendritic cell activator from the nanoparticle before reaching its target (which is the TME, e.g., DCs or their vicinity). In some embodiments, a suitable moiety for modulating the release of said at least one dendritic cell activator is a polypeptide, or a copolymer, preferably a pH sensitive copolymer. By way of a specific example, the moiety modulating the release of said at least one dendritic cell activator is albumin, e.g., bovine serum albumin (BSA) or human serum albumin (HSA). The moiety modulating the release of said at least one dendritic cell activator is interchangeably referred to as “release-modulating moiety”.

[0073] In order to allow the release of the dendritic cell activator from the nanoparticle as herein defined at its target, the moiety modulating the release of said at least one dendritic cell activator is coupled to the exterior surface of said nanoparticle via at least one cleavable linker, such as a linker sensitive to cleavage in the tumor microenvironment, for example but not limited to an acid-labile linker, an enzyme- cleavable linker, a reduction sensitive linker or a reactive oxygen spices (ROS)-sensitive linker.

[0074] It is known that anti-tumor immune response depends on the presence of DCs within the TME that can support the activation, recruitment, and / or expansion of T cells at the vicinity of their targets, e.g., tumors. However, subsets of DCs also induce immune- tolerance, leading to tumor immune escape. For example and as detailed above, Programmed Death ligand- 1 (PD-L1), a known immune checkpoint inhibitor, is abundantly expressed on DCs at the TME, and by binding to the programmed cell death 1 (PD-1) receptor positive T cells it suppresses their immune response. In addition, binding of PD-L1 on DCs to CD80 (B7.1) on the same cell via cis-interactions further prevents CD80 from activating T cells against cancer cells via CD28 co -stimulation. Blocking PD-L1 on DCs may not only prevent PD-l-mediated T cell inactivation, but further promote the molecular interaction between CD80 and CD28 and increase T cell activation.

[0075] Immune checkpoints as known in the art engage when proteins on the surface of

[0076] T cells recognize and bind to partner proteins on other cells, such as some tumor cells. When these immune checkpoints, also termed immune checkpoint proteins, bind together, they send an “off’ signal to the T cells, preventing the immune system from destroying, for example, cancer cells. By the term immune checkpoint inhibitor it is referred to any molecule (e.g., a small molecule, peptide or a polypeptide, particularly a polypeptide that is not an antibody) that can block the binding or interaction of immune checkpoints. For example, checkpoint inhibitors may be directed against the checkpoint protein called CTLA-4. Other immune checkpoint inhibitors are directed against PD-1 or its partner protein PD-L1. The PD-1 (programmed cell death- 1) receptor is expressed on the surface of activated T cells. Its ligands, PD-L1 and PD-L2, are expressed on the surface of dendritic cells, macrophages or a broad range of cancer cells. Testing the suitability of certain immune checkpoint inhibitors may be performed by methods in the art, for example by monitoring cytokines and enzymes secretion by treated T cells which are typical to T cell actiovation, etc.

[0077] Suitable immune checkpoint inhibitors may be any one of a peptide, a polypeptide, an oligonucleotide, a polynucleotide, or a small molecule inhibitor. In particular, any immune checkpoint inhibitor that is not an antibody. The immune checkpoint inhibitors as herein defined may be directed against any immune checkpoints known in the art, for example, any one of the immune checkpoints PD-1 / PD-L1, TIM- 3 / Ceacam-l / Galectin-9, LAG-3 / Galectin-3 / LSECtin / MHC-II, CTLA- 4 / CD80(B7.1) / CD86(B7.2) or DIGIT / CD 155 / CD 112, in particular directed against the immune checkpoints PD-L1 or PD-1, e.g., PD-L1.

[0078] For example, the immune checkpoint inhibitor applicable to the present disclosure comprises a PD-L1 binding peptide. Any PD-L1 binding peptide known in the art is suitable for coupling to the nanoparticle-based delivery system of the present disclosure.

[0079] The term "peptide" as herein defined refers to a molecular chain of amino acid residues, which, if required, can be modified at each one of its amino acid residues, for example by mannosylation, glycosylation, amidation (for example C-terminal amides), carboxylation or phosphorylation. The peptide may be obtained synthetically, through genetic engineering methods, expression in a host cell, or through any other suitable means. Methods for producing peptides are well known in the art. The term "amino acid" as used herein, refers to naturally occurring and / or synthetic amino acid residues, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.

[0080] The term “amino acid” also encompasses D-amino acids, which are mirror images of L-amino acids, where the chirality at carbon alpha has been inverted. In specific embodiments, the PD-L1 binding peptide as herein defined has at least one amino acid residue in a D configuration (“D amino acid residue”), at least about 25%, about 50% or about 75% D amino acid residues and in certain specific embodiments, the PD-L1 binding peptide contemplated is an all D amino acid residue peptide (i.e., all of the amino acid residues thereof are in D configuration).

[0081] Furthermore, the terms "amino acid sequence" or "peptide sequence" also relate to the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins. The sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group.

[0082] In certain specific embodiments, the PD-L1 binding peptide of the present disclosure comprises an amino acid sequence which is at least 80% identical to the amino acid sequence NYSKPTDRQYHF (Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His- Phe) denoted by SEQ ID NO. 1, specifically, the PD-L1 binding peptide comprises or consists of the amino acid sequence Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His- Phe, denoted by SEQ ID NO. 1. In further specific embodiments, the PD-L1 binding peptide comprises or consists of the amino acid sequence Asn-Tyr-Ser-Lys-Pro-Thr-Asp- Arg-Gln-Tyr-His-Phe, denoted by SEQ ID NO. 1 in which at least one amino acid residue is in a D configuration or all amino acid residues are in D configuration.

[0083] Alternatively, the PD-L1 binding peptide comprises or consists of an amino acid sequence which is at least 80% identical to the amino acid sequence CNYSKPTDRQYHF (Cys-Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe), denoted by SEQ ID NO. 2 that is also referred to herein as “cysteine-terminated PDL1bp” (or “N-terminal cysteine - harboring peptide”). In specific embodiments, the PD-L1 binding peptide comprises or consists of the amino acid sequence Cys-Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr- His-Phe (CNYSKPTDRQYHF denoted by SEQ ID NO. 2). In further embodiments, the PD-L1 binding peptide comprises or consists of the amino acid sequence denoted by SEQ ID NO. 2 in which at least one amino acid residue is in a D configuration or all amino acid residues are in D configuration.

[0084] It should be therefore understood that the present disclosure encompasses a PD- L1 binding peptide comprising the amino acid sequences denoted by SEQ ID NO. 1 or SEQ ID NO. 2 in which all amino acid residues are in L (native) or D configuration.

[0085] By the term “identical” or percent “ identity" in the context of two or more amino acid sequences, it is referred to two (or more) sequences that are the same or have a specified percentage of amino acid residues that are the same, in the context of the present disclosure, share about 80% identity, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region when compared.

[0086] That is to say, the peptide of the present disclosure (i.e., the PD-L1 binding peptide) may be a variant of the sequences defined and exemplified herein. By the term “variant” it is meant sequences of amino acids different from the sequences specifically identified herein, in which one or more amino acid residue(s) are deleted, substituted or added. It should be appreciated that by the term “added” as used herein it is meant any addition(s) of amino acid residues (natural or synthetic) to the sequences described herein. For example, the peptides of the present disclosure may be extended at their N-terminus and / or C-terminus with various identical or different amino acid residues.

[0087] Variants also encompass various amino acid substitutions. An amino acid “substitution” is the result of replacing one amino acid with another amino acid which has similar or different structural and / or chemical properties. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved. Amino acid substitutions may also be made by replacing a specific amino acid derivative by another, e.g., replacing an acetylated amino acid residue by another type of amino acid derivative. Variants also encompass conservative amino acid substitutions. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0088] Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).

[0089] The immune checkpoint inhibitor as herein defined may be coupled to the exterior surface of the nanoparticles of the present disclosure, optionally by a cleavable linker as herein defined, in particular, a cleavable linker sensitive to cleavage in the tumor microenvironment.

[0090] In order to specifically target DCs, the exemplified nanoparticles were surface- modified with mannose, which is a ligand of a dendritic cell. Ligands of any dendritic cell receptor are encompassed by the present disclosure. In particular embodiments, the dendritic cell receptor of the present disclosure is a mannose receptor or CD11c. In further particular embodiments, at least one ligand of a dendritic cell receptor is a mannose moiety or a peptide ligand of the DC receptor CD11c the peptide ligand having the amino acid sequence FYPSYHSTPQRP (denoted by SEQ ID NO. 11). For example, the ligand of a dendritic cell receptor is a mannose moiety.

[0091] Additional suitable ligands for the mannose receptor are: fucose, quinic acid and its analogs, N-acetylglucosamine (GlcNAc), oligosaccharides (fucoidan, chitosan, chondroitin sulfate or sulphated Lewisxand Lewisastructures) and fucosylated and mannosylated glycoproteins.

[0092] In certain further embodiments, the immune checkpoint inhibitor is coupled to the exterior surface of the nanoparticle delivery system via a linker attached to the ligand of a dendritic cell receptor (e.g., mannose) that is coupled to the exterior surface of said nanoparticle delivery system.

[0093] As used herein, “nanoparticle” refers to a microscopic particle of less than about 1 micron in diameter. In some embodiments, the nanoparticles range in size from about 1 nm to about 1,000 nm diameter, or alternatively between about 10 nm to about 1000 nm, between about 10 nm to about 900 nm, between about 10 nm to about 800 nm, between about 10 nm to about 700 nm, between about 10 nm to about 600 nm, between about 10 nm to about 500 nm, or alternatively between about 20 nm to about 1000, between about 20 nm to about 800 nm, between about 20 nm to about 700 nm, between about 20 nm to about 600 nm, between about 20 nm to about 500 nm; or alternatively between about 30 nm to about 1000 nm, between about 30 nm to about 900 nm, between about 30 nm to about 800 nm, between about 30 nm to about 700 nm, or alternatively between about 50 nm to about 1000 nm, between about 50 nm to about 900 nm, between about 50 nm to about 800 nm, between about 50 nm to about 700 nm, between about 50 nm to about 600 nm, between about 50 nm to about 500 nm, between about 50 nm to about 400 nm, or alternatively between about 100 nm to about 900 nm, between about 200 nm to about 1000 nm, between about 300 nm to about 1000 nm, between about 400 nm to about 1000 nm, between about 500 nm to about 1000 nm; between about 600 nm to about 1000 nm; between about 700 nm to about 1000 nm; between about 800 nm to about 1000 nm; or alternatively between about 900 nm to about 1000 nm; or alternatively between about 10 nm to about 300 nm, between about 20 nm to about 300 nm, between about 30 nm to about 300 nm, between about 40 nm to about 300 nm, between about 50 nm to about 300 nm, between about 60 nm to about 300 nm, between about 70 nm to about 300 nm, or between about 80 nm to about 300 nm, or alternatively between about 100 nm to about 300 nm, or alternatively between about 200 nm to about 600 nm, or alternatively between about 300 nm to about 600 nm, or alternatively between about 500 nm to about 800 nm. In certain embodiments, the nanoparticles described herein have a mean particle size of between about 20 nm to about 1000, between about 20 to 800 nm in diameter, preferably between about 50 to 400 nm in diameter, more preferably wherein the nanoparticles have a mean particle size of about 80 to 300 nm in diameter.

[0094] Nanoparticles are commercially available or can be prepared using various starting materials and by methods well-known to those skilled in the art. The nanoparticles may for example present a porosity adapted to accommodate the molecules to be loaded or an internal space (or core) capable of encapsulating such molecules. Thus the nanoparticles of the present disclosure may be hollow or porous nanoparticles, such as a mesoporous silica nanoparticle, a hollow silica nanoparticle, a liposome, a micelle, a vesicle, etc. In particular, the nanoparticles of the present disclosure are biocompatible and suitable for carrying functional groups (i.e., at least one dendritic cell activator, at least one immune checkpoint inhibitor and at least one ligand of a dendritic cell receptor) and for being administered to a mammalian subject, specifically a human subject.

[0095] The method for preparing the nanoparticles of the present disclosure is chosen by a skilled artisan based on the starting materials being used (e.g., polymers, lipids, silica, etc.), the desired physical properties, for example, the diameter, pore or core size, coatings, etc., as well as based on the functional groups intended to be carried by the nanoparticles and the linkers required for attaching the various functional groups.

[0096] By way of example, the nanoparticle of the present disclosure is an inorganic (e.g., metallic particles, oxide particles such as silica, alumina, zirconia and the like) or organic nanoparticle (e.g., a polymer-based nanoparticle, such as polylactic-co-glycolic acid (PLGA) or polycaprolactone (PCL)), polymeric micelles, such as PEGylated polymers, or lipid-based nanoparticles, such as liposomes, or any combination thereof.

[0097] Mesoporous silica nanoparticles can be prepared using sol-gel methods such as Stober’s method (such as using TEOS as exemplified below). Micelles-based nanoparticles can be prepared by preparing a block copolymer having hydrophilic and hydrophobic groups. Liposomes-based nanoparticles are commercially available or can be prepared and loaded according to any method known in the art.

[0098] In particular embodiments, the nanoparticles of the present disclosure are metal nanoparticles, silica nanoparticles (e.g., mesoporous silica nanoparticles), polymer-based nanoparticles (such as, polylactic-co-glycolic acid (PLGA) or polycaprolactone (PCL)), polymeric micelles (such as PEGylated polymers), polymer conjugates (such as polymer- drug conjugates, polymer-ligand conjugate) or lipid-based nanoparticles (such as liposomes, stealth liposomes, vesicles or solid lipid nanoparticles), or a combination thereof.

[0099] The nanoparticles of the present disclosure may be first prepared and then functionalized (i.e., loaded with the various functions as described herein) or alternatively they may be prepared by linking, polymerizing or co -polymerizing building blocks that have been previously functionalized with a suitable linker and / or ligand in the context of the invention.

[0100] By another aspect thereof, the present disclosure provides a method of preparing a nanoparticle-based delivery system comprising at least one dendritic cell activator, at least one immune checkpoint inhibitor and at least one ligand of a dendritic cell receptor.

[0101] For example, a method for preparing the nanoparticle-based delivery system of the present disclosure comprises at least one of the following steps: a) providing organic or inorganic nanoparticles, b) encapsulating within the nanoparticles or attaching thereto at least one dendritic cell activator optionally using at least one cleavable linker, c) optionally coating the exterior surface of the nanoparticles with at least one moiety modulating the release of said at least one dendritic cell activator, d) attaching to said nanoparticles at least one immune checkpoint inhibitor, and at least one ligand of a dendritic cell receptor to the exterior surface of the nanoparticles, optionally using at least one cleavable linker.

[0102] In particular embodiments, the nanoparticles are porous, mesoporous or hollow nanoparticles, or based on a micelle, a vesicle or a liposome. In further particular embodiments, the nanoparticles are porous, mesoporous or hollow nanoparticles or based on a micelle, a vesicle or a liposome and the at least one dendritic cell activator is encapsulated or embedded therein. In certain specific embodiments, the method of preparation further comprises coating the exterior surface of said nanoparticles with at least one moiety modulating the release of said at least one dendritic cell activator. In some embodiments, the release-modulating moiety is a degradable polymer or copolymer, such as a pH sensitive copolymer. In certain embodiments the release- modulating moiety is a polypeptide, e.g., an albumin such as bovine serum albumin (BSA). In some embodiments, said release-modulating moiety may be further functionalized.

[0103] Therefore, in certain embodiments, the nanoparticles are silica nanoparticles, for example, the nanoparticle-based delivery system according to present disclosure may be based on mesoporous silica nanoparticles (MSNP).

[0104] As used herein, the term “loading”, “encapsulating” or “embedding” refers to the introduction of a molecule such as an active agent (in the context of the present disclosure, in particular, at least one dendritic cell activator such as clotrimazole) into the pores or into the hollow space of suitable nanoparticles.

[0105] As used herein, “linker” refers to a group of atoms that is used to couple a nanoparticle or nanoparticle precursor to another function or group to spatially separate the two entities. Thus, a linker of this invention has an essentially longitudinal axis, that is, it is essentially linear rather than highly branched or clumped, although the structure will, of course, not be exactly linear due to the angular constraints placed on the structure by required bond angles between covalently bonded atoms.

[0106] Examples of linkers include, but are not limited to, straight and branched alkyl and alkenyl groups containing functional groups such as carboxyl, amino, hydroxyl, and thiol, through which covalent bonds can be formed to connect the linker to the polymer and to other components, e.g., a short alkyl chain (CH2)nwhere n=2-10, terminated by two amino groups or two carboxyl groups or one amino group and one carboxyl group. A linker may also be a short peptide chain (H — [NHCHR — CO]n-OH) where n is 1-20, or alternatively 1-18, 1-16, 1-14, 1-12, 2-14, 2-12, 3-20, 4-18, 5-20, or alternatively from 5-18, and R is the same or different for each of the n amino acids, and is one of the 22 side groups known to be present in natural amino acids. Linkers may be the same or different. A peptide linker can be incorporated into the construct by one of the peptide condensation reactions (producing an amide bond) that are known in the art.

[0107] In particular examples, a suitable linker is any linker known in the art that is sensitive to cleavage in the tumor microenvironment. Suitable linkers that are sensitive to cleavage in the tumor microenvironment are any one of an acid-labile linker (e.g., a hydrazone), an enzyme-cleavable linker (e.g., a peptide, for example glutathione (GSH), cathepsine protease- sensitive linkers, and glycosidic linker), reduction sensitive linker (e.g., a disulfide linker), reactive oxygen spices (ROS)-sensitive linker (e.g., H2O2- sensitive linkers, such as 4-mercaptophenyl boronic acid or light sensitive linker, to name but a few. In particular examples, a suitable linker is reactive oxygen spices (ROS)- sensitive linker. In further embodiments the cleavable linker sensitive to cleavage in the tumor microenvironment as herein defined comprises 4-mercaptophenyl boronic acid, a peptide, a disulfide bond, a pH-sensitive hydrazone bond or a hydrogen peroxide sensitive linker.

[0108] When the nanoparticle is an organic -based nanoparticle, the loading of the dendritic cell activator may be, e.g., simultaneous to the formation of the nanoparticle.

[0109] In some embodiments, such as when the nanoparticle is a porous solid particle (e.g., a mesoporous silica nanoparticle), the dendritic cell activator is loaded after the formation of the nanoparticle.

[0110] In some embodiments, the invention comprises at least one step of post- functionalization of the nanoparticle.

[0111] The at least one ligand of a dendritic cell may be attached to the release- modulating moiety, optionally via a linker. Furthermore, the at least one immune checkpoint inhibitor may be attached to the release-modulating moiety, optionally through a cleavable linker. In some particular embodiments, the invention comprises attaching said at least one immune checkpoint inhibitor to said ligand of a dendritic cell, optionally through a cleavable linker, where optionally, the at least one ligand of a dendritic cell may be attached to the release-modulating moiety.

[0112] In certain embodiments, the nanoparticle-based delivery system according to the present disclosure is based on mesoporous or hollow nanoparticles, e.g., wherein said nanoparticle is a silica nanoparticle, for example a mesoporous silica nanoparticle (MSNP).

[0113] Preparing mesoporous or hollow nanoparticles is known in the art. In some exemplary embodiment, the present invention relates to a method for preparing said delivery system, the method comprising:

[0114] -providing mesoporous or hollow nanoparticles,

[0115] -loading (or encapsulating) at least one dendritic cell activator such as clotrimazole within said nanoparticle to form dendritic cell activator-loaded nanoparticles,

[0116] -coating said dendritic cell loaded nanoparticles with a moiety modulating the release of the dendritic cell activator, such as a polypeptide, e.g., BSA,

[0117] - attaching at least one ligand of a dendritic cell, such as mannose to said moiety modulating the release of the dendritic cell activator,

[0118] -attaching at least one immune checkpoint inhibitor to said ligand of a dendritic cell, optionally through a cleavable linker.

[0119] In some exemplary embodiments, the present invention relates to a method for the preparation of a polymeric nanoparticle -based delivery system such as a micelle -based delivery system, said method comprising the following steps:

[0120] -preparing a block copolymer adapted for micelle formation such as a block polycaprolactone-PEG copolymer,

[0121] -attaching at least one ligand of a dendritic ell such as mannose, optionally through a linker, to one end of the block copolymer (such as the PEG end), -attaching at least one immune checkpoint inhibitor (such as a PD-L1 peptide inhibitor) to said one end of the block copolymer and / or to said ligand of a dendritic cell, optionally through a linker, such as a cleavable linker,

[0122] -adding the previously prepared block copolymer to a solution comprising at least one dendritic cell activator such as clotrimazole in conditions appropriate for micelle formation to obtain a polymeric micelles loaded with said dendritic cell activator and functionalized with said ligand of a dendritic cell and said immune checkpoint inhibitor.

[0123] As used herein, “degradable polymer” refers to a polymer which can be broken down under specific conditions (e.g., under conditions typical to the TME) to smaller units. In one aspect, repeated degradation of the polymer units in situ (in the body) allows for small fragments to be excreted or otherwise eliminated.

[0124] In certain embodiments, the nanoparticle-based delivery system according to the present disclosure is wherein the at least one immune checkpoint inhibitor is a PD-L1 binding peptide (e.g., a peptide comprising the amino acid sequence denoted by SEQ ID NO. 1 or by SEQ ID NO. 2), coupled to the exterior surface of said nanoparticles via a cleavable linker, wherein said at least one dendritic cell activator is clotrimazole, embedded within said nanoparticles, and wherein said exterior surface of said nanoparticles is decorated with a mannose moiety. In the above and other embodiments, the PD-L1 binding peptide is attached to the nanoparticles via the ligand of the dendritic cell receptor (mannose moiety), optionally through a cleavable linker. In still further specific embodiments, the mannose moiety is attached to the nanoparticle via the moiety modulating the release of the dendritic cell activator (clotrimazole).

[0125] In certain embodiments clotrimazole is loaded in an amount of about 2% to 20% by weight of the weight of the nanoparticles.

[0126] The nanoparticle -based delivery system according to the present disclosure may further comprise an imaging group. Any imaging group which may be linked to the nanoparticles of the present disclosure is suitable. By way of example, the imaging agent is fluorescein 5 isothiocyanate (FITC), indocyanine green (ICG), Cyanin dyes, Rhodamine dyes, Alexa-fluor dyes, or brilliant violet dyes. The imaging group may be linked to the nanoparticles directly or indirectly by a spacer or a liker (e.g., a cleavable linker). The nanoparticle-based delivery system as herein defined further comprising an imaging group may be useful for imaging, in particular, diagnostic purposes of various tumors.

[0127] By a further aspect thereof the present disclosure provides a pharmaceutical composition comprising (as an active agent) the nanoparticle -based delivery system as herein defined optionally with at least one carrier, diluent, excipient or a mixture thereof. In particular, the present disclosure further provides a pharmaceutical composition comprising the nanoparticles as herein defined, namely, the nanoparticles loaded with the at least one dendritic cell activator, and surface decorated with at least one immune checkpoint inhibitor and at least one ligand of a dendritic cell receptor. The pharmaceutical composition of the present disclosure may further comprise an additional therapeutic agent (e.g., an anti-cancer agent or an immunotherapeutic agent).

[0128] The “ pharmaceutical composition" of the present disclosure generally comprises the nanoparticles as herein defined as herein defined and a buffering agent, an agent which adjusts the osmolarity of the composition and optionally, one or more pharmaceutically acceptable carrier, diluent and / or excipient as known in the art. As used herein the term " harmaceutically acceptable carrier, diluent, excipient" includes any and all solvents, dispersion media, coatings, and the like, as known in the art. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.

[0129] The pharmaceutical composition of the present disclosure may be in a liquid or in a solid dosage form, e.g., in the form of a pellet, a tablet, a capsule, a solution, a suspension, a dispersion, an emulsion, an elixir, a gel, an ointment, a cream, an aqueous solution or a suppository. The pharmaceutical composition of the present disclosure may be a controlled release composition or an immediate release composition. As detailed in the Examples below, PDL1bp was released from the nanoparticles under conditions simulating the TME, tethered to PD-L1 to reverse CD80 sequestration on DC2.4 cells and increased the molecular interactions with recombinant CD28 (rCD28). ELISA and confocal imaging analysis indicated an increase in DC2.4 and EL4 T cell interaction and IL-2 secretion following MSNP-MaN-PDL1bp treatment. In addition, an increased uptake of the CLT-loaded MSNPs (also termed herein “MSNP- MaN / CLT”) by DC2.4 cells and MHC Il-mediated presentation of hen egg lysozymes (HEL) antigen. Increased IL-2 production from HEL antigen-primed 3A9 T cells was further demonstrated following co-culturing of DC2.4 and 3A9 T cells with MSNP- MaN / CLT and PDL1bp, indicating a high antigen presentation and T cell stimulatory efficacy.

[0130] Overall, the results presented herein confirm the ability of PD-L1-decorated and CLT-loaded MSNPs to modulate DC and T cell activity within the TME. MSNP-MaN- PDL1bp / CLT were significantly superior over MSNP-MaN / CLT when tested in an animal model (as shown in Fig. 21), which demonstrates the crucial role of PDL1bp in achieving an effective immune mediated control of tumor growth. The anti -tumor effect obtained with MSNP-MaN-PDL1bp / CLT treatment suggests the ability of the NPs to drain to the lymph node where they can be taken-up by DCs in vivo, rather than affecting directly on tumor cells. This highlights the opportunity to overcome some of the shortcomings of traditional cancer immunotherapy (i.e., poor penetration and heterogeneous distribution in solid tumors) to achieve enhanced therapeutic efficacy.

[0131] Therefore by a further aspect thereof the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of the nanoparticle-based delivery system or the pharmaceutical composition as defined herein.

[0132] The method of treatment as herein define may be affected by administering a therapeutically effective amount of the nanoparticle-based delivery system as herein defined, or the pharmaceutical composition comprising thereof, and may be determined by such consideration as are known in the art in order to prevent, cure or at least ameliorate or reduce or delay the onset of at least one clinical indication resulting from a disease or condition as herein defined. For any preparation used in the methods of the invention, the dosing of the therapeutically effective amount can be estimated based on in vitro cell assay, for example, as detailed in the Examples, or based on animal models as known in the art.

[0133] By a further aspect thereof the present disclosure provides the nanoparticle -based delivery system or the pharmaceutical composition comprising thereof as defined herein for use in a method of treating a disease or disorder in a subject in need thereof.

[0134] Optionally, the above method may further comprise administrating anti-cancer therapy.

[0135] It is to be understood that the terms “treat”, “treating”, “treatment” of forms thereof as used herein mean preventing, curing or at least ameliorating or reducing or delaying the onset of at least one clinical indication resulting from a disease or condition as herein defined.

[0136] In particular embodiments, the disease or disorder as herein defined is a proliferative disease or disorder or an immune -related disease or disorder. By the term “proliferative disease or disorder” it is referred to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non- solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. In general, the methods and compositions of the present invention may be used in the treatment of non-solid and / or solid tumors. In specific embodiments, the disease or disorder as herein defined is cancer. In further specific embodiments, the present disclosure encompasses cancer that is melanoma, lung cancer (both non-small cell lung cancer (NSCLC) and small cell lung cancer), kidney cancer, bladder cancer or head and neck squamous cell carcinoma (HNSCC).

[0137] Administered may be performed by any suitable route. For example, a pharmaceutical composition in a form suitable for oral, intravenous, intraarterial, intramuscular, intracranial, intranasal, subcutaneous, parenteral, transmucosal, transdermal, or topical administration.

[0138] Monitoring the efficacy of the nanoparticle-based delivery system as herein defined may be performed by any method known in the art, by a skilled physician, for example, using an animal model as described herein below, e.g., by monitoring parameters associated with activation of the immune effector cells as known in the art (e.g., by following secretion of cytokines, Granzyme B etc.)

[0139] The term “subject" as used herein means warm-blooded animals, such as for example rats, mice, dogs, cats, guinea pigs, primates and humans. The present disclosure is pertinent to a nanoparticles and methods for their preparation and use thereof as herein defined which are suitable in particular for the treatment of a subject which is a mammal, for example, a human. A skilled physician is able to diagnose a subject in need thereof, namely, a subject inflicted by any one of the diseases or disorders as herein defined.

[0140] As detailed above, when decorated with PDL1bp, the MSNPs inhibited PD- Ll:CD80 cis interactions on DCs, and released CD80 to facilitate co-stimulatory molecular interactions with CD28 on T cells. The results presented below collectively show that the decorated MSNPs may manipulate signal pathways while demonstrating a synergistic benefit, employing both PDL1bp and CLT mediated DC activation to increase T cell stimulation.

[0141] Therefore, by a further aspect thereof the present disclosure provides a method of immunomodulation in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of the nanoparticle-based delivery system as defined herein or the pharmaceutical composition comprising thereof. In particular, the present disclosure provides a method of at least one of dendritic cell activation and T cell activation.

[0142] By the term immunomodulation it is meant alteration of the subject’s immune response to foreign agents and pathogens either by stimulation (immune- stimulation) or inflammation suppression (immunosuppressant) in order to maintain the desired level of host immune-protection.

[0143] By the term “at least one” it is meant one, two, three, four, five or more of the indicated substances, depending on the context.

[0144] As used herein, the term about when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ± 10%, more preferably ± 5%, even more preferably ± 1%, and still more preferably ± 0.1% from the specified value, as such variations are appropriate to perform the disclosed invention.

[0145] EXAMPLES

[0146] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0147] Materials

[0148] Cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), 3- aminopropyl trimethoxysilane (APTMS), Aldrithiol-2, l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-Hydroxy succinamide (NHS) and clotrimazole (CLT) were purchased from Alfa Asear. D-mannosamine (MaN) and Bovine serum albumin (BSA) were purchased from Sigma-Aldrich. The PD-L1 binding peptide having the all D amino acid residue sequence Cys-Asn-Tyr-Ser-Lys-Pro- Thr-Asp-Arg-Gln-Tyr-His-Phe (denoted herein by SEQ ID NO. 2) in all D amino acid configuring was ordered from GL Biochem (Shanghai) Ltd. DMEM and RPMI 1640 cell culture media, fetal bovine serum (FBS), L-glutamine, penicillin / streptomycin and HEPES buffer were purchased from Biological Industries / Sartorius. The amino acid sequences of peptides described herein as well as nucleic acid sequences of primers described below are also listed in Table 2 below. Methods

[0149] Synthesis of mesoporous silica nanoparticles (MSNPs)

[0150] In the typical synthesis procedure, 0.45 g of CT AB and 1.8 mL of sodium hydroxide (2M) were added to 240 mL of water and the mixture was stirred at a temperature of 80°C. After several minutes (X-Y minutes), 2.4 mL of TEOS was added drop wise to the resulting solution and allowed to stir vigorously for 2 hours to achieve the as- synthesized MSNPs. The formed MSNPs were washed with ethanol and de-ionized water multiple times. For CT AB removal, MSNP-NH2 was stirred in a mixture composed of HC1 (37%, 6 mL) and ethanol (54 mL) at 65°C for 8 hours. To introduce the amine group, 300 mg of as-prepared MSNPs were added to methanol containing 50pL of triethanolamine, followed by 75 μ oLf APTMS, and the reaction was stirred for 12 hours. The formed MSNP-NH2 was washed several times with the ethanol, precipitated and dried. Then, the calcinated MSNP-NH2 was washed several times with distilled water and ethanol.

[0151] Loading of clotrimazole (CLT) and coating with bovine serum albumin (BSA)

[0152] For the loading of CLT, 100 mg of MSNP-NH2 was suspended in 15 mL of ethanol and allowed to stir for 30 minutes. Later, 6 mg of CLT was added to the ethanolic suspension and the product was allowed to stir for 24 hours. After completion of the reaction, the loaded MSNP-NH2 / CLT was centrifuged (5000 rpm) and the supernatant was collected for CLT spectroscopic analysis, and the MSNP was dried. Then, BSA was chemisorbed on the MSNP (i.e., on the nanoparticle also termed herein MSNP-NH2 / CLT) to close the orifices using the carbodiimide chemistry. The COOH-group of BSA (5.5 mg) was pre- activated using EDC (4 mg) and NHS (2 mg) in ImL phosphate-buffered saline (PBS) solution. After 30 minutes of incubation, the activated BSA molecules were allowed to interact with the MSNP-NH2 / CLT (50 mg in 15 mL PBS solution) for 8 hours. The resulting product, MSNP-BSA / CLT, was washed multiple times with PBS to remove the unreacted BSA from the silica surface. FITC incorporated MSNPs were synthesized by replacing CLT with FITC (6 mg) and following the above mentioned steps to generate MSNP-BSA / FITC or MSNP-MaN-PDL1bp / FITC. Attachment of MaN to MSNP-BSA / CLT

[0153] The MSNP-BSA / CLT was conjugated with D-mannosamine (MaN) in order to achieve active targeting of dendritic cells (DCs). The free carboxyl groups in BSA (50 mg of MSNP-BSA / CLT) were activated with EDC (6 mg) / NHS (4 mg) and allowed to stir for 30 minutes in PBS. Later, MaN (7 mg) was added and the resulting mixture was stirred for 6 hours. Afterwards, the desired product (MSNP-MaN / CLT) was washed with PBS multiple times, to remove any free residual MaN.

[0154] Attachment of the 4-merceptophenyl boronic acid to MSNP-MaN / CLT

[0155] The vicinal diols of the mannose structure are suitable for molecular association with boronic acid derived species. The MSNP-MaN / CLT (25 mg) was allowed to stir in 10 mL PBS solution for 20 minutes. Later, 0.5 mL of 4-mercaptophenyl boronic acid solution (5 mg / mL in DMSO) was added and the resulting solution was stirred over night to form the boronate ester bond to generate MSNP-MaN-BA / CLT. Then, the particles were washed multiple times with water and used for the attachment of PDL1bp as further detailed below.

[0156] Activation of MSNP-MaN-BA / CLT and conjugation of PDLlbp

[0157] MSNP-MaN-BA / CLT (40 mg) were added to a solution of Aldrithiol-2 (125 mg) in 10 mL PBS. Following stirring for 12 hours at room temperature (RT), the resulting material, i.e., 2- pyridinyldisulfanylpropyl-functionalized MSNP-MaN-BA / CLT, was generated and washed with ethanol and cold PBS many times. Then, the purified material was again suspended to a 10 mL PBS solution containing cysteine-terminated PDL1bp (5 mg) having the amino acid sequence Cys-Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His- Phe (denoted herein by SEQ ID NO. 2) and stirred in closed conditions for 12 hours to obtain the desired product, MSNP-MaN-PDL1bp / CLT.

[0158] Drug loading content and drug loading efficiency

[0159] Drug loading content (DLC) was equivalent to 5.6% and the drug loading efficiency (DLE) was 85.3% as determined by the formulae reported below. Characterization of the Nanoparticles by spectroscopy

[0160] The MSNPs functionalized as detailed above were sonicated in ethanol solution (1 mg / mL) and dried over a copper grid before being imaged with an FEI Tecnai 12, 200kV high-resolution transmission electron microscope (TEM). Thermal degradation analysis (TGA) was performed using a PerkinElmer Pyris TG analyzer with nitrogen as an oxidant and continuous heating from 30-800°C at a heating rate of 10°Cmin-1. A Nicolett 6700 FTIR spectrometer was used to measure FTIR spectra. TECAN Infinite M200 microplate reader was used for UV / Vis spectroscopy and fluorescence measurements. Surface areas of MSNPs were assessed in a Quantachrome surface area analyzer at 77K using nitrogen adsorption / desorption. Dynamic light scattering (DLS; ZetaSizer Nano -ZS 90, Malvern) with a He-Ne laser (633nm) and a scattering angle of 90° was used to determine the zeta potentials and particle size. The MSNPs were diluted with PBS (pH 7.4) to O.lmg mL"1, then sonicated for Imin and measured at predefined time intervals.

[0161] Release of the PDLlbp in the simulated TME condition (in vitro)

[0162] MSNP-MaN-PDL1bp carry H2O2and redox (GSH) responsive linkages, as shown in Figure 1. In order to test the release of the peptide, the particles (1 mg / mL) were incubated in a solution of DTT (10 mM), H2O2(10mM) or PBS for 1, 2, 4, 6, 8 or 12 hour time periods. Later, the particles were spun and the supernatant was collected and incubated with 100 μL Fluorescamine (Thermo Fisher Scientific) solution in acetone (4 mg / mL) for 2 hours at 4°C to detect primary amine (Lys) in the peptide sequence. Finally, the fluorescence intensity was measured (excitation at 390 nm and emission at 460 nm) using the TECAN microplate reader. The amount of the PDL1bp attached to the MSNP surface through disulfide bond was calculated to be 105 pg / mg of the nanoparticles, on average.

[0163] Release of CLT from the CLT -loaded MSNPs in simulated enzymatic conditions (in vitro) In order to investigate the amount of CLT released from MSNP-BSA / CLT, 10 mg of the nanoparticles (containing 520 pg of CLT) were dispersed in 5 mL of ethanokPBS (20:80) mixture with or without Trypsin (2.5%) and stirred (170 rpm, 37°C). A sample was collected (100 μL) at predetermined time intervals (0.5, 1, 2, 4, 8, 12 & 24 hours), and the volume was replaced with 100 μL of fresh ethanolic:PBS mixture (with / without Trypsin). To the sample was added 400 μL of cold acetone and it was incubated for 1 hour at -18°C. After 1 hour, the medium was centrifuged (14,000 rpm, 10 min) to remove Trypsin and chopped BSA (as pellet) and the supernatant was dried and resuspended in a mixture of ethanol and aqueous dibasic potassium hydrogen phosphate solution (70:30). The supernatant was then analyzed by a validated HPLC (Waters Alliance 2695) method using Phenomenex Kinetex (C18 100A, 250 mm x 4.60 mm; 5pL) and the amount of CLT released from nanoparticles was determined with reference to a standard curve.

[0164] Cell culture

[0165] DC2.4, 3A9 and EL4 cells were purchased from the American Type Culture Collection (ATCC Manassas, VA, USA) and cultured at 37°C in a humidified atmosphere with 5% CO2. DC2.4 and EL4 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES buffer, 100 lU / mL penicillin, and 100 μg / mL of streptomycin. The 3A9 cells were cultured in DMEM supplemented with 5% FBS, 4 mM L-glutamine, 0.05mM β-mercaptoethanol, 100 lU / mL penicillin, and 100 μg / mL streptomycin.

[0166] XTT Assay

[0167] The cytotoxicity of PDL1bp-decorated and CLT-loaded MSNPs was measured by the Cell proliferation Kit (XTT based, Biological Industries / Sartorius) after incubation of1x104DC2.4 cells per well with MSNP formulations at concentrations ranging from 10- 50 μg / mL for 24 hours.

[0168] Cellular uptake and binding studies

[0169] For cellular uptake studies, DC2.4 cells (1x105) were added to a 24 well plate and activated for 24 hours with lipopolysaccharide (LPS, Sigma Aldrich, 250ng / mL) and interferon-gamma (IFNy, PeproTech, 250 ng / mL). Then, different FITC-loaded MSNP conjugates were incubated with activated / unactivated DC2.4 cells at a concentration of 20 μg / mL. After 24 hours of incubation, the cells were washed three times with PBS, then fixed with 4% paraformaldehyde (PFA) and visualized by Zeiss Celldiscoverer 7. Ten frames were acquired for each treatment group, which consisted of roughly 160-200 cells, and the median intensity was plotted. The image processing was done in CellProfiler ver 4.2.2 with custom designed script. All measurements were exported as CSV and further data processing was done in R ver 4.1 in RStudio (ver 2022.12.0). In order to assess cellular binding of functionalized MSNPs, DC2.4 cells (1x105) were seeded in a 12 well plate, and activated as described above. After activation for 24 hours, the DC2.4 cells were treated with MSNP-MaN-PDL1bp / FITC (equivalent to 25μg / mL free peptide) for 45 minutes at 4°C to inhibit active cellular internalization. Following that, the supernatant was removed and the cells were washed three times with PBS, trypsinized, washed again and re-suspended in PBS for analysis by flow cytometry.

[0170] Determining receptor expression levels on DC2.4 cells

[0171] DC2.4 cells (1x106) were seeded in six well plates, and treated with or without the activating agents LPS and IFNy as described above, and allowed to grow in the medium for 24 hours. Next, the cells were washed with PBS, trypsinized and suspended in 3% BSA solution in PBS for 30 minutes at RT. Then, the cells were incubated with the anti- PD-L1 primary antibody (Biolegend, 1:100 dilution) at 4°C for 30 minutes. Subsequently, the cells were washed with 1% BSA in PBS solution followed by the incubation with secondary antibody (donkey anti-rat IgG Alexa Fluor 647, Jackson ImmunoResearch) for another period of 30 minutes. The cells were washed, centrifuged, resuspended in PBS solution and the fluorescence was analyzed by flow cytometry (Guava® easyCyte HT Systems, Millipore). CD80 expression was detected by anti-CD80-AlexaFluor647 (Biolegend, 1:100 dilution).

[0172] Surface expression of MHC II (anti-I-A / I-E-APC-Fire750, Biolegend), CDl lc (anti- CDl lc-PE, Biolegend) and CD80 on DC2.4 cells after incubation with MSNPs was assessed accordingly, after the cells were incubated with 27.5 μM CLT or equivalent concentrations of functionalized MSNPs for 24 hours.

[0173] Co-localization assay ofPD-Ll

[0174] DC2.4 cells (1x105) were seeded in a 24-well plate on cover slips and then treated for 24 hours with / without LPS and IFNy, as described above. The cells were then fixed with 4% PFA (for 2-3min) and blocked with 3% BSA in PBS for 30 minutes and 10 μg / mL Fc blocker (anti-CD 16 / 32, Biolegend) for 10min, before staining for PD-L1 (anti-PD-L1 primary antibody + Alexa Fluor488 Goat anti-rat IgG secondary antibody, Jackson ImmunoResearch) and CD80 (anti-CD80-AlexaFluor 647). Later, the cover slips were mounted on glass slides with the DAPI mounting medium (Fluoromount-G, SouthernBiotech) and the cells were visualized by confocal laser scanning microscope (LSM 800, Carl Zeiss) at 488nm and 647nm laser excitation. Images from all groups were acquired with same photomultiplier gain settings and analyzed using image analysis software ZEN Microscopy. The intensity profile plots were generated by taking the intensity values for both CD80 and PD-L1 for the indicated region in the figure and then plotted in the origin. rCD28 molecular interactions with DC2.4

[0175] The DC2.4 cells (1x105) were seeded in a 12 well plate, and activated by treating with the LPS and IFNy as mentioned above. The cells were then treated with free PDL1bp (25μg / mL), MSNP-MaN or MSNP-MaN-PDL1bp (equivalent to 25μg / mL free peptide) for 45min at 4°C, then washed 3 times with cold PBS followed by BSA (3% in PBS) blocking. 5(6)-carboxyfluorescein-labeled recombinant mouse CD28-Fc (rCD28) at a concentration of 1μg / mL (20nM) or 2 μg / mL (40nM) was added to bind with the available CD80 at 4°C. After 30min incubation, the cells were centrifuged, washed, and resuspended in PBS for the fluorescent binding studies by flow cytometry. The labeling of rCD28 was performed with the 5(6)-carboxyfluorescein succinimidyl ester (Abeam) according to the manufacture protocol.

[0176] Immunological Synapse imaging

[0177] The DC 2.4 cells (1x104) were seeded on eight-well glass bottom slides (Ibidi). The cells were activated to generate the mDC2.4 as mentioned above. The mDC2.4 were then incubated with free PDL1bp (5μg / mL), or MSNP-MaN-PDL1bp (equivalent to 5μg / mL free peptide) for 45min at 4°C and then washed thrice the cells with PBS to remove the unconjugated particles. EL4 T cells were then added at a DC:T cell ratio of 1:5 and co- cultured for Ih at 37°C. The cells were blocked with 3% BSA solution at RT and then treated with Fc blocker (10μg / mL) for 10min at 4°C. Later the cells were incubated with antibodies for the surface markers CD80-Alexa flour 647 and CD28-FITC (Biolegend) and then fixed with 4% PFA solution. The staining was observed with confocal laser scanning microscope (LSM 800, Carl Zeiss). Digital images were captured with 488nm and 647nm laser excitation. For the Zeiss Cell Discoverer 7 sample, briefly, 2x104DC2.4 cells were placed in a 24 well plate and activated subsequently as described. The cells were then treated for 45min at 4°C with various treatments (free PDL1bp, MSNP-MaN, and MSNP-MaN-PDL1bp, equivalent to 10μg / mL peptide). The cells were then rinsed to eliminate any remaining unconjugated particles. After the EL4 cells (1x105) were added in a 1:5 ratio to the 24 well plate and left to interact for 4h at 37 °C, the subsequent steps were repeated as described above. The imageJ software was used to count the number of the synapses. It should be noted that we did not count the EL4 cells that were passively trapped in the DC2.4 cluster.

[0178] IL-2 concentration in EL4 cell co-culture supernatants

[0179] Activated DC2.4 cells (2x 104) were incubated with MSNP formulations (equivalent to 10μg / mL PDL1bp) and co-cultured with EL4 cells (1x105) for 24h as described above. After co-culture, the medium was collected, centrifuged to remove T cells and stored at - 20°C. ELISA MAX Standard Set Mouse IL-2 (Biolegend) was used to determine the IL- 2 concentration in the cell culture supernatant, in accordance with the manufacturer's instructions.

[0180] HEL antigen uptake of DCs

[0181] DC2.4 (1x105cells) were seeded on cover slips in the 24-well plate and incubated for 12h. The cells were then treated with the free CLT (27.5μM) or MSNP-MaN / CLT (27.5μM CLT equivalent) for Ih, then washed and pulsed with 5(6)-carboxyfluorescein-labelled Hen Egg lysosome (HEL, Sigma- Aldrich, 150 μg / mL) for 2h. The cells were then washed twice with PBS followed by the addition of the LysoTracker™ Red DND-99 (Invitrogen, 75nM in RPMI medium) and allowed for 30min of incubation. Later, the sample was rinsed, fixed with 4% PFA, and mounted on a glass slide for confocal microscopy analysis. The HEL was labeled with the 5(6)-carboxyfluorescein succinimidyl ester as described above.

[0182] IL-2 concentration in 3A9 cell co-culture supernatants

[0183] DC2.4 (1x105) were seeded in the round -bottom 96 well plate, and then treated with free CLT, MSNP-BSA-MaN / CLT and MSNP-BSA-MaN (27.5μM CLT equivalent) for 1h before being pulsed with HEL (150μg / mL) for 20h, followed by careful washing by PBS and co-culture with the 3A9 cells (1x105) in serum free DMEM for 24h. The supernatant was collected after 24h and the IL-2 concentration was measured by ELISA.

[0184] To assess the activation by stimulating both pathways, 1X105DC2.4 cells were seeded in the round-bottom 96 well plate and then treated with free CLT, free PDL1bp, free PDL1bp plus free CLT, MSNP-MaN-PDL1bp, MSNP-BSA-MaN / CLT and MSNP- BSA-MaN / CLT plus free PDL1bp (equivalent to 27.5μM CLT and 25μg / mL PDL1bp) in the presence of HEL (150μg / mL) for Ih, then washed with PBS and co-cultured with the 3A9 cells (1x105cells) in serum free DMEM for 36h. The supernatant was collected and the IL-2 concentration was determined by ELISA.

[0185] Mice

[0186] Female C57BL / 6 mice were obtained from Envigo CRS (Ness-Ziona, Israel). All animal experiments were approved and performed in compliance with the standards of the Ben- Gurion University of the Negev (BGU) Institutional Animal Care and Use Committee (IACUC).

[0187] B16 tumor model

[0188] C57BL / 6 mice at 6-7 weeks of age were inoculated with 5x105B16-F10 melanoma cells subcutaneously (s.c.) above the right flank under isoflurane anesthesia. The mice received the nanoparticle construct (MSNP) or control treatments at doses equivalent to 40 mg / kg CLT and equivalent peptide doses twice weekly, starting from day 3 after cancer cell inoculation, for a total of 4 treatments. The treatments were administered s.c. at the lateral tail base to allow lymphatic drainage to the inguinal lymph nodes. A control treatment of 200 pg per mouse anti-PD-L1 antibody (BioXcell, Lebanon, NH, USA) was administered intraperitoneally (i.p.) on days 7 and 10 post cancer cell inoculation.

[0189] Tumor volumes were assessed when tumors were measurable and calculated according to the formula: length x width2x 0.52. Body weight was monitored twice per week. Mice were euthanized around 16 days after tumor inoculation, and tumors were excised and weighted. Tissue samples were snap frozen. Analysis of gene expression in tumor tissue

[0190] Total RNA was extracted from frozen B16-F10 tumor tissues using the TRI Reagent® according to the manufacturer's protocol. RNA quantity and purity were assessed by BioDrop Spectrophotometer (BioDrop, Cambridge, UK). cDNA was synthesized using the qScriptTM cDNA Synthesis Kit (QuantaBio, Beverly, MA, USA). Quantitative real- time PCR was performed in triplicates by the Real-time PCR Instrument QuantS tudioTMl (Applied Biosystems, Waltham, MA, USA) using the SYBR Green Master Mix (Applied Biosystems) according to the manufacturer's instructions. The primers were obtained from Syntezza Bioscience Ltd. (Jerusalem, Israel). Sequences of the primers are as follows: For GAPDH: forward, 5'- GTGGAGTCATACTGGAACATGTAG-3' (denoted herein by SEQ ID NO. 3), reverse, 5'- AATGGTGAAGGTCGGTGTG-3’ (denoted herein by SEQ ID NO. 4). For TNFa: forward 5’-AGGCTGCCCCGACTACGT-3’ (denoted herein by SEQ ID NO. 5), reverse 5’-GACTTTCTCCTGGTATGAGATAGCAAA-3’ (denoted herein by SEQ ID NO. 6). For IFNy: forward 5’-CTGAGACAATGAACGCTACACA-3’ (denoted herein by SEQ ID NO. 7), reverse 5’-TCCACATCTATGCCACTTGAG-3’ (denoted herein by SEQ ID NO. 8). For Granzyme B: forward 5’-AAGAGAGCAAGGACAACACTC-3’ (denoted herein by SEQ ID NO. 9), reverse 5’-CATGTCCCCCGATGATCTC-3’ (denoted herein by SEQ ID NO. 10).

[0191] Statistical Analysis

[0192] One-way ANOVA and Tukey's test were used to determine statistical significance. Differences between groups were considered significant at a p-value <0.05. Statistical analysis was performed by GraphPad Prism 5 for Windows (GraphPad Software, San Diego, CA, USA).

[0193] EXAMPLE 1

[0194] Synthesis and characterization of functionalized mesoporous silica nanoparticles

[0195] Spherical MSNPs, at the average diameter size in the range of 50-80 nm as visualized by transmission electron microscopy (TEM, Fig. 1 A), were synthesized by sol- gel chemistry as detailed above. The surface of the MSNPs was aminated with 3- aminopropyl silane (MSNP-NH2), as verified by Fourier-transform infrared spectroscopy (FTIR) analysis of that indicated specific bending vibrations of the N-H groups at 1520cm-1(Fig. IB). The aminated MSNPs were then loaded with CLT and surface-coated with BSA via carbodiimide chemistry, as verified by the emergence of the 1365 cm-1peak indicating the presence of C-N bond stretching vibrations of the BSA-conjugated nanoparticles (MSNP-BSA, Fig. IB). D-mannosamine (MaN) was then conjugated to BSA (MNSP-MaN) and the successful modification was confirmed by the emergence of a new peak at 1427cm-1due to C-O- bond frequency of the mannose ring (Fig. IB). The hydroxyl groups of mannose were then capped with 4-mercaptophenyl boronic acid (4- MBA). The successful annexation of 4-MBA was also confirmed by FTIR, showing the presence of the new thiol (-SH group) peak at 2625cm-1(Fig. IB & Fig. 2). UV absorption measurements confirm the successful loading of CLT into the porous MSNP-NH2, with the CLT absorption maximum detected at 254 nm (Fig. 2C).

[0196] A schematic presentation of the step-wise synthesis and functionalization of the MSNPs prepared as described above along with a model of the mechanism of action of the MSNPs are shown in Figure 3.

[0197] It was previously reported that PD-L1 and CD80 on dendritic cells (DCs) interact with PD-1 and CD28 (respectively) on T cells to regulate T cell priming, which is mandatory for initiating cell differentiation and effective immune response. However, PD-L1 expression on immature and mature DC (iDCs & mDCs respectively) in tumor and peripheral tissues of cancer patients is elevated, and their interaction with PD-1 positive T cells in trans impairs their costimulatory function with respect to T cell activation. Furthermore, PD-L1 expression on DCs further sequesters CD80 in cis, lowering DC costimulatory signaling.

[0198] The MSNPs prepared as described above were functionalized with a short all D amino acid residue PD-L1 binding peptide (also termed herein “cysteine terminated PDL1bp”) denoted by SEQ ID NO. 2 that binds to the PD-L1 IgV domain with high affinity (Chang, H. N. et al., Angew. Chem. Int. Ed. 2015, 54 (40), 11760 -11764) in order to inhibit PD-L1 mediated interaction. The surface area and pore size of the MSNPs were analyzed by Brunauer-Emmett- Teller (BET) and Barrett-Joyner-Halenda (BJH) analysis, respectively, as detailed above. BSA conjugation to seal the pores of MSNP-NH2 reduces the BET calculated surface area from 463.37 m2 / g to 58.467m2 / g (as shown in Fig. ID) and sealed the 3.2 nm size pores on the MSNP-NH2 (as shown in Fig. IE). Thermal degradation analysis (TGA) further confirmed the successful surface modification of the MSNPs as shown in Fig. IF. MSNP- BSA show significant degradation at the range of 200-400°C, due to decomposition of the amino acids of BSA. The MSNP-MaN showed a typical carbohydrate degradation pattern between 450°C and 550°C. PDL1bp conjugation to MSNP-MaN increased weight loss between 200-400°C due to the higher content of peptide. The weight loss at low temperatures up to 100°C can be explained by the presence of water in the void spaces in all formulations. The hydrodynamic radius of MSNP-NH2, MSNP-BSA and MSNP- MaN-PDL1bp were about 80 nm, 110 nm and 254 nm, respectively (as shown in Fig. 1G). The increase in the size of MSNP-MaN-PDL1bp relative to MSNP-NH2 can be explained by hydrophobic interactions and the association of the peptide decorated nanoparticle (NPs). Zeta potential measurements further confirmed the successful modification of the NPs surface. BSA functionalization switched the zeta potential of MSNP-NH2 from +18.3 mV +1.50 mV to -12.8 mV+ 1.07 mV (as shown in Fig. 1H) (owing to the overall negative charge of BSA at pH 7). Mannose conjugation slightly increased the zeta potential, most likely due to the decrease in the number of free carboxylic groups in the BSA structure. 4-MBA conjugation switches back the zeta potential to a slightly positive potential (+4.87 mV +0.77 mV) due to the decrease of free hydroxyl groups on mannose. Finally, the PDL1bp conjugation via disulfide bonds reduced the zeta potential to -2.8 mV + 0.58 mV, due to the presence of the peptide with isoelectric point at physiological pH.

[0199] As indicated above, the PDL1bp was linked to the mannose-terminated MSNPs via boronate ester and disulfide bonds that can be cleaved by H2O2and GSH, respectively, both of which are abundant at the tumor microenvironment (TME). The release profile of PDL1bp was evaluated in 10 mM H2O2or 10 mM DTT to simulate in vitro the conditions at the TME. Fig. 4A shows a fast and continuous release of PDL1bp over time in the presence of DTT and H2O2. After 12 hours of incubation, more than 60% of the PDL1bp was released from the NPs incubated with either DTT or H2O2. Similarly, Trypsin treatment cleaved the conjugated BSA and reversed the closure of the pores, allowing for a progressive and continuous release of CLT from MSNPs as shown in Fig. 4B. The rate of CLT release from MSNPs after 24 hours of incubation with trypsin was about 4-times higher relative to non-treated NPs. Without wishing to be bound by theory, the slow release of CLT from non-treated MSNPs is most probably due to free diffusion of CLT to the media.

[0200] Blocking the PD-L1 pathway by the release of PDL1bp is intended to promote a dual effect on DCs, i.e., by reducing the PD-1 / PD-L1 inhibitory signal as well as by releasing PD-L1 / CD80 sequestration for robust costimulatory signaling on T cells via CD28. In addition, the released clotrimazole (CLT) is intended to stimulate T cell activity via the lysosomal pathway.

[0201] Various MSNPS carrying only two functions i.e., MSNPs carrying only mannose and CLT, MSNPs carrying only mannose and PD-L1bp, etc., were also prepared using the methods described above, as intermediate products.

[0202] EXAMPLE 2

[0203] Co-localization and molecular binding studies of PDLlbp -decorated MSNPs to DC2.4

[0204] The murine dendritic cells DC2.4 were used for proliferation assays, cellular uptake studies, and PD-L1 and CD80 expression assays after stimulation with LPS (250 ng / mL) and IFNy (250 ng / mL) for 24 hours, following the procedures described above. The results are shown in Fig. 5 and in Fig. 6, as follows.

[0205] As shown in Fig. 5, the impact of PD-L1bp-decorated and CLT-loaded NPs (MSNP-MaN-PDL1bp / CLT) on dendritic cells (DC2.4) proliferation was first evaluated. The MSNP by themselves are not harmful to the cells, and the CLT-loaded MSNPs slightly increased cell proliferation at lower concentrations. Similarly, a lower concentration of free CLT (10 μg / mL, 27.5 μM) stimulated cellular growth, whereas CLT decreased cell viability at a dose of 20 μg / mL (55μM). Free PDL1bp did not induce any toxic effect on the DC2.4 cells. MSNPs were taken-up more significantly by matured DC2.4 cells (mDC2.4, activated by IFNy at 250 ng / mL and LPS at 250 ng / mL for 24 hours) when compared to the immature, non-activated DC2.4 cells (iDC2.4) as shown in Fig. 6. Cell activation was confirmed by observing structural morphological changes under microscope (as shown in Fig. 7). MSNP-MaN / FITC were taken-up more significantly into mDC2.4 and iDC2.4 cells, relative to non-mannosylated MSNPs (Fig. 6). The uptake of the PD-L1bp decorated NPs (MSNP-MaN-PDL1bp / FITC) by mDC2.4 and iDC2.4 cells was decreased, without wishing to be bound by theory, probably due to less efficient endocytosis compared to particles carrying only the mannose receptor (Fig. 6).

[0206] Next, the expression of the PD-L1 molecule on mDC2.4 cells was assessed (at this stage, in the absence of the functionalized nanoparticles, as a control). While iDC2.4 cells express relatively low levels of PD-L1, approximately 94% of mDC2.4 cells were positive for PD-L1 and expressed high levels of the PD-L1 molecule (Fig. 8A). Additionally, LPS / IFNy activation of mDC2.4 resulted in a rise of the surface expression of CD80, compared to iDC2.4 cells (Fig. 8B).

[0207] In order to investigate the PD-L1:CD80 cis interaction, PD-L1 and the CD80 co- localization on DC2.4 cells were visualized. To this end, activated mDC2.4 and inactivated iDC2.4 were fluorescently labeled for PD-L1 and CD80 molecules. The level of PD-L1 and CD80 expression was significantly higher in mDC2.4 relative to iDC2.4, as indicated by confocal images (Fig. 9A, Fig 9B, Fig. 10). For mDC2.4, the increased expression levels of PD-L1 and CD80 detected by flow cytometry correlated with enhanced fluorescent staining observed in the confocal microscope (as shown in Fig. 9B). The intensity plots along the diagonal line marked on the images revealed that on mDC2.4 the spatial distribution of fluorescence of PD-L1 and CD80 were correlated, indicating their co-localization on the cells (Fig. 9B), while for iDC2.4 cells, this was not observed (Fig. 9A). Thus, it appears that both receptors are highly expressed on mDC2.4 cells and do co-localize on the cell surface.

[0208] Next, in order to assess the binding affinity of the functionalized MSNPs to iDC2.4 and mDC2.4, DCs were treated with various types of functionalized FITC-loaded MSNPs. MSNP-MaN-PDL1bp / FITC showed significant binding to the cell surface of both iDC2.4 and mDC2.4 as shown in Fig. 9C and Fig. 9D. The binding of MSNP-MaN- PDL1bp / FITC to mDC2.4 was 3-folds higher relative to the binding to iDC2.4 (Fig. 9E, Fig. 9F), which correlates to the increase of PD-L1 molecules on the surface of mDC2.4 (Fig. 9A, Fig. 8A). The binding of the control, boronic acid-terminated MSNPs (MSNP- MaN-BA / FITC) without PDL1bp is caused by non-specific interactions with the DC2.4, as in these NPs the mannose residues are masked by 4-MBA, thereby restricting receptor- specific interactions with the DCs. Overall, the PDL1bp -decorated MSNPs showed high binding activity to DCs, and more specifically to mDCs, which overexpress the PD-L1 receptor.

[0209] EXAMPLE 3

[0210] Inhibition of cis PD-L1:CD8O interaction enhances recombinant CD28 binding

[0211] PD-L1 blockade by PDL1bp is expected to relieve CD80 cis sequestration by PD- Ll, making it available to interact with CD28. To assess the availability of CD80 for molecular interaction with recombinant CD28 (rCD28), the binding of fluorescently- labeled rCD28 to DCs in the presence or absence of MSNP-MaN-PDL1bp was tested and the results are presented in Fig. 11 A. CD80 expression levels were higher in mDCs than in iDCs, corresponding to approximately 73% and 60%, respectively, of cells making a molecular connection to rCD28 as shown in Fig. 11B. In addition, rCD28 showed enhanced binding to mDCs that were pre-treated with free PDL1bp (25 μg / mL) (Fig. 11C).

[0212] Similarly, an increase in rCD28-binding to mDCs was observed following anti- PD-L1 antibody treatment (up to 74%) (Fig. 12). These results indicate that PD-L1 blockade disrupts PD-LLCD80 cis interactions to release CD80, and thus promotes molecular interactions between CD80 / CD28, as described previously. The MSNPs decorated with an equivalent amount of PDL1bp (i.e., relative to 25μg / mL free PDL1bp), promote rCD28-binding in the same way as the unconjugated peptide, reaching to 88% of labeled cells as shown in Fig. 1 IB). The control MSNPs without PDL1bp (MSNP- MaN) did not affect the amount of available CD80 receptor on mDC2.4 cells (Fig. 11C). Then, while maintaining the same concentration of PDL1bp or equivalent MSNP treatments, the molecular interaction on mDC2.4 was examined with a higher concentration of rCD28. We infer from the results displayed in Fig. S8 that there was an approximate 20% increase in the rCD28 binding with a twice higher concentration. Binding of rCD28 did not increase proportionally with the increase of concentration; however saturation of binding capacities may limit the effects of increased rCD28 concentrations and dose-dependency. Overall, the peptide-decorated MSNPs were a highly effective treatment to restore CD80 availability to enhance CD80 / CD28 interactions.

[0213] EXAMPLE 4

[0214] Blocking PD-L1 improves cellular interactions and co-stimulation of EL4 cells

[0215] Next, it was investigated whether inhibiting PD-L1:CD80 cis connections can improve DCs contacts with CD28-expressing T cells and their subsequent activation. Murine EL4 T cells, which express the CD28 receptor (as shown in Fig. 14), were used in order to study the cellular interactions between mDC2.4 and T cells. mDC2.4 cells were treated with MSNP-MaN-PDL1bp before co-culturing with EL4 cells (Fig. 15A), and after fluorescent labeling (CD80 & CD28), the cells were fixed and imaged under confocal microscopy. When mDC2.4 cells were treated with free PDL1bp or MSNP- MaN-PDL1bp, more contacts between the EL4 cells and the DC2.4 cells were observed than with untreated mDC2.4 cells (Fig. 15B). Treatment with mannosylated MSNPs without a peptide (MSNP-MaN) resulted in a slight increase of cellular interactions between DCs and EL4 cells, most likely because of the mannose-decoration of MSNPs over mDC2.4, increasing cellular contacts.

[0216] The DCs / T cells interactions were further investigated on a larger scale by using Zeiss Cell discoverer 7. Approximately 500 cells over the course of 45 frames for each treatment group were imaged to assess the degree of interactions between the EL4 and mDC2.4 cells. Free PDL1bp or the peptide-conjugated MSNP treatment significantly increased the cellular contacts between the EL4 T cells and DC2.4 (Fig. 15C). The number of total cellular interactions counted in all frames was 45:68: 105:83 for untreated: MSNP-MaN: free PDL1bp: MSNP-MaN-PDL1bp-treated mDC2.4, respectively (Fig. 15D). The comparable results obtained for the free PDL1bp and the MSNP-MaN- PDL1bp particle demonstrate the accessibility of the MSNP-delivered peptide.

[0217] The interaction of CD80 on DCs with CD28 on T cells is a co-stimulatory pathway to activate T cells and induce IL-2 secretion. To test the ability of the MSNPs to stimulate T cell activity, we pre-treated mDC2.4 cells with MSNP-MaN-PDL1bp and then co- cultured the cells with EL4 cells at 1:5 DCs:T cells cellular ratio. The level of IL-2 secretion was measured by ELISA. As shown in Figure 15Ctreatment of mDC2.4 with MSNP-MaN-PDL1bp increased the amount of IL-2 secretion from EL4 T cells nearly to the same level as with free PDL1bp. Despite of having more cellular contacts in the MSNP-MaN group (Fig 15C), the mannosylated MSNPs did not trigger IL-2 production of T cells, without being bound by theory, perhaps since the PD-1 / PD-L1 pathway reduces CD28 signaling. It is important to note that mDC2.4 were not exposed to any antigen during treatment to preclude concerns regarding alternative activation pathways of T cells. Thus, inhibition of PD-L1:CD8O cis interactions by PDL1 bp-decorated MSNPs manifests an augmented cellular interaction and subsequent EL4 T cell activation.

[0218] EXAMPLE 5

[0219] CLT-mediated antigen presentation and DC maturation

[0220] Next, the uptake of Hen egg lysozyme (HEL) antigen and its subsequent presentation via MHC II on DC2.4 following pre-treatment with CLT-loaded MSNPs (Fig. 16A) was investigated. As detailed above, clotrimazole (CLT) is known to activate DC2.4 cells, as evidenced by the reported increased surface expression of the MHC II, CDl lc, and CD80 receptors. Surprisingly, the results shown in Figure 16B-16D demonstrate that the MHC II, CDl lc, and CD80 expression levels of DC2.4 cells treated with mannosylated MSNPs loaded with CLT (MSNP-MaN / CLT) were significantly higher when compared to free CLT treatment at a drug-equivalent dose (i.e., 27.5μM) (Fig. 16B - Fig. 16D, S 10). The mannose-tagged MSNPs were taken-up more rapidly into cells via receptor-mediated endocytosis, resulting in better cellular activation. Cells that were treated with mannosylated NPs without a drug (MSNP-MaN) showed basal levels of surface marker expression that were almost identical to untreated control cells. In general, iDC cells have a more pronounced phagocytic ability as compared to mDCs. Therefore, iDC2.4 cells were pre-treated for 1 hour with MSNP-MaN / CLT, free CLT or MSNP-MaN and then exposed to fluorescently labeled HEL. As demonstrated in Figure 16E, HEL antigen was taken-up more significantly by cells that were pre-treated with MSNP-MaN / CLT or with free CLT compared to control groups and was localized mainly in lysosomal compartments. Similarly, Manders colocalization coefficient (MCC) in MSNP-MaN / CLT and free CLT was nearly 0.71 and 0.52 respectively greater than in the untreated group (Fig. 18). This suggests that a higher proportion of HEL antigen will be presented as stable peptide-MHC II complex after CLT treatment. An increase of the number of lysosomes in DC cells that were treated with free and MSNP-loaded CLT was also noted (Fig. 19), possibly speeding up the process of antigen presentation. When the HEL-treated DCs were co-cultured with 3A9 T cells, a cell line primed against the HEL peptide, a significant increase of IL-2 secretion was detected when the DC2.4 cells were pre-treated with MSNP-MaN / CLT. The IL-2 secretion of 3A9 cells after co-culture with free CLT-treated DCs was enhanced, though to a substantially lesser extent than after MSNP-MaN / CLT treatment (Fig. 16F).

[0221] To test the benefit of the nanoparticle formulation for activating simultaneously both pathways, i.e., DC activation for antigen presentation together with T cell priming by interfering with PD-L1 mediated interactions, the enhancement of antigen presentation by CLT in parallel to PD-L1 blockade by PDL1bp was tested. To this end, DC2.4 cells were first incubated with the NP construct MSNP-MaN / CLT along with free PDL1bp and simultaneous exposure to HEL for 1 hour, before co-culturing with 3A9 T cells. This experimental setting simulates the scenario in which PDL1bp is cleaved under the tumor microenvironment (TME) conditions and binds to the cells as free peptide, while exposing the targeting MaN-decoration. As shown in Figure 20, a significant increase in IL-2 release from the treated 3A9 T cells was noted following the combination of MSNP- MaN / CLT + free PDL1bp, which can be attributed to improved CLT-mediated antigen presentation and the suppression of PD-L1 cis interaction with CD80 on DCs or with PD- 1 on T cells. The enhancement of IL-2 secretion following treatment with free CLT, free PDL1bp or their combination was low as compared to MSNP-MaN / CLT + free PDL1bp. It is important to note that the effect of the MSNPs, even without CLT (MSNP-MaN- PDL1bp), was higher when compared to the free peptide alone (Figure 20). These results highlight the ability to modulate the immune checkpoint using drug-free nanoparticles (i.e., a nanoformulation without a conventional drug) and the importance of the multivalent presentation of PDL1bp on the MSNPs to improve T cell activation. Overall, our MSNP-based carrier may function as an efficient immuno-modulator for priming T cells at the TME through treated DCs.

[0222] EXAMPLE 6

[0223] Simultaneous activation of DCs and PD-L1 blockade inhibits murine melanoma tumor growth

[0224] Finally, it was tested whether blocking of PD-L1-mediated activity while simultaneously enhancing antigen presentation by DCs may inhibit tumor growth in mice. To this end, immunocompetent C57BL / 6 mice bearing s.c. B16-F10 tumors were treated subcutaneously (by tail base injection, to allow lymphatic drainage to the inguinal lymph node) with MSNP-MaN-PDL1bp / CLT or the various control treatments (among others, with intermediate MSNP constructs) at doses equivalent to 40 mg / kg CLT and equivalent peptide doses (1 mg / kg PDL1bp) twice weekly, starting from day 3 after subcutaneous cancer cell inoculation, for a total of 4 treatments (Fig. 21 A). A control treatment of 200 pg per mouse with anti-PD-L1 antibody was administered i.p. on days 7 and 10 post cancer cell inoculation (the effective dose and dosing frequency that has been successful in B16-F10 melanoma model).

[0225] As shown in Fig. 21B it was found that MSNP-MaN-PDL1bp / CLT significantly (P<0.001) inhibited B16-F10 melanoma growth in mice compared to peptide-decorated NPs without CLT (MSNP-MaN-PDL1bp) or CLT-loaded NPs without peptide decoration (MSNP-MaN / CLT) treatment (Fig. 21B). In addition, MSNP-MaN-PDL1bp / CLT treatment showed a clear trend toward higher tumor suppression when compared to anti- PD-L1 antibody, although the differences were not significant. Moreover, the weight of tumor mass in the MSNP-MaN-PDL1bp / CLT group at the end of the experiment was significantly lower than in MSNP-MaN-PDL1bp- or MSNP-MaN / CLT-treated groups, demonstrating that MSNP-MaN-PDL1bp / CLT could induce anti-tumor immune responses in the melanoma mouse model (Fig. 21C). The stability of body weight throughout the study (Figure 22) reflects lack of systemic toxicity and the general safety of the treatment.

[0226] Notably, it was found that the mice treated with MSNP-MaN-PDL1bp / CLT showed the highest levels of TNFa, IFNy and Granzyme B mRNA in tumor tissues (Fig. 21D - Fig. 21F). As known in the art, fFNy is typically associated with enhanced antigen priming and presentation by antigen presenting cells and serves as a predictor of antigen- specific cytotoxic CD8+T-lymphocyte (CTL)-mediated responses. Therefore the results above suggest that the exemplified MSNPs (i.e., MSNP-MaN-PDL1bp / CLT) play a major role in priming antigen specific T cells response. Additionally, IFNy and GrzmB are T-cell effector molecules, and the higher GrzmB levels indicate increased cytolytic function of immune cells (mostly T-cells and NK cells).

[0227] As detailed above, it has been recently reported that CLT can potentiate the anti- tumor efficacy of anti-PDl by stimulating tumor immune microenvironment in tumor- bearing mouse models (Wang, Z. et al., Journal for Immunotherapy of Cancer 2021, 9 (5)). The above results show that the combination of free CLT with anti-PD-L1 therapy (i.e., free PDL1bp) does not enhance antitumor immunity but rather promotes tumor growth (Fig. 21C).

[0228] EXAMPLE 7

[0229] PD-Llbp-PCL-b-PEG polymeric micelles (PM) enhance molecular interactions between DCs and T cells

[0230] Synthesis of PEG-b-PCL-IR806 and PCL-b-PEG-PD-Llbp wherein X is PDL1bp; R is a dye molecule (e.g., IR806); and wherein m and n are the degree of polymerization of PCL and PEG blocks, respectively. Synthesis of PEG-b-PCL-IR806

[0231] PEG-b-PCL-IR806 was prepared by conjugating carboxylic acid-functionalized IR780 dye with maximal absorption at 806 nm (designated IR-806) to poly(ethylene glycol)-b-poly(ε-caprolactone) ( PEG-b-PCL) di-block copolymer (PEG average Mn2000, PCL average Mn5000, Sigma-Aldrich) by esterification with dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) as a coupling reagent (c.f Fig 23A). IR806 (10 mg, 0.012 mmol), DCC (5.2 mg, 0.025 mmol), and DMAP (3.1 mg, 0.025 mmol) were dissolved in 2 mL of dry CH2CI2and stirred at 0°C for 2 h. Next, PEG-b-PCL-OH (89 mg, 0.012 mmol) was dissolved in dry CH2CI2(2mL) and added to the above reaction mixture with continued stirring for 22 h at room temperature. The formed dicyclourea (DCU) by-product was filtered-off and the polymer conjugate was precipitated by cold diethyl ether. PEG- / 2-PCL-IR8O6 was concentrated under vacuum, purified by LH20 column with methanol as eluent, and dried. This di- block copolymer was used to prepare PM and study the interaction between DC and T cells by confocal microscopy.

[0232] Synthesis of PCL-PEG-PD-L1bp conjugate

[0233] The NHS group in PCL-b-PEG-NHS (PEG average Mn2000, PCL average Mn5000, Creative PEGworks) was first converted into maleimide (MAL) to give PCL-6- PEG-MAL that was then conjugated to N-terminal cysteine harboring PD-L1bp by thiol- maleimide click reaction. PCL-b-PEG-NHS (0.0035 mmol, 0.025 g) was first dissolved in dry DMF (250 μL) Amino ethyl maleimide (0.0042 mmol, 0.001 g) and TEA (0.0128 mmol, 0.00179 mL) were mixed separately in dry DMF (250 μL), and slowly added to the PCL-b-PEG-NHS solution and stirred for 17 h at 30°C on water bath. Then, the product was dialyzed (MWCO: 3.5 kDa) against deionized water for 72 h to remove unreacted reactants and impurities. The PCL-b-PEG-MAL conjugate was lyophilized and characterized as detailed below. PD-L1bp (CNYSKPTDRQYHF denoted by SEQ ID NO.2) was then conjugated to PCL-b-PEG-MAL (c.f. Fig. 23B). PCL-b-PEG-MAL (0.0021 mmol, 0.015 g) was dissolved in dry DMF (400 μL). PD-L1bp (0.0042 mmol, 0.0071 g) and TEA (0.0021 mmol, 0.00029 mL) were added to the PCL-b-PEG-MAL solution. The reaction mixture was stirred at 37°C for 24 h on water bath. The block copolymer-peptide conjugate was purified by dialysis (MWCO: 3.5 kDa) against deionized water for 72 h and lyophilized. Yield: 73.38%.

[0234] The synthesis of PEG-b-PCL-Dye and PCL-PEG-PD-L1bp conjugates is schematically presented in Fig. 23 and represented by the above structure formula.

[0235] Characterization of di-block polymers

[0236] 1H-NMR experiment was performed using a Bruker Avance 500 MHz instrument at 298 K using CDCI3, DMSO-d6and D2O as solvents. Chemical shifts (δ) are reported in ppm using deuterated solvent as an internal standard. The polymers and micelle X-ray diffraction (XRD) data were recorded with a Bruker D8 Advance diffractometer using Cu Ka radiation from 5° to 60° at room temperature. X-ray photoelectron spectroscopy (XPS) experiment (ESCALAB 250, Thermofisher) was performed on PCL-b-PEG-PD- Llbp and PEG-b-PCL to verify peptide conjugation to the polymer. PD-L1bp conjugation to polymer was further investigated using fluorescamine assay. The PD-L1bp, PCL-b- PEG-PD-L1bp, and PCL-b-PEG samples were dissolved in DMF, then different concentrations of the sample were added to 100 of flu μorLescamine (4 mg / mL in acetone). After two minutes of incubation, the fluorescence intensity of the samples was measured at an excitation wavelength of 390 nm and an emission wavelength of 475 nm (data not shown).

[0237] Preparation and characterization of polymeric micelle (PM)

[0238] Characterization of polymeric micelle (PM) is shown in Fig. 24. Specifically, micelles with different composition ratios of PCL-b-PEG-PD-L1bp and PEG-b-PCL-IR806 block copolymer were prepared first to identify the polymeric micelles (PM) formulation with optimal size, morphology, and polydispersity. To prepare the PM, PEG-b-PCL-IR806 and PCL-b-PEG-PD-L1bp were dissolved in 1 mL acetone in weight ratios of 50:50, 70:30, and 90:10. The solution was then added drop wisely into the 3 mL of deionized water and kept stirring for 17 h to allow evaporation of the acetone and form the solution. Dynamic light scattering (DLS) was used to measure the size of the micelle at a fixed scattering angle (90°). The morphology of the micelle was examined by Transmission electron microscopy (TEM). The Critical micelle concentration (CMC) was measured using hydrophobic fluorescent pyrene as a probe. The physicochemical characteristics of PM are shown in Table 1 below.

[0239] Table 1: The physicochemical characteristics of PM

[0240] PM prepared at the ratio 70:30 of PEG-b-PCL-IR806: PCL-b-PEG-PD-L1bp showed the best physicochemical properties (spherical morphology, small size and narrow PD) and this ratio was thus selected for preparing the control PM with a peptide but without a dye (PCL-Zt-PEGiPCL-Zt-PEG-PD-L1bp), or the blank PM with a dye and without a peptide (PEG-b-PCL-IR806:PCL-b-PEG).

[0241] Micelles prepared with 70% of PEG-b-PCL-IR806 were at the smallest size (around 80 nm) and show a narrow distribution, suggesting optimum interaction between the hydrophobic PCL segment in PD-L1bp-PEG-b-PCL and IR806 dye in PEG-b-PCL- IR806. Control PM that lacks IR806 in PEG-b-PCL exhibited larger hydrodynamic size (~ 200 nm, Table 1), indicating the contribution of IR806 to the compaction of PMs. PM with 90% of PEG-b-PCL-IR806 were larger in size relative to micelle with 70%. The zeta potential analysis results show that PM with higher contents of the PD-L1bp (with net charge +1) on the PM exhibit higher (slightly positive) zeta potential (Table 1). The blank PM that lacks PD-L1bp shows a slight negative zeta potential (Table 1, Fig. 24B). Near- neutral zeta potential value is desirable to avoid potential interaction with serum proteins in the circulation. The TEM image shown in Fig. 24C indicated that PM composed of PEG-b-PCL-IR806 block copolymer show spherical morphology. The hydrophobic PCL chain and the presence of IR806 promote the formation of micelle spheres. At the absence of IR806 dye, a mixture of spheres and rod-shaped structured PM were obtained (Fig. 24D). The assembly of the block copolymers into a micelle structure was validated by XRD analysis indicating almost complete masking of the sharp crystal peaks of PCL and IR806 following micelle formation. The slight intensity observed at 21° & 23°, may be attributed to the crystallinity of the PEG (data not shown).

[0242] To assess the availability of CD80 for molecular interaction with recombinant CD28 (rCD28), the binding of fluorescently-labelled rCD28 to DCs in the presence or absence of PDL1bp-PM was tested. As shown in Fig. 25, a significant increase in rCD28-binding to mature (activated) DCs was observed following PDL1bp-PM treatment relative to blank PM. PDL1bp in an equivalent amount of PDL1bp-PM promote rCD28-binding to the same extent as PDL1bp-PM. The extent of free PD-L1 and PM binding was similar to iDC2.4 cells. The results suggest that PD-L1-PM can restore CD80 availability to enhance CD80 / CD28 interactions. Confocal images show more contacts between the EL4 cells and the DC2.4 cells following treatment with PD-L1bp and PD-L1bp-PM as shown in Fig. 26. This confirms that PD-L1bp-PM releases CD80 sequestration in cis by PDL1, thus allowing CD80 to interact with CD28 on T cells. Fig. 27 indicates that the binding of PD-L1bp-PM to PDLl-overexpressing dendritic and cancer cells was significantly higher relative to non-stimulated cells or control PM without PD-L1bp decoration.

[0243] Table 2 Amino add and nucleic acid sequences of peptides and primers described above

Claims

CLAIMS:

1. A nanoparticle-based delivery system comprising at least one dendritic cell activator, at least one immune checkpoint inhibitor and at least one ligand of a dendritic cell receptor.

2. The nanoparticle-based delivery system according to claim 1, wherein said at least one dendritic cell activator is embedded within the nanoparticle or coupled to said nanoparticle by at least one cleavable linker.

3. The nanoparticle-based delivery system according to claim 1, wherein the nanoparticle is a hollow or porous nanoparticle, and wherein said at least one dendritic cell activator is embedded within the nanoparticle.

4. The nanoparticle-based delivery system according to claim 1 or claim 2, wherein said at least one dendritic cell activator is attached to said delivery system by a cleavable linker sensitive to cleavage in the tumor microenvironment.

5. The nanoparticle-based delivery system according to any one of the preceding claims, wherein the exterior surface of said nanoparticle is coated with at least one moiety modulating the release of said at least one dendritic cell activator.

6. The nanoparticle -based delivery system according to claim 5, wherein said moiety modulating the release of said at least one dendritic cell activator is coupled to the exterior surface of said nanoparticle via at least one cleavable linker sensitive to cleavage in the tumor microenvironment, preferably an acid-labile linker, an enzyme-cleavable linker, a reduction sensitive linker or a reactive oxygen spices (ROS)-sensitive linker.

7. The nanoparticle-based delivery system according to claim 5 or claim 6, wherein said moiety modulating the release of said at least one dendritic cell activator is a polypeptide, or a copolymer, preferably a pH sensitive copolymer.

8. The nanoparticle-based delivery system according to any one of claims 5 to 7, wherein said moiety modulating the release of said at least one dendritic cell activator is albumin, such as bovine serum albumin (BSA) or human serum albumin (HSA).

9. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said at least one dendritic cell activator is an agent enriching lysosomal pathway-associated genes, an inflammatory cytokine, a chemokine or an oligonucleotide.

10. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said at least one dendritic cell activator is clotrimazole.

11. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said at least one immune checkpoint inhibitor is directed against any one of PD-1 / PD-L1, TIM-3 / Ceacam-l / Galectin-9, LAG-3 / Galectin-3 / LSECtin / MHC-II, CTLA-4 / CD80(B7.1) / CD86(B7.2) or DIGIT / CD155 / CD112.

12. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said at least one immune checkpoint inhibitor is directed against PD-L1 or PD- 1.

13. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said at least one immune checkpoint inhibitor is directed against PD-L1.

14. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said at least one immune checkpoint inhibitor comprises a peptide, a polypeptide, an oligonucleotide, a polynucleotide, or a small molecule inhibitor.

15. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said at least one immune checkpoint inhibitor is not an antibody.

16. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said at least one immune checkpoint inhibitor comprises a PD-L1 binding peptide.

17. The nanoparticle -based delivery system according to claim 16, wherein said PD- L1 binding peptide comprises an amino acid sequence which is at least 80% identical to the amino acid sequence Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe, denoted by SEQ ID NO. 1 or to the amino acid sequence Cys-Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg- Gln-Tyr-His-Phe, denoted by SEQ ID NO. 2.

18. The nanoparticle -based delivery system according to claim 16 or claim 17, wherein said PD-L1 binding peptide comprises or consists of the amino acid sequence Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe, denoted by SEQ ID NO. 1 or the sequence Cys-Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe, denoted by SEQ ID NO. 2.

19. The nanoparticle-based delivery system according to any one of claims 16 to 18, wherein said PD-L1 binding peptide comprises or consists of an amino acid sequence which is an all D amino acid configuration of the amino acid sequence Asn-Tyr-Ser-Lys- Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe denoted by SEQ ID NO. 1, or an amino acid sequence which is an all D amino acid configuration of the amino acid sequence Cys-Asn-Tyr-Ser- Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe, denoted by SEQ ID NO. 2.

20. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said at least one immune checkpoint inhibitor is coupled to the exterior surface of said functionalized nanoparticle, optionally by a cleavable linker.

21. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said at least one ligand of a dendritic cell receptor is coupled to the exterior surface of said nanoparticle delivery system, optionally by a cleavable linker.

22. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said immune checkpoint inhibitor is coupled to the exterior surface of said nanoparticle delivery system indirectly, via a cleavable linker attached to the at least one ligand of a dendritic cell receptor that is coupled to the exterior surface of said nanoparticle delivery system.

23. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said dendritic cell receptor is a mannose receptor or CD11c.

24. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said dendritic cell receptor is a mannose receptor.

25. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said at least one ligand of a dendritic cell receptor is a mannose moiety or a peptide ligand of the DC receptor CD 11c, the peptide ligand having the amino acid sequence FYPSYHSTPQRP denoted by SEQ ID NO. 11.

26. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said at least one ligand of a dendritic cell receptor is a mannose moiety.

27. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said nanoparticles are selected from metal nanoparticles, silica nanoparticles (e.g., mesoporous silica nanoparticles), polymer-based nanoparticles (such as, polylactic-co-glycolic acid (PLGA) or polycaprolactone (PCL), polymeric micelles (such as PEGylated polymers), polymer conjugates (such as polymer-drug conjugates, polymer-ligand conjugate) or lipid-based nanoparticles (such as liposomes, stealth liposomes, vesicles or solid lipid nanoparticles), or a combination thereof.

28. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said nanoparticle is a silica nanoparticle.

29. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said nanoparticle is a mesoporous silica nanoparticle (MSNP).

30. The nanoparticle-based delivery system according to any one of the preceding claims, wherein the nanoparticles have a mean particle size of between about 20 nm to about 1000, between about 20 to 800 nm in diameter, preferably between about 50 to 400nm in diameter, more preferably wherein the nanoparticles have a mean particle size of about 80 to 300 nm in diameter.

31. A nanoparticle-based delivery system according to claim 1, wherein said nanoparticles are porous or hollow nanoparticles, said at least one immune checkpoint inhibitor is a PD-L1 binding peptide, coupled to the exterior surface of said nanoparticles via a cleavable linker, wherein said at least one dendritic cell activator is clotrimazole, embedded within said nanoparticles, and wherein said exterior surface of said nanoparticles is decorated with a mannose moiety.

32. The nanoparticle-based delivery system according to claim 31, wherein said clotrimazole is loaded in an amount of about 2% to 20% by weight of the weight of the nanoparticles.

33. The nanoparticle -based delivery system according to claim 31 or claim 32, wherein said PD-L1 binding peptide comprises the amino acid sequence denoted by SEQ ID NO. 1 or the amino acid sequence denoted by SEQ ID NO. 2.

34. The nanoparticle -based delivery system according to claim 31 or claim 32, wherein said PD-L1 binding peptide comprises an amino acid sequence which is an all D amino acid configuration of the amino acid sequence denoted by SEQ ID NO. 1 or the amino acid sequence denoted by SEQ ID NO. 2.

35. The nanoparticle-based delivery system according to any one of the preceding claims, wherein said nanoparticle -based delivery system further comprises an imaging group.

36. A pharmaceutical composition comprising the nanoparticle-based delivery system according to any one of the preceding claims and at least one carrier and / or excipient.

37. A method of treating a disease or disorder in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of thenanoparticle-based delivery system as defined in any one of claims 1 to 35 or the pharmaceutical composition according as defined in claim 36.

38. The method according to claim 37, wherein said disease or disorder is a proliferative disease or disorder or an immune -related disease or disorder.

39. The method according to claim 37 or claim 38, wherein said disease or disorder is cancer.

40. The method according to claim 39, wherein said cancer is melanoma, lung cancer (both non-small cell lung cancer (NSCLC) and small cell lung cancer), kidney cancer, bladder cancer or head and neck squamous cell carcinoma (HNSCC).

41. A method of immunomodulation in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of the nanoparticle-based delivery system as defined in any one of claims 1 to 35 or the pharmaceutical composition according as defined in claim 36.

42. The method of immunomodulation according to claim 41, for at least one of dendritic cell activation and T cell activation.

43. The nanoparticle-based delivery system as defined in any one of claims 1 to 35 or the pharmaceutical composition as defined in claim 36 for use in a method of treating a disease or disorder in a subject in need thereof.

44. The nanoparticle-based delivery system as defined in any one of claims 1 to 35 or the pharmaceutical composition according as defined in claim 36 for use in a method of immunomodulation in a subject in need thereof.

45. A method for preparing the nanoparticle-based delivery system of any one of claims 1-35, said method comprising at least one of the following steps: a) providing organic or inorganic nanoparticles, b) encapsulating within the nanoparticles or attaching thereto at least one dendritic cell activator optionally using at least one cleavable linker,c) optionally coating the exterior surface of the nanoparticles with at least one moiety modulating the release of said at least one dendritic cell activator, d) attaching to said nanoparticles at least one immune checkpoint inhibitor, and at least one ligand of a dendritic cell receptor to the exterior surface of the nanoparticles, optionally using at least one cleavable linker.

46. The method of claim 45, wherein said nanoparticles are porous or hollow nanoparticles and said at least one dendritic cell activator is encapsulated therein, and wherein said method further comprises coating the exterior surface of said nanoparticles with at least one moiety modulating the release of said at least one dendritic cell activator.

47. The method of claim 45, wherein said at least one of the immune checkpoint inhibitor, dendritic cell activator, and ligand of a dendritic cell receptor are attached to the surface of the nanoparticle through a cleavable linker.

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