Nanovesicles for engineering immune cells

Abstract

The present invention concerns, among others, a nanovesicle comprising an aqueous core and a shell formed from a plurality of conjugates, each conjugate comprising a head portion, a tail portion and an intermediate portion in-between, wherein the head portion contains a first drug component, the tail portion contains a polymer component and optionally a second drug component and / or a targeting ligand, and the intermediate portion contains a cleavable linker, and wherein the head portion has a higher hydrophobicity than the tail portion.

Description

[0001] Nanovesicles for engineering immune cells

[0002] Technical field

[0003] The invention pertains to the field of engineering immune cells. In particular, nanovesicles are described that are capable of entering immune cells without conventional targeting moi- eties. By delivering immune receptor agonists highly efficient activation of immune cells can be achieved. Furthermore, the immune cells are matured into powerful antigen-presenting cells. By this strategy, nanovesicle-engineered B cells are able to prime and activate both helper and cytotoxic T cells which are able to recognize and kill cancer cells or infected cells. Additionally, the nanovesicles can promote B cell differentiation into plasma cells which release antibodies after vaccination, and reduce the unwanted immunoregula- tory phenotypes of B cells.

[0004] Background of the invention

[0005] B cells are crucial in anti-virus immunity and anti-cancer immunity. They play a crucial role as antigen-presenting cells, activating both cytotoxic and helper T cells against cancer cells. Moreover, B cells differentiate into plasma cells, releasing anti-cancer antibodies binding to cancer cells and destroy them. Beyond cancer therapy, B cells are indispensable in anti-infection immunity and vaccination, exemplified in the context of fighting COVID-19 infection antibody response after vaccination. Despite their pivotal role, the activation of B cells is limited. This limitation arises from a lack of comprehensive understanding of B cell immunity and a scarcity of prior research on engineering therapeutic agents to activate B cells. Therefore, therapeutic agents activating B cells are lacking.

[0006] Due to these limitations, it was an object of the present invention to provide means and methods suitable for the activation of immune cells, in particular B cells.

[0007] This problem is solved by the present invention as defined in the appended claims and described in the following.

[0008] Summary of the invention

[0009] In a first aspect of the present invention, a nanovesicle comprises an aqueous core and a shell formed from a plurality of conjugates, each conjugate comprising a head portion, a tail portion and an intermediate portion in-between, wherein the head portion contains a first drug component, the tail portion contains a polymer component and optionally a second drug component or a targeting ligand, the intermediate portion contains a cleavable linker, and wherein the head portion has a higher hydrophobicity than the tail portion.

[0010] A conjugate comprising a head portion, a tail portion and an intermediate portion in-between, wherein the head portion contains a first drug component, the tail portion contains a polymer component and optionally a second drug component and / or a targeting ligand, the intermediate portion contains a cleavable linker, and wherein the head portion has a higher hydrophobicity than the tail portion, forms a second aspect of the present invention.

[0011] According to a preferred embodiment of the invention, the polymer component is linear, branched, blocked or dendritic; is selected from the group consisting of polyethylene glycol (PEG), dextran, polyglycerol, polyvinyl alcohol, polymethacrylates, polymethacrylamides, polyacrylates, polyacrylamides, polymethacrylic acid, polyacrylic acid, polyvinylpyrrolidone, polysarcosines, poly(amino acid)s, poly(thioglycidyl glycerol) and derivatives of the above hydrophilic segments. It is further preferred that the polymer component has a molecular weight ranging from 200 Da to 100,000 Da, preferably 2,000 Da to 8,000 Da, more preferably 3,000 Da to 7,000 Da, most preferably 4,000 Da to 6,000 Da.

[0012] According to a further preferred embodiment of the invention, the linker is enzymatically cleavable, preferably by enzymes present in endosomes and lysosomes. More preferably, the linker is a glucuronide-containing linker being cleavable by p-glucuronidase, or a pep- tide-based linker being cleavable by a peptidase, or an ester bond being cleavable by an esterase, or an ester-based linker being cleavable by an esterase, or a combination of two or more linkers listed above. Most preferably, the linker is or includes: (also referred to herein as “GL2”).

[0013] In the above structure, R1 stands for the first drug component and R2 for the polymer component.

[0014] According to a further preferred embodiment of the invention, the first drug component includes an antigen, preferably being directly connected to the linker and containing a T cell epitope; and / or the first drug component includes an immune modulating agent, preferably selected from the group consisting of agonists to toll-like receptors, stimulator of interferon genes (STING), nucleotide-binding oligomerization domain (NOD)-like receptors, retinoic acid-inducible gene-1 (RIG-l)-like receptors, C-type lectin receptors and scavenger receptors.

[0015] In a further preferred embodiment of the invention, the first drug component includes SIINFEKL, OVA323-339, ErBb2; VYDFFVWL; E75, GP2; AE37; MT288-296, or Cy3.

[0016] In other preferred embodiments of the invention, the first drug component includes a peptide epitope derived from RNF43, KOC1 , DEPDC1 , MPHOSPH1 , TTK, URLC10, CypB, NRPL, p56Lck, ppMAPkkk, SART3, UBE2V, WHSC2, PPV, WT1 , KIF20A, VEGFR1 / 2, TS, SART3, Cyclophilin B, p56lck, ppMAPkkk, WHSC2, UBE2V, HNRPL, SART2, MRP3, PAP, PSA, EGF-R, IEX-1 , p-tublin5, HER2, DEPDC1 , MPHOSPH1 .

[0017] In a yet further preferred embodiment of the invention, the second drug component or targeting ligand, if present, contains an antigen directly connected to the polymer component that is recognized by immune cells, preferably antigen-presenting cells, more preferably B cells, macrophages and / or T-cells, most preferably B cells.

[0018] According to another preferred embodiment ofthe invention, the surface ofthe nanovesicle is functionalized with the second drug component and / or targeting ligand.

[0019] A third aspect of the present invention pertains to the use of a nanovesicle or a conjugate as described herein as a drug delivery vehicle. Preferably, the use is for delivering to immune cells, preferably antigen-presenting cells, more preferably B cells, macrophages and / or dendritic cells.

[0020] A fourth aspect of the invention relates to the use of a nanovesicle or a conjugate as described herein for activation of immune cells, preferably antigen-presenting cells, more preferably B cells, macrophages, dendritic cells and / or T-cells, most preferably B cells, macrophages and dendritic cells. Preferably, the activation of the immune cells involves induction of antigen presentation to T cells by B cells, dendritic cells and / or macrophages.

[0021] A fifth aspect of the invention provides a nanovesicle or conjugate as described herein for use in vaccination and / or immune therapy. Preferably the immune therapy is selected from the group consisting of therapies of cancers, infectious diseases, auto immune diseases, transplant rejections, and cardiovascular diseases.

[0022] According to a sixth aspect of the present invention, a method for producing a nanovesicle comprises the steps: providing a plurality of conjugates as defined herein; dispersing the plurality of conjugates in an organic phase; and adding the dispersed conjugates dropwise to an aqueous phase while stirring. Other possible preparation methods including especially microfluidics, (micro-) emulsion, thin-film rehydration, spray drying and high-pressure homogenization are mentioned further below.

[0023] In one embodiment, the method further comprises: functionalizing the surface of the nanovesicle.

[0024] A method of producing a conjugate as defined herein, according to a seventh aspect of the present invention, comprises the steps: providing a polymer component, a linker and a first drug component; coupling the first polymer component to the linker by click chemistry to provide a pre-conjugate comprising the first polymer component and the linker; activating a free end group of the linker contained in the pre-conjugate; coupling the first drug component to the activated end group to provide the conjugate comprising the first polymer component, the linker and the drug component; and optionally providing a targeting ligand and / or a second drug component, and coupling the targeting ligand and / or the second drug component to a free end group of the polymer component.

[0025] According to a further preferred embodiment of the method, the polymer component is as defined herein; and / or the linker is as defined herein; and / or the first drug component is as defined herein; and / or the targeting ligand and / or the second drug component is as defined herein.

[0026] Further aspects, embodiments and advantages of the invention will be apparent from the detailed description and the experimental section together with the drawings and the claims.

[0027] Detailed description of the invention

[0028] 1 . Nanovesicles

[0029] A nanovesicle as defined herein comprises an aqueous core and a shell formed from a plurality of conjugates. Each conjugate comprises a head portion, a tail portion and an intermediate portion between the head portion and the tail portion. The head and tail portions may form opposing end portions of the conjugate. The head portion includes a first drug component. The tail portion includes at least a polymer component. The intermediate portion includes a cleavable linker. The linker couples the first drug component and the polymer component to each other. The head portion has a higher hydrophobicity than the tail portion. Due to the amphiphilic nature of the conjugates, the tail portions may tend to orient radially inwards to the aqueous core or radially outwards, although imperfections of this structure are possible and encompassed by the present invention.

[0030] The nanovesicles are constructed from conjugates (described in more detail below) having properties similar to prodrugs. Hence, the conjugates can be referred to as polymer-pro- drug conjugates. Without being restricted thereto, the conjugates may include small molecule immune receptor agonists, pharmacologically active drugs (e.g. chemotherapeutics), antigens specific to tumors or infectious pathogens, and targeting ligands that are specifically recognized by B cell receptors. The thus defined nanovesicles of the present invention show a number of remarkable characteristics. They are capable of entering primary B cells without conventional targeting moieties. Highly efficient activation of immune cells can be achieved by nanovesicles containing single or combinations of immune receptor agonists. Furthermore, the nanovesicles are capable of engineering immune cells into powerful antigen-presenting cells. By this strategy, nanovesicle-engineered immune cells are able to prime and activate both helper and cytotoxic T cells which are able to recognize and kill cancer cells or infected cells. Additionally, the nanovesicles can promote B cell differentiation into plasma cells which release antibodies after vaccination, and reduce the unwanted immunoregulatory phenotypes of B cells.

[0031] Different conjugates may be present in the same nanovesicle. For example, a first subset of the conjugates may comprise a targeting ligand, whereas a second subset of the conjugates may have the same structure as the first subset conjugates except that the targeting ligand is lacking. In another example, a first subset of the conjugates may comprise a first antigen as the first drug component, whereas a second subset of the conjugates may comprise a second antigen as the first drug component different from the first antigen.

[0032] Preferred nanovesicles have a size ranging from 10 nm to 1000 nm, preferably 20 nm to less than 1000 nm but more than 21 nm, more preferably 50 nm to less than 1000 nm but more than 51 nm.

[0033] 2. Conjugates useful for forming the nanovesicles

[0034] The present invention relates not only to the nanovesicles incorporating the conjugates but also to the conjugates as such. A plurality of conjugates can be used to form nanovesicles as described herein.

[0035] As mentioned above, the conjugate comprises a head portion, a tail portion and an intermediate portion between the head portion and the tail portion. The head and tail portions form opposing end portions of the conjugate. The head portion includes a first drug component. The tail portion includes a polymer component. The intermediate portion includes a cleavable linker. The linker couples the first drug component and the polymer component to each other. The head portion has a higher hydrophobicity than the tail portion.

[0036] It is preferred that the conjugate consists of the head, tail and intermediate portions, and / or that the head portion consists of the first drug component and / or that the intermediate portion consists of the linker. The tail portion may, however, either consist of the polymer com- ponent or contain one or more additional moieties / components. For instance, the tail portion, in addition to the polymer component, may include a targeting ligand and / or a second drug component.

[0037] The following conjugate structures are particularly preferred:

[0038] As mentioned above, the head portion is more hydrophobic than the tail portion. That is, the tail portion is more hydrophilic than the head portion. The expression “more hydrophilic" means having a greater affinity for water or being more attracted to water. Hydrophilic substances are typically polar or have typically a high polarity. This makes them soluble or dispersible in water. Materials that are more hydrophilic tend to interact more readily with water and dissolve or disperse in it easily. Hydrophobic substances, in turn, tend to repel or not mix with water. This is because hydrophobic molecules are typically non-polar or have typically a low polarity, meaning they have a relatively equal distribution of electrons and don't readily form hydrogen bonds with water molecules. Instead, they tend to aggregate together, excluding water molecules and forming droplets or separate phases in water.

[0039] Preferably, the hydrophobicity I hydrophilicity is determined on the basis of solubility in water under standard conditions, i.e. at standard temperature (25°C) and standard pressure (101 ,3325 kPa). Accordingly, the head portion is considered more hydrophobic than the tail portion, if the solubility of the components making up the head portion in water under standard conditions is determined to be lower than the solubility of the components making up the tail portion. It is further preferred that the ratio of the head portion solubility to the tail portion solubility is 1 :2 or less, preferably 1 :5 or less, more preferably 1 :10 or less, most preferably 1 :20 or less. The solubility referred to herein pertains to the thermodynamic (equilibrium) solubility. It is the saturation solubility of a compound at the end of the dissolution process, where the dissolved compound is in equilibrium with the undissolved material in excess.

[0040] In a preferred embodiment, the head portion is hydrophobic, whereas the tail portion is hydrophilic. A component is considered hydrophobic, if the solubility in water under standard conditions is 0.1 g per 100 mL water or less, preferably 0.01 g per 100 mL water or less, more preferably 0.001 g per 100 mL water or less. Moreover, a component is considered hydrophilic, if the solubility in water under standard conditions is 0.1 g per 100 mL water or more, preferably 1 g per 100 mL water or more, more preferably 10 g per 100 mL water or more.

[0041] When comparing the hydrophobicity and / or hydrophilicity of the head portion and the tail portion, it may not be sufficient to consider only the first drug component and the polymer component, but further components that are included in the head and tail portions have to be taken into account.

[0042] The term “coupling” denotes a connection between two components so that they are hold together and understood as belonging to the same entity. The connections include covalent bonds, ionic bonds, hydrogen bonds, van der Waals forces and coordination bonds. Preferably, the first drug component is covalently bonded to a first end of the linker, and the polymer component is covalently bonded to a second end of the linker. If a second drug component or a targeting ligand is included in the conjugate, the second drug component or the targeting ligand is coupled, preferably covalently bonded, to the polymer component opposite to the linker.

[0043] In a preferred embodiment of the invention, the polymer component is selected from the group consisting of polyethylene glycol (PEG), dextran, polyglycerol, polyvinyl alcohol, poly meth acrylates, polymethacrylamides, polyacrylates, polyacrylamides, polymethacrylic acid, polyacrylic acid, polyvinylpyrrolidone, polysarcosine, poly(amino acid), poly(thioglyc- idyl glycerol) and derivatives thereof. Preferably, the polymer component is PEG.

[0044] As regards the size of the polymer component, it is preferred that its molecular weight ranges from 200 Da to 100,000 Da, preferably 2,000 Da to 8,000 Da, more preferably 3,000 Da to 7,000 Da, most preferably 4,000 Da to 6,000 Da. It shall be understood that each specific type of polymer is combinable with each specifically mentioned molecular weight range. For example, the polymer component may be PEG having a molecular weight ranging from 200 Da to 100,000 Da, preferably PEG having a molecular weight ranging from 2,000 Da to 8,000 Da, more preferably PEG having a molecular weight ranging from 3,000 Da to 7,000 Da, most preferably PEG having a molecular weight ranging from 4,000 Da to 6,000 Da, or the polymer component may be dextran having a molecular weight ranging from 200 Da to 100,000 Da, preferably PEG having a molecular weight ranging from 2,000 Da to 8,000 Da, more preferably PEG having a molecular weight ranging from 3,000 Da to 7,000 Da, most preferably PEG having a molecular weight ranging from 4,000 Da to 6,000 Da, etc.

[0045] The linker is arranged between the polymer component and the first drug component, and is cleavable. In a preferred embodiment of the invention, the linker is enzymatically cleavable, preferably by enzymes present in endosomes and / or lysosomes. A particular preferred linker contains glucuronide that is cleavable by beta-glucuronidase. Another preferred linker is peptide-based that is cleavable by a peptidase. Another preferred linker is ester-based that is cleavable by an esterase. A particular preferred linker is or includes wherein R1 stands for the first drug component and R2 for the polymer component.

[0046] The linker may include one or more cleavage sites. For example, the linker may include more than one cleavable glucuronide bonds / functions, more than one cleavable peptide bonds / functions, or more than one cleavable ester bonds / functions. Different types of cleavable bonds / functions may be combined with each other. By way of example, the linker may include one or more cleavable glucuronide bonds / functions and one or more cleavable peptide bonds / functions, etc. The first and second drug components as understood herein include substances that, when taken into the body, alters physiological functions or produces a biochemical effect. The drug components include typically substances used to diagnose, prevent, treat, or cure a disease or medical condition. Preferably, the drug components include a therapeutic, prophylactic or diagnostic drug meaning that the drug component has therapeutic, prophylactic or diagnostic potential / value.

[0047] According to one embodiment, the first drug component includes an immune active component. A preferred immune active agent is an antigen or an immune modulating agent. The antigen is preferably a T cell epitope. The immune modulating agent is preferably selected from the group consisting of agonists to toll-like receptors, stimulator of interferon genes (STING), nucleotide-binding oligomerization domain (NOD)-like receptors, retinoic acid-inducible gene-l (RIG-l)-like receptors, C-type lectin receptors and scavenger receptors. In particular, the immune active component includes an epitope selected from the group consisting of SIINFEKL, OVA323-339, ErBb2; VYDFFVWL; E75, GP2; AE37; MT288-296, and Cy3.

[0048] In other preferred embodiments of the invention, the first drug component includes a peptide epitope derived from RNF43, KOC1 , DEPDC1 , MPHOSPH1 , TTK, URLC10, CypB, NRPL, p56Lck, ppMAPkkk, SART3, UBE2V, WHSC2, PPV, WT1 , KIF20A, VEGFR1 / 2, TS, SART3, Cyclophilin B, p56lck, ppMAPkkk, WHSC2, UBE2V, HNRPL, SART2, MRP3, PAP, PSA, EGF-R, IEX-1 , p-tublin5, HER2, DEPDC1 , MPHOSPH1 .

[0049] The second drug component, if present, may also include an immune active component as described above.

[0050] The targeting ligand, if present, helps the nanovesicle to be delivered to a specific target in vivo. By coupling it to the polymer component, the targeting ligand is assumed to be positioned on the outer or inside surface of the shell. Preferred targets of the nanovesicle includes immune cells, preferably antigen-presenting cells, more preferably B cells, macrophages and / or T cells. To this end, the targeting ligand may be an antigen recognized by immune cells, preferably antigen-presenting cells, more preferably B cells, macrophages and / or T cells.

[0051] 3. Uses of the nanovesicles and conjugates The nanovesicles and conjugates as defined herein have a variety of uses. The inventors’ studies demonstrated the usefulness of the nanovesicles to activate B cells and provoke their immune functions. This can be realized by efficient induction of immune receptor agonists and antigens into B cells through the use of the nanovesicles. This induction may result in a significant activation of B cells, particularly CD86 expression and IL6 production, both being important immunoactivation markers. Furthermore, a substantial antigen presentation capability of B cells is obtained after treatment with the nanovesicles, and the nanovesicle-treated B cells are able to prime both cytotoxic and helper T cells. In vivo experiments illustrated that the system effectively activates B cells and T cells in the presence of specific antigens. Additionally, the capability of nanovesicles to reduce immunosuppres- sive / regulatory functions (e.g., expression ofTGF-p) of activated B cells and to differentiate B cells into plasma cells was shown.

[0052] In accordance with these observations, a first use is as a drug delivery vehicle. Preferably, the use involves delivering the conjugates in the form of nanovesicles or as such to immune cells, preferably antigen-presenting cells, more preferably B cells, macrophages and / or dendritic cells. Another use of the nanovesicles or conjugates is for activation of immune cells, preferably antigen-presenting cells, more preferably B cells, macrophages, dendritic cells and / or T-cells, most preferably B cells, macrophages and dendritic cells. A further use lies in vaccination and / or immune therapy. The immune therapy may be selected from the group consisting of therapies of cancers, infectious diseases, auto immune diseases, transplant rejections, and cardiovascular diseases.

[0053] A plurality of nanovesicles may be comprised in a (common) formulation. Preferred formulations are therapeutic or diagnostic formulations. It is further preferred that the nanovesicles in the formulation have a mean particle size ranging between 10 nm and less than 1000 nm but more than 10 nm, preferably between 20 nm and less than 1000 nm but more than 20 nm, more preferably between 30 nm and less than 1000 nm but more than 30 nm, yet more preferably between 40 nm and less than 1000 nm but more than 40 nm, most preferably between 50 nm and less than 1000 nm but more than 50 nm.

[0054] 4. Production of the nanovesicles and the conjugates

[0055] The nanovesicles as described herein may be prepared from the conjugates using a wide variety of methods known in the art. For example, nanovesicles can be formed by microfluidics, (micro-) emulsification, thin-film hydration, spray drying and high-pressure homog- enization, spray drying, nanoprecipitation, flow focusing using (micro-) fluidic channels, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, and other methods well known to those of ordinary skill in the art.

[0056] In one aspect of the invention, the nanovesicles are produced by a method comprising the following steps: providing a plurality of conjugates as defined herein; dispersing the plurality of conjugates in an organic phase; and adding the dispersed conjugates dropwise to an aqueous phase while mixing the aqueous phase.

[0057] The volume ratio of the organic phase to the aqueous phase preferably ranges from 1 :10,000 to 10,000:1 . The concentration of the conjugates in the organic phase may range from 0.000,000,1 pg / mL to 1 ,000,000 kg / mL. The temperature of the aqueous phase may range from 0 °C to 100 °C.

[0058] The method may further comprise the step of functionalizing the surface of the nanovesicle. In other words, formed nanovesicles are post-modified with a functional ligand such as a drug or a targeting ligand. To this end, the conjugates forming the nanovesicle may have a reactive group at a free end of the tail portion. After forming the nanovesicle, the reactive group is located on the outer surface of the nanovesicle, or at least close enough to the outer surface such that it is accessible for coupling with a functional ligand.

[0059] If the second drug component is small enough, it can be directly coupled to the polymer component in the conjugate before nanovesicle formation.

[0060] A method for producing a conjugate as defined herein comprises the steps of: providing a polymer component, a linker and a first drug component; coupling the first polymer component to the linker by click chemistry to provide a pre-conjugate comprising the first polymer component and the linker; activating a free end group of the linker contained in the preconjugate; coupling the first drug component to the activated end group to provide the conjugate comprising the first polymer component, the linker and the drug component; and optionally providing a targeting ligand and / or a second drug component, and coupling the targeting ligand and / or the second drug component to a free end group of the polymer component. With respect to preferred features of the polymer component, the linker, the first drug component and the targeting ligand and / or the second drug component, reference is made to the above.

[0061] The present invention will now be further described with reference to selected examples and the accompanying drawings, which follow after the brief description of the drawings.

[0062] Brief description of the drawings

[0063] Fig. 1 HPLC chromatograms of GL2-IMDQ and PEG5K-GL2-IMDQ at 254 nm wavelength.

[0064] Fig. 2 HPLC chromatograms of STING agonist diABZI, GL2-diABZI and PEG5K-GL2- diABZI at 254 nm wavelength.

[0065] Fig. 3 HPLC chromatograms of Cy3, PEG5K-GL2 and PEG5K-GL2-Cy3 at 550 nm wavelength.

[0066] Fig. 4 Nanovesicle formulation design, synthesis of building blocks and in vitro characterizations. (A) Schematic depiction of the nanovesicle composition, building block synthesis, formulation and intracellular release. (B) Size of nanovesicles based on different PEG- prodrug conjugates. (C) Transmission electron microscopy (TEM) image capturing nanovesicle morphology. (D) Enzyme-mediated drug release kinetics from the nanovesicles.

[0067] Fig. 5 Active Uptake of Nano-Sized Antigens by human and mouse primary B Cells. (A) Efficient uptake of nanovesicles (NVs) by human primary B cells compared to a soluble antigen (HAS). (B) Confirmation of NV uptake and intracellular presence through confocal microscopy. (C-E) Significant NV uptake by mouse primary B cells, contrasting with negligible uptake of soluble natural models (OVA and HAS) and a synthetic model antigen (PEG).

[0068] Fig. 6 Enhanced B Cell Activation by TLR7 / 8 Agonist-Loaded nanovesicles (NVs). (A1-3) Activation of human B cells by NVs containing TLR7 / 8 agonist. NVs exhibited significantly stronger B cell activation, evident in increased CD86 expression compared to the free compound. (B and C) Further examination of IL-6 mRNA expression and production also demonstrated heightened activation in response to NVs. (D1-3) Mouse B Cell Response to TLR7 / 8 Agonist-Loaded NVs. Similar to human B cells, mouse B cells exhibited heightened activation marked by increased CD86 expression when exposed to NVs containing the TLR7 / 8 agonist. (E1-3) Synergistic Activation with TLR7 / 8 and STING Agonists in NVs. I ntriguingly, the combination of TLR7 / 8 and STING agonists within NVs resulted in a remarkable nearly 8-fold upregulation of CD86 expression in human B cells, showcasing a synergistic effect on activation. (F1-2) Age-dependent analysis revealed that NV-induced B cell activation remained consistent across three different age groups. Fig. 7 Nanovesicle-Engineered B Cell-Antigen Presentation to Prime Helper and Cytotoxic T Cells. (A) Schematic depiction illustrating the utilization of nanovesicles (NVs) to induce B cell-antigen presentation to T cells. (B) Detection of the OVA epitope SIINFEKL presentation on MHC-I of B cells treated with NVs. Notably, while OVA alone demonstrates inefficiency in uptake, processing, and presentation on B cells, NVs exhibit significantly enhanced antigen presentation. The addition of a TLR7 / 8 agonist further augments presentation efficiency. (C) Inhibition of Beta-GUS activity substantially reduces antigen presentation on B cells treated with NVs. The light grey curve depicts the reduction in beta-GUS activity achieved by the inhibitor. (D1-2) In a B and CD8 cytotoxic T cell co-culture system, NV-treated B cells induce significant activation of T cells (CD69) compared to OVA alone. The addition of a TLR7 / 8 agonist to the NVs does not result in further enhancement of activation. (E1-3) Proliferation of T cells activated by B cells treated with NVs. NVs demonstrate significantly higher T cell activation compared to OVA alone. (F1-2) Addition of the beta-GUS inhibitor during B cell treatment significantly reduces the expression of T cell activation markers and inhibits proliferation. (G1-2) NVs induce significant cytokine release in T cell activation, including TNF-alpha and IFN-gamma. (H-1-2) Helper T cell priming, proliferation and CD69 expression, by B cell treated with NVs.

[0069] Fig. 8 (A) Regulation of the immunosuppressive factor TGF-p expressed by B cells. The TGF-p level was decreased when losartan co-treatment within the nanovesicles was applied. (B and C) The morphology of naive and activated B cells measured by TEM. The activated B cells were treated with NVs and anti-IgM F(ab’)2. The activated B cells exhibited properties consistent with plasma cells, including the presence of obvious networks of dilated rough endoplasmic reticulum (filaments) in the cytoplasm, a higher ratio of cytoplasm to nucleus, and the close adherence of heterochromatin (black) to the inner nuclear membranes.

[0070] Fig. 9 (A) Schematic representation of developed approach to post-modify formed nanovesicles with drug components. (B) Formation of conjugate with a reactive group. (C) Coupling of drug component to reactive group on the surface of the nanovesicle. Examples

[0071] The present disclosure relates to nanovesicles and their uses. The feasibility of the new approach was demonstrated by engineering B cells as an example using the nanovesicles described herein. Materials have been synthesized, and their suitability proven by biological and immunological testing of the nanovesicles.

[0072] 1 . Synthesis of conjugates

[0073] In the following, formation of exemplary nanovesicles from different types of conjugates differing in their first drug component, polymer component and their second drug component. To this end, four strategies were developed to synthesize three distinct types of pol- ymer-prodrug conjugates, encompassing small molecule immune receptor agonists, antigens specific to tumors or viruses, and targeting ligands for B cell receptors. This synthesis methodology enabled the formulation of nanovesicles with diverse components. Notably, with different types of conjugates, an endless number of nanovesicles can theoretically be formulated by mixing the conjugates with different peptide antigens and different ratios between the conjugates.

[0074] Regarding the synthesis of polymer-prodrug conjugates, PEG5k-GL2-Cy3, PEG5k-GL2- SIINFEKL, PEG5k-GL2-OVA323~339, click chemistry was performed firstly after obtaining the double spacer compound 16-2. The detailed procedures are outlined in Scheme 1 . After a 24 h click reaction between PEG5K and compound 16-2, pure PEG5K-GL2 conjugates were obtained by dialysis. Subsequently, the end group of spacer moiety in PEG5K-GL2 compound was activated to conjugate with Cy3, SIINFEKL, or OVA323-339. This strategy makes the purification easier, as the purification of each step can be done by dialysis instead of column chromatography. Yield: PEG5k-GL2-Cy3 75.6%, PEG5k-GL2-SIINFEKL 68.9%, PEG5k-GL2-OVA323~339 65%.

[0075] Regarding the synthesis of polymer-prodrug conjugates of small molecule immune receptor agonists (e.g., TLR7 / 8 receptors and the STING receptor). The small molecule prodrugs of TLR7 / 8 agonist IMDQ and the STING agonist diABZI were synthesized firstly through conjugating IMDQ or diABZI to double spacer compound 17-2 (Scheme 2). The detailed procedures are following:

[0076] Synthesis of IMDQ prodrugs with alkyne group (Scheme 2) Firstly, 0.49 mmol of compound 17-2 was dissolved in 14 mL of DMF. To this solution, 0.4 mmol of IMDQ 2HCI and 113 pL (0.8 mmol) of TEA were added. After an overnight stirring at room temperature, the crude mixture was purified by column chromatography (20% methanol / ethyl acetate) to obtain IMDQ prodrugs of GL2-IMDQ. Synthesis of diABZI prodrugs (Scheme 2)

[0077] Compound 17-2, with a concentration of 0.02 mmol was dissolved in 300 pL of DMF. To this solution, 0.0066 mmol of diABZI and 0.0066 mmol of 4-dimethylaminopyridine (DMAP) were added. The reaction mixture was left to react overnight at room temperature. The crude products of diABZI prodrugs of GL2-diABZI were purified by column chromatography (20% methanol / DCM).

[0078] Subsequently, the purified prodrug GL2-IMDQ or GL2-diABZI was conjugated to PEG by click chemistry (Scheme 2). The detailed procedures are following: Azide functionalized PEG (1 mmol) and prodrug compounds (1.2 mmol), GI2-IMDQ or GL2-diABZI, were dissolved in 200 pL of dried DMSO. To the solution, 0.5 mg (0.25 mmol) of Cui and 1 mg (0.5 mmol) of sodium ascorbate were added. The reaction was kept at 40 °C for 24 hours. The pure PEG-GL2-IMDQ and PEG-GL2-diABZI were then obtained by dialysis.

[0079]

[0080] Scheme 1. Synthesis of polymer-prodrug conjugates PEG-GL2-SIINFEKL, PEG-GL2-OVA323-339 and PEG-GL2-Cy3.

[0081] Scheme 2. Synthesis of polymer-prodrug conjugates, including PEG-GLn-IMDQ and PEG-GLn-diABZI.

[0082] Synthesis of NP-s-Aminocaproyl-PEG5k-N3 (NP-PEG5k-N3) compound (Scheme 3)

[0083] The compound of NP-PEG5k-Ns was synthesized through a simple substitution reaction. Firstly, 16.3 mg of NP-s-Aminocaproyl-OSu and 100 mg of NH2-PEG5k-N3 were dissolved in 1 mL of DMSO. Next, 0.202 mg of TEA was added as a catalyst to the above mixture, keeping the reaction for 24 h of stirring at room temperature. The products were then purified by dialysis in DMSO to obtain pure NP-PEG-N3 conjugates. Yield: 94%.

[0084] Synthesis of NP modified polymer-prodrug conjugates (NP-PEG5k-GL2-IMDQ, Scheme 3)

[0085] The polymer-prodrug conjugates of NP-PEG-GL2-IMDQ were also synthesized by click chemistry. The procedure was similar with the synthesis of polymer-prodrug conjugates of PEG5k-GL2-IMDQ. Yield: 70%

[0086] Synthesis of NP modified polymer-prodrug conjugates (NP-PEG5k-GL2-OVA323~339, Scheme 3)

[0087] The synthesis procedures of polymer-prodrug conjugates of NP-PEG5k-GL2-OVA323~339 were similar with PEG5k-GL2-OVA323~339 preparation. Yield: 52.4%. Synthesis of maleimide modified polymer-prodrug conjugates (MAL-PEG5k-GL2-IMDQ, Scheme 4)

[0088] The synthesis procedures of maleimide modified polymer-prodrug conjugates of MAL- PEG5k-GL2-IMDQ were similar with PEG5k-GL2-IMDQ preparation. Yield: 70.2%.

[0089]

[0090]

[0091] Scheme 3. Synthesis of NP modified double spacer polymer-prodrug conjugates of NP-PEG-GL2-IMDQ and NP-PEG-GL2-OVA323-339

[0092]

[0093] Scheme 4. Synthesis of maleimide modified double spacer polymer-prodrug conjugates of MAL-PEG-GL2-IMDQ

[0094] Characterization of prodrugs and polymer-prodrug conjugates

[0095] GL2-IMDQ

[0096] 1H NMR (600 MHz, DMSO-cfe) 6 10.06 (s, 1 H, Ar-NH), 9.77 (s, 1 H, Ar-NH), 7.81 - 7.68 (m, 2H, Ar), 7.60 - 6.98 (m, 14H, Ar), 6.97 (s, 1 H, COONH), 6.53 (s, 2H, -NH2), 5.83 (s, 2H, Ar-CH2), 5.51 - 5.40 (m, 3H, Glu 1-H, Glu-OH), 5.36 (d, J = 4.5 Hz, 1 H, Glu-OH), 5.06 (s, 2H, Ar-CH2), 4.91 (s, 2H, Ar-CH2), 4.83 - 4.69 (m, 2H, O-CH2-C CH), 4.12 (d, J = 6.1 Hz, 2H, Ar-CH2-NH), 3.95 (d, J = 9.3 Hz, 1 H, Glu 5-H), 3.59 (t, J = 2.4 Hz, 1 H, -CECH), 3.28 - 3.19 (m, 3H, Glu 2,3,4-H), 2.89 (t, J = 7.7 Hz, 2H, -CH2), 1 .73 - 1 .67 (m, 2H, -CH2), 1 .40 - 1.34 (m, 2H, -CH2), 0.86 (t, J = 7.3 Hz, 3H, -CH3). m / z [M+H]+= 916.3 (theoretical), found = 916.3.

[0097] GL2-diABZI

[0098] 1H NMR (600 MHz, DMSO-cfe) 6 12.79 (d, J = 15.8 Hz, 2H, Ar-CO-NH2), 10.03 (s, 1 H, Ar- NH), 9.76 (s, 1 H, Ar-NH), 7.99 - 7.85 (m, 2H, Ar-CO-NH2), 7.68 (d, J = 8.5 Hz, 1 H, CO- NH), 7.62 (d, J = 8.6 Hz, 1 H, CO-NH), 7.51 - 7.16 (m, 13H, Ar), 6.50 (d, J = 10.0 Hz, 2H, NCCH), 5.96 (d, J = 15.4 Hz, 1 H, NCH2CH-CHCH2N), 5.70 (d, J = 15.4 Hz, 1 H, NCH2CH- CHCH2N), 5.50 - 5.41 (m, 3H, Glu 1-H, Glu-OH), 5.31 (d, J = 4.6 Hz, 1 H, Glu-OH), 5.06 (s, 2H, NCH2CH), 5.04 (s, 2H, NCH2CH), 4.96 (s, 2H, Ar-CH2), 4.91 (s, 2H, Ar-CH2), 4.78 - 4.70 (m, 2H, O-CH2-C CH), 4.54 - 4.46 (m, 4H, NCH2CH3), 4.13 (t, J = 6.2 Hz, 2H, Ar-O- CH2CH2CH2), 4.06 (t, J = 5.9 Hz, 2H, Ar-O-CH2CH2CH2), 3.93 (d, J = 9.2 Hz, 1 H, Glu 5-H), 3.57 (t, J = 2.4 Hz, 1 H, -C CH), 3.37 - 3.32 (m, 3H, Glu 2,3,4-H), 2.08 (d, J = 7.1 Hz, 6H, 2NCCH3), 1.96 - 1.89 (m, 2H, Ar-O-CH2CH2), 1.23 (dd, J = 9.4, 4.6 Hz, 6H, 2NCH2CH3). m / z [M+H]+= 1307.3 (theoretical), found = 1307.5.

[0099] PEG5k-GL2-IMDQ

[0100] 1H NMR (600 MHz, DMSO-cfe) 6 10.05 (s, 1 H, Ar-NH), 9.77 (s, 1 H, Ar-NH), 8.18 (s, 1 H, triazyl), 7.87 - 7.66 (m, 2H, Ar), 7.62 - 7.00 (m, 14H, Ar), 6.98 (s, 1 H, OCONH), 6.97 (s, 1 H, COONH), 6.54 (s, 2H, -NH2), 5.83 (s, 2H, Ar-CH2-N), 5.50 - 5.40 (m, 3H, Glu 1-H, Glu- OH), 5.34 (d, J = 5.2 Hz, 1 H, Glu-OH), 5.06 (s, 2H, Ar-CH2), 4.91 (s, 2H, Ar-CH2), 4.57 (t, J = 5.5 Hz, 1 H, (O-CH2-CH2-)II2-OH), 4.35 (t, J = 7.1 Hz, 2H, COOCH2), 4.12 (d, J = 6.2 Hz, 2H, Ar-CH2-NH), 3.91 (d, J = 9.3 Hz, 1 H, Glu 5-H), 3.87 (s, 2H, -CH2-triazyl), 3.65 - 3.59 (m, 2H, OCH2O), 3.55 - 3.44 (m, 448H, (O-CH2-CH2-)n2), 3.25 - 3.20 (m, 3H, Glu 2,3,4-H), 3.15 - 3.09 (m, 2H, -CH2), 2.89 (t, J = 7.8 Hz, 2H, -CH2), 2.04 - 1.92 (m, 2H, - CH2-CH2-triazyl), 1 .73 - 1 .64 (m, 2H, -CH2), 1 .42 - 1 .30 (m, 2H, -CH2), 0.86 (t, J = 7.4 Hz, 3H, -CH3). Yield: 86.7%.

[0101] The HPLC chromatograms of GL2-IMDQ and PEG5K-GL2-IMDQ at 254 nm wavelength is depicted in Fig. 1 .

[0102] PEG5K-GL2-diABZI

[0103] 1H NMR (600 MHz, DMSO-cfe) 6 12.80 (d, J = 16.9 Hz, 2H, Ar-CO-NH2), 12.54 (s, 1 H, 2H, Ar-CO-NH2), 10.02 (s, 1 H, Ar-NH), 9.70 (s, 1 H, Ar-NH), 8.16 (s, 1 H, triazyl), 7.95 (s, 1 H, OCONH), 7.91 (s, 1 H, COONH), 7.84 - 7.65 (m, 2H, Ar), 7.68 (d, J = 8.5 Hz, 1 H, CO-NH), 7.62 (d, J = 8.6 Hz, 1 H, CO-NH), 7.56 - 7.18 (m, 11 H, Ar), 6.49 (d, J = 15.0 Hz, 2H, NCCH), 5.96 (d, J = 15.4 Hz, 1 H, NCH2CH-CHCH2N), 5.70 (d, J = 15.4 Hz, 1 H, NCH2CH-CHCH2N), 5.50 - 5.42 (m, 3H, Glu 1-H, Glu-OH), 5.32 (d, J = 4.6 Hz,1 H, Glu-OH), 5.17 (s, 2H, NCH2CH), 5.03 (s, 2H, NCH2CH), 4.96 (s, 2H, Ar-CH2), 4.90 (s, 2H, Ar-CH2), 4.55 (t, J = 5.5 Hz, 1 H, (O-CH2-CH2-)H2-OH), 4.54 - 4.46 (m, 4H, 2NCH2CH3), 4.33 (s, 2H, COOCH2), 4.13 (d, J = 6.2 Hz, 2H, Ar-CH2-NH), 4.06 (t, J = 6.2 Hz, 2H, Ar-O-CH2CH2CH2), 3.99 (t, J = 5.9 Hz, 2H, Ar-O-CH2CH2CH2), 3.89 (d, J = 9.2 Hz, 1 H, Glu 5-H), 3.85 (s, 2H, -CH2- triazyl), 3.62 - 3.58 (m, 2H, OCH2O), 3.53 - 3.44 (m, 448H, (O-CH2-CH2-)n2), 3.25 - 3.20 (m, 3H, Glu 2,3,4-H), 2.08 - 1.92 (m, 2H, -CH2-CH2-triazyl), 2.08 (d, J = 7.0 Hz, 6H, 2NCCH3), 1 .96 - 1 .89 (m, 2H, Ar-O-CH2CH2), 1 .23 (dd, J = 14.8, 8.2 Hz, 6H, 2NCH2CH3).

[0104] The HPLC chromatograms of STING agonist diABZI, GL2-diABZI and PEG5K-GL2-diABZI at 254 nm wavelength is shown in Fig. 2.

[0105] The HPLC chromatograms of Cy3, PEG5K-GL2 and PEG5K-GL2-Cy3 at 550 nm wavelength is shown in Fig. 3.

[0106] OAC-PEG5K-GL2

[0107] 1H NMR (600 MHz, DMSO-cfe) 6 10.02 (s, 1 H, Ar-NH), 9.65 (s, 1 H, Ar-NH), 8.16 (s, 1 H, triazyl), 7.81 (s, 1 H, OCH2ONH), 7.52 - 7.32 (m, 8H, Ar), 5.50 - 5.40 (m, 3H, Glu 1-H, Glu- OH), 5.31 (d, J = 5.2 Hz, 1 H, Glu-OH), 5.06 (s, 2H, Ar-CH2), 5.03 (s, 2H, Ar-CH2), 4.33 (t, J = 7.2 Hz, 2H, COOCH2), 4.12 (d, J = 6.2 Hz, 2H, Ar-CH2-NH), 4.08 (dd, J = 5.5, 4.1 Hz, 1 H, Ar-CH2OH), 3.89 (d, J = 9.4 Hz, 1 H, Glu 5-H), 3.85 (s, 2H, -CH2-triazyl), 3.65 - 3.59 (m, 2H, OCH2O), 3.55 - 3.44 (m, 448H, (O-CH2-CH2-)n2), 3.38 - 3.35 (m, 3H, Glu 2,3,4- H), 2.01 - 1.92 (m, 3H, -OAc). Activated end group of spacer moiety of OAc-PEG5K-GL2

[0108] 1H NMR (600 MHz, DMSO-cfe) 6 10.02 (s, 1 H, Ar-NH), 9.84 (s, 1 H, Ar-NH), 8.35 - 8.26 (m, 2H, Ar), 8.16 (s, 1 H, triazyl), 7.80 (s, 1 H, OCH2ONH), 7.61 - 7.18 (m, 8H, Ar), 5.50 - 5.40 (m, 3H, Glu 1-H, Glu-OH), 5.20 (t, J = 6.3 Hz, 1 H, Glu-OH), 5.10 (s, 2H, Ar-CH2), 5.06 (s, 2H, Ar-CH2), 4.32 (t, J = 7.2 Hz, 2H, COOCH2), 4.12 (d, J = 6.2 Hz, 2H, Ar-CH2-NH), 3.90 (d, J = 9.3 Hz, 1 H, Glu 5-H), 3.85 (s, 2H, -CH2-triazyl), 3.69 - 3.59 (m, 2H, OCH2O), 3.55 - 3.44 (m, 448H, (O-CH2-CH2-)n2), 3.19 - 3.10 (m, 3H, Glu 2,3,4-H), 2.01 - 1.92 (m, 3H, - OAc).

[0109] OAC-PEG5K-GL2-SIINFEKL

[0110] 1H NMR (600 MHz, DMSO-cfe) 6 12.48 (s, 1 H, CH-COOH), 12.06 (s, 1 H, CH2-COOH), 10.02 (s, 1 H, Ar-NH), 9.84 (s, 1 H, Ar-NH), 9.74 (s, 1 H, CH2-NH-COOCH2), 8.16 (s, 1 H, triazyl), 8.12 - 7.84 (m, 7H, CO-NH-CH), 7.80 (s, 2H, -NH2), 7.71 - 7.19 (m, 13H, Ar), 6.93 (s, 2H, -NH2), 5.50 - 5.40 (m, 3H, Glu 1-H, Glu-OH), 5.31 (d, J = 5.1 Hz, 1 H, Glu-OH), 5.20 (s, 1 H, NHCHCO), 5.19 (s, 1 H, NHCHCO), 5.15 (s, 1 H, NHCHCO), 5.13 (s, 2H, Ar-CH2), 5.04 (s, 2H, Ar-CH2), 4.88 (s, 1 H, NHCHCO), 4.55 - 4.46 (m, 2H, 2NHCHCO), 4.38 (d, J = 5.4 Hz, 1 H, -OH), 4.33 (t, J = 7.0 Hz, 2H, COOCH2), 4.20 (s, 2H, CH2-OH), 4.12 (s, 2H, Ar- CH2-NH), 4.09 - 4.06 (m, 5H, CH-NH), 4.00 (s, 1 H, NH-NH2), 3.90 (d, J = 9.3 Hz, 1 H, Glu 5-H), 3.85 (s, 2H, -CH2-triazyl), 3.55 - 3.44 (m, 448H, (O-CH2-CH2-)n2), 3.19 - 3.10 (m, 3H, Glu 2,3,4-H), 3.07 - 2.97 (m, 2H, Ar-CH2), 2.97 - 2.87 (m, 4H, 2CH2), 2.81 (s, 2H, CH2), 2.59 (s, 1 H, CH), 2.36 (s, 2H, CH2), 2.01 - 1 .92 (m, 3H, -OAc), 1 .77 (s, 1 H, CH), 1 .63 (s, 2H, CH2), 1 .48 (m, 2H, CH2), 1 .36 (d, J = 7.6 Hz, 4H, 2CH2), 1 .21 (s, 2H, CH2), 1 .03 (m, 6H, 2CH-CH3), 0.88 - 0.70 (m, 6H, 2CH-CH3), 0.66 (m, 6H, 2CH2-CH3).

[0111] OAC-PEG5K-GL2-OVA323~339

[0112] 1H NMR (600 MHz, DMSO-cfe) 6 12.48 (s, 1 H, CH-COOH), 12.06 (s, 1 H, CH2-COOH), 10.77 (s, 2H, 2NCH-NH), 10.01 (s, 1 H, Ar-NH), 9.82 (s, 1 H, Ar-NH), 9.74 (s, 1 H, CH-NH- COOCH2), 9.65 (s, 2H, 2N-CH-NH), 9.10 (s, 2H, 2N-CH), 8.21 (s, 1 H, CH2NH), 8.16 (s, 1 H, triazyl), 8.12 - 7.75 (m, 16H, CO-NH-CH), 7.56 - 7.24 (m, 8H, Ar), 7.18 (d, J = 8.4 Hz, 2H, CO-NH2), 7.04 (d, J = 8.5 Hz, 2H, CO-NH2), 6.99 (s, 1 H, COONH), 6.95 (d, J = 8.5 Hz, 1 H, C-NH), 6.81 - 6.69 (m, 2H, 2CO-NH-CH), 6.62 (d, J = 8.5 Hz, 2H, 2CO-NH-CH), 5.73 (s, 2H, 2CO-NH-CH), 5.45 - 5.37 (m, 3H, Glu 1-H, Glu-OH), 5.31 (d, J = 5.1 Hz, 1 H, Glu-OH), 5.20 (d, J = 12.6 Hz, 1 H, NHCHCO), 5.16 (d, J = 7.5 Hz, 1 H, NHCHCO), 5.13 (s, 1 H, NHCHCO), 5.04 (s, 4H, 2Ar-CH2), 4.57 - 4.46 (m, 4H, 4NHCHCO), 4.38 (d, J = 5.4 Hz, 3H, 3NHCHCO), 4.28 (s, 1 H, NHCHCO), 4.25 (dd, J = 14.2, 7.1 Hz, 2H, 2NHCHCO), 4.21 (s, 2H, NHCHCO), 4.16 (s, 2H, CH2-OH), 4.10 (d, J = 5.9 Hz, 2H, Ar-CH2-NH), 4.00 (dd, J = 14.1 , 7.1 Hz, 2H, 2NHCHCO), 3.89 (d, J = 9.4 Hz, 1 H, Glu 5-H), 3.82 (s, 2H, -CH2-triazyl), 3.78 (t, J = 5.1 Hz, 2H, CH2), 3.73 - 3.65 (m, 2H, CH2), 3.63 - 3.58 (m, 2H, CH2), 3.57 - 3.45 (m, 448H, (O-CH2-CH2-)n2), 3.39 - 3.33 (m, 2H, CH2), 3.23 - 3.12 (m, 3H, Glu 2,3,4- H), 2.99 (dd, J = 12.8, 6.8 Hz, 3H, CH2, CH), 2.21 (s, 2H, CH2), 2.19 (s, 2H, -CH2-CH2- triazyl), 2.11 - 2.04 (m, 2H, CH2), 2.01 - 1 .92 (m, 3H, -OAc), 1 .75 - 1 .61 (m, 4H, 2CH2), 1.59 (s, 4H, 2CH2), 1.44 (dt, J = 15.2, 7.6 Hz, 6H, 2CH3), 1.35 (dt, J = 14.8, 7.4 Hz, 4H, 2CH2), 1 .29 - 1.11 (m, 17H, 5CH3, CH2), 0.90 - 0.68 (m, 12H, 4CH3).

[0113] NP-PEG5K-GL2

[0114] 1H NMR (600 MHz, DMSO-cfe) 6 10.78 (s, 1 H, Ar-OH), 10.01 (s, 1 H, Ar-NH), 9.64 (s, 1 H, Ar-NH), 8.77 (s, 3H, Ar), 8.13 (s, 1 H, triazyl), 8.10 (d, J = 8.4 Hz, 1 H, Ar-CH2O-NH), 7.99 (s, 1 H, CH2ONH), 7.78 - 7.45 (m, 8H, Ar), 5.74 - 5.42 (m, 3H, Glu 1-H, Glu-OH), 5.16 (d, J = 7.6 Hz, 1 H, Glu-OH), 5.13 (s, 2H, Ar-CH2), 5.05 (s, 2H, Ar-CH2), 4.54 - 4.45 (m, 2H, COOCH2), 4.39 (d, J = 6.2 Hz, 2H, Ar-CH2-NH), 3.89 (d, J = 9.4 Hz, 1 H, Glu 5-H), 3.82 (s, 2H, -CH2-triazyl), 3.62 - 3.59 (m, 2H, OCH2O), 3.56 - 3.47 (m, 448H, (O-CH2-CH2-)n2), 3.23 - 3.15 (m, 3H, Glu 2,3,4-H), 3.05 - 2.95 (m, 2H, -CH2), 2.09 - 2.06 (m, 2H, -CH2-CH2- triazyl), 2.05 - 1.98 (m, 2H, -CH2), 1.44 (d, J = 4.9 Hz, 2H, -CH2), 1.41 - 1.31 (m, 2H, - CH2), 1.19 (dd, J = 20.3, 11 .0 Hz, 2H, -CH2).

[0115] Activated end group of spacer moiety of NP-PEG5K-GL2

[0116] 1H NMR (600 MHz, DMSO-cfe) 6 10.76 (s, 1 H, Ar-OH), 10.01 (s, 1 H, Ar-NH), 9.83 (s, 1 H, Ar-NH) , 8.37 - 8.29 (m, 2H, Ar), 8.13 (s, 1 H, triazyl), 8.10 (s, 1 H, Ar-CH2O-NH), 7.98 (s, 1 H, CH2ONH), 7.90 - 7.29 (m, 8H, Ar), 7.18 - 7.04 (m, 2H, Ar), 5.50 - 5.40 (m, 3H, Glu 1-

[0117] H, Glu-OH), 5.30 (d, J = 5.0 Hz, 1 H, Glu-OH), 5.16 (s, 2H, Ar-CH2), 5.13 (s, 2H, Ar-CH2), 4.58 - 4.45 (m, 2H, COOCH2), 4.38 (d, J = 5.4 Hz, 2H, Ar-CH2-NH), 3.89 (d, J = 9.4 Hz, 1 H, Glu 5-H), 3.82 (s, 2H, -CH2-triazyl), 3.63 - 3.58 (m, 2H, OCH2O), 3.56 - 3.47 (m, 448H, (O-CH2-CH2-)n2), 3.38 - 3.34 (m, 3H, Glu 2,3,4-H), 3.2 - 3.1 (m, 2H, -CH2), 3.05 - 2.99 (m, 2H, -CH2), 2.10 - 1 .96 (m, 2H, -CH2-CH2-triazyl), 1 .43 (dd, J = 15.1 , 7.4 Hz, 2H, -CH2),

[0118] I .35 (dd, J = 14.7, 7.4 Hz, 2H, -CH2), 1.19 (dd, J = 15.2, 8.2 Hz, 2H, -CH2).

[0119] NP-PEG5K-GL2-IMDQ1H NMR (600 MHz, DMSO-cfe) 6 10.01 (s, 1 H, Ar-NH), 9.74 (s, 1 H, Ar-NH), 8.10 (s, 1 H, triazyl), 7.98 (s, 1 H, Ar), 7.74 (m, 2H, Ar), 7.59 - 7.17 (m, 16H, Ar), 7.01 (d, J = 7.6 Hz, 1 H, Ar-CH2O-NH), 6.96 (s, 1 H, COONH), 6.50 (s, 2H, -NH2), 5.81 (s, 2H, Ar-CH2-N), 5.50 - 5.40 (m, 3H, Glu 1-H, Glu-OH), 5.31 (d, J = 5.1 Hz, 1 H, Glu-OH), 5.17 (dd, J = 35.1 , 12.7 Hz, 2H, Ar-CH2-O-NH), 5.04 (s, 2H, Ar-CH2), 4.89 (s, 2H, Ar-CH2), 4.52 - 4.43 (m, 1 H, Ar- OH), 4.10 (d, J = 5.9 Hz, 2H, Ar-CH2-NH), 3.89 (d, J = 9.3 Hz, 1 H, Glu 5-H), 3.82 (s, 2H, - CH2-triazyl), 3.63 - 3.57 (m, 2H, OCH2O), 3.55 - 3.44 (m, 448H, (O-CH2-CH2-)n2), 3.39 - 3.33 (m, 2H, -CH2), 3.23 - 3.12 (m, 3H, Glu 2,3,4-H), 2.99 (d, J = 6.2 Hz, 1 H, -CH2), 2.91 - 2.84 (m, 2H, -CH2), 2.01 (t, J = 7.4 Hz, 2H, -CH2-CH2-triazyl), 1 .75 - 1 .61 (m, 2H, -CH2), 1.50 - 1.40 (m, 2H, -CH2), 1.35 (s, 2H, -CH2), 1.19 (d, J = 7.3 Hz, 2H, -CH2), 0.84 (t, J = 7.3 Hz, 3H, -CH3).

[0120] NP-PEG5K-GL2-OVA323~339

[0121] 1H NMR (600 MHz, DMSO-cfe) 6 12.48 (s, 1 H, CH-COOH), 12.06 (s, 1 H, CH2-COOH), 10.77 (s, 2H, 2NCH-NH), 10.01 (s, 1 H, Ar-NH), 9.82 (s, 1 H, Ar-NH), 9.74 (s, 1 H, CH-NH- COOCH2), 9.65 (s, 2H, 2N-CH-NH), 9.10 (s, 2H, 2N-CH), 8.21 (s, 1 H, CH2NH), 8.11 (s, 1 H, triazyl), 8.03 - 7.65 (m, 10H, CO-NH-CH), 7.54 - 7.22 (m, 11 H, Ar), 7.18 (d, J = 8.4 Hz, 2H, CO-NH2), 7.04 (d, J = 8.6 Hz, 2H, CO-NH2), 6.99 (s, 1 H, COONH), 6.95 (d, J = 8.5 Hz, 1 H, C-NH), 6.81 - 6.69 (m, 2H, 2CO-NH-CH), 6.62 (d, J = 8.5 Hz, 2H, 2CO-NH-CH), 5.74 (s, 2H, 2CO-NH-CH), 5.45 - 5.37 (m, 3H, Glu 1-H, Glu-OH), 5.31 (s, 1 H, Glu-OH), 5.20 (d, J = 12.6 Hz, 1 H, NHCHCO), 5.16 (d, J = 7.6 Hz, 1 H, NHCHCO), 5.13 (s, 1 H, NHCHCO), 5.03 (s, 4H, 2Ar-CH2), 4.53 - 4.46 (m, 4H, 4NHCHCO), 4.38 (d, J = 4.9 Hz, 3H, 3NHCHCO), 4.28 (s, 1 H, NHCHCO), 4.25 (dd, J= 14.2, 7.1 Hz, 2H, 2NHCHCO), 4.21 (s, 1 H, NHCHCO), 4.16 (s, 2H, CH2-OH), 4.10 (d, J = 5.9 Hz, 2H, Ar-CH2-NH), 4.00 (dd, J = 14.1 , 7.1 Hz, 2H, 2NHCHCO), 3.89 (d, J = 9.4 Hz, 1 H, Glu 5-H), 3.82 (s, 2H, -CH2-triazyl), 3.78 (t, J = 5.1 Hz, 4H, 2CH2), 3.73 - 3.65 (m, 2H, CH2), 3.63 - 3.58 (m, 2H, CH2), 3.57 - 3.45 (m, 448H, (O-CH2-CH2-)n2), 3.39 - 3.33 (m, 2H, -CH2), 3.23 - 3.12 (m, 3H, Glu 2,3,4-H), 3.06 (s, 2H, CH2), 2.99 (dd, J = 12.7, 7.0 Hz, 5H, 2CH2, CH), 2.91 - 2.84 (m, 4H, 2CH2), 2.21 (s, 2H, CH2), 2.08 (s, 2H, CH2), 2.01 (t, J = 7.4 Hz, 2H, -CH2-CH2-triazyl), 1.92 (s, 5H, 2CH2, CH), 1 .75 - 1 .61 (m, 6H, 3CH2), 1 .59 (s, 4H, 2CH2), 1 .44 (dt, J = 15.2, 7.6 Hz, 6H, 2CH3), 1 .35 (dt, J = 14.8, 7.4 Hz, 4H, 2CH2), 1.3 - 1.09 (m, 17H, 5CH3, CH2), 0.90 - 0.68 (m, 12H, 4CH3). The nanovesicle (NV) including the building blocks, and their mechanism of action is schematically depicted in Fig. 4A. The mean particle size of the resulting nanovesicles ranged between 100 nm and about 1000 nm (cf. Fig. 4B). The nanovesicles had a spherical morphology, as determined by transmission electron microscopy (TEM) (cf. Fig. 4C). By enzymatic inhibition experiments it could be shown that the release of the drug component is enzyme-mediated (cf. Fig. 4D).

[0122] 2. Biological and immunological testing

[0123] Using nanovesicles formed from PEG-GL2-Cy3 conjugates in human and mouse primary B cell cultures it was shown that the nanovesicles were actively taken up by the cells. The uptake of nanovesicles by human primary B cells was higher as compared to a soluble antigen (HAS) (cf. Fig. 5A). Nanovesicle uptake and their intracellular presence was confirmed through confocal microscopy (cf. Fig. 5B). Significant nanovesicle uptake by mouse primary B cells contrasted with negligible uptake of soluble natural models (OVA and HAS) and a synthetic model antigen (PEG) (cf. Figs. 5C-E).

[0124] In the case of TLR7 / 8 agonist loaded nanovesicles an enhanced B cell activation was observed. The nanovesicles exhibited significantly stronger B cell activation, evident in increased CD86 expression compared to the free compound (cf. Fig. 6A1-3). Further examination of IL-6 mRNA expression and production also demonstrated heightened activation in response to the nanovesicles (cf. Fig. 6B-C). Mouse B cell response to TLR7 / 8 Agonist- loaded nanovesicles is shown in Fig. 6D1-3. Similar to human B cells, mouse B cells exhibited heightened activation marked by increased CD86 expression when exposed to NVs containing the TLR7 / 8 agonist. As can be seen in Fig. 6E1-3, activation with TLR7 / 8 and STING Agonists in nanovesicles was synergistic. Intriguingly, the combination of TLR7 / 8 and STING agonists within NVs resulted in a remarkable nearly 8-fold upregulation of CD86 expression in human B cells, showcasing a synergistic effect on activation. Age-dependent analysis revealed that nanovesicle induced B cell activation remained consistent across three different age groups (cf. Fig. 6F1-2).

[0125] Referring to Fig. 7A, the utilization of nanovesicles (NVs) to induce B cell-antigen presentation to T cells (prime helper and cytotoxic T Cells) is schematically depicted. OVA epitope SIINFEKL presentation on MHC-I of B cells treated with nanovesicles was detected (cf. Fig. 7B). Notably, while OVA alone demonstrated inefficiency in uptake, processing, and presentation on B cells, the nanovesicles exhibited significantly enhanced antigen presentation. Addition of a TLR7 / 8 agonist further augmented presentation efficiency. As shown in Fig. 7C, inhibition of beta-GUS activity substantially reduced antigen presentation on B cells treated with the nanovesicles. The lower curve depicts the reduction in beta-GUS activity achieved by the inhibitor. In a B and CD8 cytotoxic T cell co-culture system, nanovesicle treated B cells induced significant activation of T cells (CD69) compared to OVA alone (cf. Fig. 7D1-2). The addition of a TLR7 / 8 agonist to the NVs did not result in further enhancement of activation. Fig. 7E1-3 demonstrates proliferation of T cells activated by B cells treated with nanovesicles. The nanovesicles demonstrated significantly higher T cell activation compared to OVA alone. Addition of the beta-GUS inhibitor during B cell treatment significantly reduced the expression of T cell activation markers and inhibited proliferation (cf. Fig. 7F1-2). Fig. 7G1-2 shows that nanovesicles induced significant cytokine release in T cell activation, including TNF-alpha and IFN-gamma. Fig. 7H-1-2 demonstrates helper T cell priming, proliferation and CD69 expression by B cell treated with nanovesicles.

[0126] Regulation of the immunosuppressive factor TGF-p expressed by B cells in response to treatment with nanovesicles is shown in Fig. 8. As can be seen in Fig. 8A, the TGF-p level was decreased when losartan co-treatment within the nanovesicles was applied. The morphology of naive and activated B cells measured by TEM is shown in Fig. 8B-C. The activated B cells were treated with NVs and anti-IgM F(ab’)2. The activated B cells exhibited properties consistent with plasma cells, including the presence of obvious networks of dilated rough endoplasmic reticulum (filaments) in the cytoplasm, a higher ratio of cytoplasm to nucleus, and the close adherence of heterochromatin (black) to the inner nuclear membranes.

[0127] In summary, the results depicted in Figs. 4 to 8 showed that the nanovesicles of the invention enter primary B cells without conventional targeting moieties. Highly efficient activation of B cells was achieved by nanovesicles containing single or combinations of immune receptor agonists. Furthermore, the nanovesicles were demonstrated to engineer B cells into powerful antigen-presenting cells. By this strategy, nanovesicle-engineered B cells were able to prime and activate both helper and cytotoxic T cells which are able to recognize and kill cancer cells or infected cells. Additionally, the nanovesicles were proven to be able to promote B cell differentiation into plasma cells which release antibodies after vaccination, and reduce the unwanted immunoregulatory phenotypes of B cells.

[0128] 3. Functionalizing nanovesicle surface Referring to Fig. 9, a strategy to post-modify formed nanovesicles with drugs is shown. In this case the conjugate has a reactive group and forms a nanovesicle. Afterwards, a drug is coupled to the surface of the nanovesicle.

[0129] Regarding the synthesis of polymer-prodrug conjugates with a second drug / targeting lig- and, a polymer with a reactive group (for example, maleimide group; NHS-activated carboxylic acid, etc.) for coupling with the second drug / targeting ligand was used to synthesize the conjugate via the approaches (first polymer conjugation with the linker and then the first drug is coupled; or the polymer was coupled to the first drug pre-modified with the linker) described above. After nanovesicle formation with the conjugate, the second drug was cou- pled to the nanovesicles, for example, a protein is coupled to the nanovesicles with maleimide containing conjugates by thiol-maleimide conjugation.

Claims

Claims1 . Nanovesicle comprising an aqueous core and a shell formed from a plurality of conjugates, each conjugate comprising a head portion, a tail portion and an intermediate portion in-between, wherein the head portion contains a first drug component, the tail portion contains a polymer component and optionally a second drug component and / or a targeting ligand, and the intermediate portion contains a cleavable linker, wherein the head portion has a higher hydrophobicity than the tail portion, wherein the linker is enzymatically cleavable, preferably by enzymes present in endosomes and lysosomes, wherein the first drug component includes an antigen, preferably being directly connected to the linker and containing a T cell epitope; and / or wherein the first drug component includes an immune modulating agent, preferably selected from the group consisting of agonists to toll-like receptors, stimulator of interferon genes (STING), nucleotide-binding oligomerization domain (NOD)-like receptors, retinoic acid-inducible gene-l (RIG-l)-like receptors, C-type lectin receptors and scavenger receptors, and wherein the second drug component or targeting ligand, if present, contains an antigen directly connected to the polymer component that is recognized by immune cells, preferably antigen-presenting cells, more preferably B cells, macrophages and / or T-cells, most preferably B cells.

2. Conjugate comprising a head portion, a tail portion and an intermediate portion inbetween, wherein the head portion contains a first drug component, the tail portion contains a polymer component and optionally a second drug component or a targeting ligand, the intermediate portion contains a cleavable linker, wherein the head portion has a higher hydrophobicity than the tail portion, wherein the linker is enzymatically cleavable, preferably by enzymes present in endosomes and lysosomes, wherein the first drug component includes (i) an antigen, preferably being directly connected to the linker and containing a T cell epitope, and / or (ii) an immune modulating agent, preferably selected from the group consisting of agonists to toll-like receptors, stimulator of interferon genes (STING), nucleotide-binding oligomerization domain (NOD)-like receptors, retinoic acid-inducible gene-1 (RIG-l)-like receptors, C-type lectin receptors and scavenger receptors, wherein the second drug component or targeting ligand, if present, contains an antigen directly connected to the polymer component that is recognized by immune cells, preferably antigen-presenting cells, more preferably B cells, macrophages and / or T-cells, most preferably B cells..

3. Nanovesicle or conjugate according to claim 1 or 2, wherein the polymer component is selected from the group consisting of polyethylene glycol (PEG), dextran, polyglycerol, polyvinyl alcohol, polymethacrylates, polymethacrylamides, polyacrylates, polyacrylamides, polymethacrylic acid, polyacrylic acid, polyvinylpyrrolidone, polysarcosine, poly(amino acid), poly(thioglycidyl glycerol) and derivatives of the above hydrophilic segments.

4. Nanovesicle or conjugate according to any of claims 1 to 3, wherein the polymer component has a molecular weight ranging from 200 Da to 100,000 Da, preferably 2,000 Da to 8,000 Da, more preferably 3,000 Da to 7,000 Da, most preferably 4,000 Da to 6,000 Da.

5. Nanovesicle or conjugate according to any of claims 1 to 4, wherein the linker is a glucuronide-containing linker being cleavable by 0-glu- curonidase, or a peptide-based linker being cleavable by a peptidase, or an ester- based linker being cleavable by a esterase, or a combination of two or more of the linkers above, more preferably wherein the linker is or includes:

6. Nanovesicle or conjugate according to any of claims 1 to 5, wherein the first drug component includes SIINFEKL, OVA323-339, ErBb2; VYDFFVWL; E75, GP2; AE37; MT288-296, or CY3, or wherein the first drug component includes a peptide epitope derived from RNF43, KOC1 , DEPDC1 , MPHOSPH1 , TTK, URLC10, CypB, NRPL, p56Lck, ppMAPkkk, SART3, UBE2V, WHSC2, PPV, WT1 , KIF20A, VEGFR1 / 2, TS, SART3, Cyclophilin B, p56lck, ppMAPkkk, WHSC2, UBE2V, HNRPL, SART2, MRP3, PAP, PSA, EGF-R, IEX-1 , p-tublin5, HER2, DEPDC1 , MPHOSPH1 .

7. Nanovesicle according to any of claims 1 and 3 to 6, wherein the surface of the nanovesicle is functionalized with the second drug component and / or targeting ligand.

8. Use of a nanovesicle or a conjugate according to any of claims 1 to 7 as a drug delivery vehicle.

9. Use of claim 8, wherein the use is for delivering to immune cells, preferably antigen- presenting cells, more preferably B cells, macrophages and / or dendritic cells.

10. Use of a nanovesicle or a conjugate according to any of claims 1 to 7 for activation of immune cells, preferably antigen-presenting cells, more preferably B cells, macrophages, dendritic cells and / or T-cells, most preferably B cells, macrophages and dendritic cells, preferably wherein the activation of the immune cells involves induction of antigen presentation to T cells by B cells, dendritic cells and / or macrophages.11 . Nanovesicle or conjugate according to any of claims 1 to 7 for use in vaccination and / or immune therapy, wherein preferably the immune therapy is selected from the group consisting of therapies of cancers, infectious diseases, auto immune diseases, transplant rejections, and cardiovascular diseases.

12. Method for producing a nanovesicle, comprising: providing a plurality of conjugates as defined in any of claims 2 to 7; dispersing the plurality of conjugates in an organic phase; and adding the dispersed conjugates dropwise to an aqueous phase while stirring.

13. Method of claim 12, further comprising: functionalizing the surface of the nanovesicle.

14. Method of producing a conjugate as defined in any of claims 2 to 7, comprising: providing a polymer component, a linker and a first drug component; coupling the first polymer component to the linker by click chemistry to provide a preconjugate comprising the first polymer component and the linker; activating a free end group of the linker contained in the pre-conjugate; coupling the first drug component to the activated end group to provide the conjugate comprising the first polymer component, the linker and the drug component; and optionally providing a targeting ligand and / or a second drug component, and coupling the targeting ligand and / or the second drug component to a free end group of the polymer component.

15. Method of claim 14, wherein the polymer component is as defined in claim 3 and / or claim 4; and / or the linker is as defined in claim 5; and / or the first drug component is as defined in claim 6; and / or the targeting ligand and / or the second drug component is as defined in claim 7.

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