Immunostimulatory compositions

Abstract

In certain embodiments, the present invention provides immunostimulatory compositions, methods for making immunostimulatory compositions, and methods for using immunostimulatory compositions. In one aspect, provided herein is an immunostimulatory composition comprising: (a) a nanoparticle (NP), (b) an activating agent operably linked to the NP, and (c) a blood dendritic cell antigen 2 (BDCA2) ligand operably linked to the NP.

Description

[0001] VHPM 17023.303WO1 / UIRF 25010

[0002] IMMUNOSTIMULATORY COMPOSITIONS

[0003] CROSS REFERENCE TO RELATED APPLICATION

[0004] This application claims priority to United States Provisional Application Number 63 / 735,715 that was filed on December 18, 2024. The entire content of the application referenced above is hereby incorporated by reference herein.

[0005] BACKGROUND

[0006] Plasmacytoid Dendritic Cells (pDCs)

[0007] Plasmacytoid Dendritic Cells (pDCs) are rare immune system cells that serve as sentinels of danger and play a central role in initiating the immune response. Activated pDCs produce large amounts of type I interferons (including IFNa) that result in secondary activation of other immune cell types. The state of activation of pDCs helps determine whether an immune response is initiated. Activating pDCs with immune stimulants can enhance the immune response. This has led to including such agents as those immune stimulants in vaccines designed to prevent infection, and in situ immunization in cancer where such immune stimulants are injected directly into, or directed to, a tumor to enhance an anti -cancer response. While these results are promising, including clinical efficacy demonstrated in early phase trials, current approaches to delivering immune stimulants to pDCs in a tumor, and to enhancing the antitumor immune response, are suboptimal.

[0008] Toll Like Receptors (TLRs) and TLR agonists

[0009] Toll Like Receptors (TLRs) are a family of pattern recognition receptors expressed on a variety of immune cells, including pDCs, that recognize pathogen-associated or danger- associated molecular patterns. A variety of TLR agonists have been identified and evaluated preclinically and clinically. One example is synthetic, unmethylated, CG-rich CpG oligodeoxynucleotides (ODNs). Such ODNs mimic prokaryotic DNA and are known to activate TLRs, such as TLR9. CpG ODN with TLR9 agonist properties have classified into three families (designated CpG A, B and C) with distinct structural and biological characteristics based on their ability to induce IFNa secretion from pDC and stimulate B cells. TLR9 agonists directly activate innate signaling pathways that then enhance an adaptive immune response and have been used as immune adjuvants in tumor Ag immunization and as systemic therapy alone or in combination with other therapeutics. Additional TLR agonists, particularly those that activate TLR7 (and TLR8) have been investigated as therapeutics. For instance, immunostimulatory RNA or small molecules (e.g., imiquimod) have been explored as therapeutics. VHPM 17023.303WO1 / UIRF 25010

[0010] Role of Blood dendritic cell antigen 2 (BDCA2)

[0011] Blood dendritic cell antigen 2 (BDCA2) is a molecule that is uniquely expressed by pDCs. BDCA2 is also known as CLEC4C or CD303. BDCA2 is a C-type lectin found selectively on plasmacytoid dendritic cells (pDCs). BDCA2 can serve as a receptor for a number of ligands, including as a receptor for a variety of proteins that express a form of a galactose-terminated biantennary N-linked glycans. These include, but are not limited to, heparin and all subsets of IgG (see, M Kiyoshi et al., “Glycosylation of IgG-Fc: a molecular perspective,” Int. Immunol. 2017 Jul 1;29(7):311-317). The BDCA2 -ligand interaction is based on glycosylation of the ligand in the form of a galactose-terminated biantennary N-linked glycans. Signaling via BDCA2, as mediated by ligands that include these glycans, inhibits the ability of pDCs to produce IFNa, particularly in response to TLR9 agonists. Based on these data, the primary role of BDCA2 on pDCs has been understood to be suppression of pDC activation and downregulation of the subsequent immune response.

[0012] A variety of ligands that bind to BDCA2 are plentiful in a variety of tissues and are known to suppress the activation of pDCs. For example, binding of these soluble ligands to BDCA2 on pDCs significantly reduces the response of pDCs to TLR9 agonists. Thus, accepted dogma is that novel approaches to activating pDCs with TLR9 agonists should avoid binding to BDCA2 in order to bypass the suppression of pDCs. Consistent with this understanding, it has been confirmed in preliminary studies that soluble heparin or IgG suppresses the response of pDCs to TLR9 agonists. Also, antibodies directed against BDCA2, such as litifilimab, are being evaluated as treatments for autoimmunity given their immunosuppressive effects.

[0013] More efficient approaches to activate pDCs, however, represent a clear unmet need.

[0014] SUMMARY

[0015] In one aspect, provided herein is an immunostimulatory composition comprising: (a) a nanoparticle (NP), (b) an activating agent operably linked to the NP, and (c) a blood dendritic cell antigen 2 (BDCA2) ligand operably linked to the NP.

[0016] In one aspect, provided herein is pharmaceutical composition comprising the immunostimulatory composition described herein and a pharmaceutically acceptable excipient.

[0017] In one aspect, provided herein is a method of activating plasmacytoid dendritic cells (pDCs), comprising administering the immunostimulatory composition described herein or the pharmaceutical composition described herein to a patient in need thereof.

[0018] In one aspect, provided herein is a method of enhancing an immune response against cancer, comprising administering the immunostimulatory composition described herein or the pharmaceutical composition described herein to a patient in need thereof. VHPM 17023.303WO1 / UIRF 25010

[0019] In one aspect, provided herein is a method of enhancing an immune response to a vaccine, comprising administering the immunostimulatory composition described herein or the pharmaceutical composition described herein to a patient in need thereof.

[0020] BRIEF DESCRIPTION OF DRAWINGS

[0021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0022] Figures 1A-1D. Schema illustrating unexpected ability of heparin-coated nanoparticles containing TLR9 agonists to activate pDCs. Figure 1A depicts the ability of soluble TLR9 agonist to induce production of moderate levels of IFNa from pDCs. Figure IB depicts how soluble heparin, by interacting with BDCA2 expressed by pDCs, reduces the ability of soluble TLR9 agonist to produce IFNa from those pDCS. Figure 1C depicts how untargeted nanoparticles containing a TLR9 agonist induce moderate levels of IFNa from pDCs. Figure ID depicts how nanoparticles containing a TLR9 agonist that are also coated with a BDCA2 ligand such as heparin, are unexpectedly potent inducers of IFNa from pDCs.

[0023] Figures 2A and 2B. Nanoparticles were produced by adding 0 or 10 pg of TLR9 agonist (G10), and then 1, 5, 10, 15, 20, or 30 pg of heparin to a reaction volume of 200 pl. Peripheral blood mononuclear cells from a normal donor were treated with nanoparticles containing varying abouts of TLR9 agonist (G10) and coated with varying amounts of heparin. IFNa in supernatant as determined by ELISA (Figure 2A) and the phenotype of the pDCs within the PBMCs was evaluated by flow cytometry (Figure 2B). Analyses were run in duplicate. Similar results were found with three different donors. Dotted line indicates level of activation induced by soluble G10. Figure 2A depicts how coating of TLR9 agonist-containing particles with low levels of heparin (1 of 5 micrograms) reduces the production of IFNa , while coating TLR9 agonist-containing particles with higher levels of heparin (10-30 micrograms) enhances production of IFNa. Figure 2B. depicts how coating of TLR9 agonist-containing particles with low levels of heparin (1 of 5 micrograms) has minimal impact on their ability to activate pDCs, while coating them with higher levels of heparin (10-30 micrograms) enhances their ability to activate pDCs as indicated by their expression of PDL1.

[0024] DETAILED DESCRIPTION

[0025] In certain aspects, the present invention provides nanoparticles (NP) that are operably linked to toll-like receptor (TLR) agonists and are operably linked with heparin or heparin-like VHPM 17023.303WO1 / UIRF 25010 molecules. In certain aspects, specific activating agents are operably linked (e.g., coated) to the NPs.

[0026] As used herein, the term “operably linked” refers to a linkage of two elements in a functional relationship. “Operably linked” refers to the association of two chemical moieties wherein the components so described are configured so as to perform their usual function. The term “operably linked” refers to a structural linkage, covalent or non-covalent, of two or more fragments in a composition. In certain embodiments, an NP may be operably linked to a nonprotein moiety, such as heparin.

[0027] Nanoparticles (NPs)

[0028] In certain aspects, the nanoparticles (NPs) are composed of lipids or polymers, or are virus-like particles (VLP).

[0029] In certain embodiments, the NP is composed of a dendrimer. Dendrimers are globular, hyperbranched polymers with a central core, repeating branches, and terminal groups. Dendrimers offer one of the most highly specific nanoparticle physical characteristics due to their stepwise branching synthesis. Each outward “layer” (also called a “generation”) of the dendrimer contains exponentially more branching points. With each synthetic generation of synthesis having exactly double the number of surface molecules, particle size and binding sites can be exactly controlled.

[0030] In certain embodiments, the NP dendrimer is composed of polyamidoamine (PAMAM). PAMAMs generally have a sphere-like shape overall with an internal molecular architecture having repetitive tree-like branched subunits. This branched architecture distinguishes PAMAMs and other dendrimers from traditional polymers, because it allows for low poly dispersity and a high level of structural control during synthesis, and gives rise to a large number of surface sites relative to the total molecular volume. PAMAM dendrimers are branched cationic polymers with polyamidoamine units on the surface with primary amino groups. PAMAM can be linked with biologically active compounds. PAMAM dendrimers have high molecular uniformity, narrow molecular weight distribution, defined size and shape characteristics and a multifunctional terminal surface.

[0031] PAMAM, similar to other dendrimers, is typically sub-classified by the number of its branched subunit layers (“generations”). In certain embodiments, certain generations of PAMAM can be used alone or in combination with other different generations as part of the NP. In certain embodiments, the PAMAM may contain chemical modifications. In the literature, PAMAM generations 1, 2, 3, 4, and 5 are most commonly used for nucleic acid (e.g., ODN) VHPM 17023.303WO1 / UIRF 25010 delivery and broadly, for nucleic acid delivery into the cells. In certain embodiments, PAMAM of generations higher than 5 are used for the same purposes.

[0032] In certain embodiments, the NPs are composed of a chemically modified, 5thgeneration PAMAM dendrimer.

[0033] In certain embodiments, a PAMAM dendrimer (generation 5, G5) is chemically modified by conjugating polyethylene glycol (PEG; average molecular weight of 2,000 Da) on PAMAM. In certain embodiments, the average molecular weights of PEG are 1,000 Da to 20,000 Da.

[0034] In certain embodiments, the PEG to PAMAM (G5) molar ratios are 0 to 64. In certain embodiments, the PEG to PAMAM (G5) molar ratio is 9. In certain embodiments, PAMAM is chemically conjugated to PEG at various molar ratios to improve the stability of the NP. In certain embodiments, PAMAM is chemically conjugated to PEG to reduce the toxicity of the NPs. In certain embodiments, PAMAM is chemically conjugated to PEG to improve the stability of the NP and reduce the toxicity of the NPs.

[0035] In certain embodiments, instead of (or in addition to) PEG, other similar polymers are used. In certain embodiments, dextran or dextran derivatives are conjugated to the NPs.

[0036] In certain embodiments, other species of positively charged dendrimers or polymers can be used in the NPs. In certain embodiments, the NPs comprise polyethyleneimine, polypropyleneimine, poly-L-lysine, poly-D-lysine, poly-L-arginine, poly-D-arginine, poly-L- histidine, poly-D-histidine, and / or chitosan.

[0037] In certain embodiments the NPs are liposomes or lipid nanoparticles constructed of cationic / ionizable lipids, neutral lipids, cholesterol and / or cholesterol analogues and PEG- conjugated lipids.

[0038] In certain embodiments the NP is a virus-like particle constructed of a virus capsid, such as the Qbeta bacteriophage capsid.

[0039] Activating Agents

[0040] In certain aspects, specific activating agents are coated and / or linked to the NPs or are contained within the NP. In certain aspects, the activating agents are incorporated into the NP.

[0041] TLR agonists

[0042] In certain aspects, TLR agonists are operably linked to or are incorporated into the NPs. In certain aspects, the TLR agonist is a TLR9 agonist. For example, studies were performed with NP containing a class A CpG ODN TLR9 agonist. In certain embodiments, the TLR9 agonist was G10 TLR9 agonist. Additional embodiments include other TLR agonists including CpG ODN of various classes (Class A, B, C), immunostimulatory RNA that activate pDCs via TLR7 or 8, and small molecule TLR agonists. VHPM 17023.303WO1 / UIRF 25010

[0043] In certain embodiments, the NPs conjugate to GIO were fabricated primarily through electrostatic interactions between positively charged amino groups on the surface of the PAMAM dendrimer and the negatively charged phosphate backbone of G10. The nitrogen / phosphate (N / P) ratio is the molar ratio of nitrogen atoms of the PAMAM molecule as compared to phosphorus atoms of the TLR agonist, can range from 3 : 1 to 10: 1 In certain embodiments, the N / P ratio is 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1. In certain aspects, the TLR9 agonists are encapsulated in NP at a loading range of 0.05 to 0.3 mg-agonists to mg-NPs, with the optimal range of 0.1 to 0.25 mg to mg NPs.

[0044] BDCA2 ligand

[0045] Heparin or Heparin-Like Molecules

[0046] In certain aspects, heparin or heparin-like molecules are operably linked to the NPs. In certain embodiments, the heparin is full length or low molecular weight heparin.

[0047] In certain aspects, the heparin or heparin-like molecules are present on the surface of the NPs at a concentration or density of 2-60% (by weight), equivalent to 0.02-0.6 mg of heparin or heparin-like molecules per 1 mg of the NPs, with the optimal range of 15-45% (by weight). The optimal range of heparin or heparin-like molecules on the NPs is required to impart BDCA2 targeting property to the NPs under physiological conditions, while maintaining the integrity of the NPs. In certain aspects, the NP is coated with heparin via electrostatic interactions with the residual amino groups of PAMAM, as well as through van der Waals interactions, including hydrophobic interactions. The amount of heparin coated on the nanoparticles (NPs) can vary.

[0048] In certain embodiments, other options such as low-molecular-weight heparin, keratan sulfate, and chondroitin sulfate can also be applied as the BDCA2 ligand. These ligands form coatings through physical absorption, and in certain embodiments, they are chemically conjugated to PAMAM for incorporation into the NP system.

[0049] In certain embodiments, the BDCA2 ligand is IgG or other glycoproteins or glycans that include a galactose-terminated biantennary N-linked glycan.

[0050] The state-of-the-art suggests targeting pDCs with TLR agonists is an effective approach to initiating an immune response. It also suggests that NP containing TLR agonists might be effective immunostimulatory agents. The art, however, teaches against targeting TLR- containing NP to pDCs by using BDCA2 ligands because the function of BDCA2 is known to suppress pDC activation, particularly in response to TLR agonists. Thus, the art suggests that it would not be an effective approach to enhancing the immune response.

[0051] Surprisingly, the present inventors discovered that contrary to what was taught in the art (Figure 2A), NP containing a TLR agonist coated with large amounts of a BDCA2 ligand (2.5- VHPM 17023.303WO1 / UIRF 25010

[0052] 7.5 pg / ml) are immunostimulatory, rather than immunosuppressive. It was discovered that NP containing TLR9 agonists that are coated with larger amounts of a BDCA2 ligand (heparin) have the opposite of the expected effect based on current art. While soluble heparin, and NP coated with low amounts of heparin (0.25-1.25 pg / ml) suppress activation of pDCs, it was found that NP coated with larger amounts of heparin are potent activators of pDCs. This activation was confirmed by assessing the ability of such particles to induce a pDC activation phenotype and to increase production of Interferon alpha (IFNa) by pDCs and was found with both human and mouse cells.

[0053] Additional agents to induce a specific immune response

[0054] In certain aspects, for some indications, additional agents are operably linked to the NP to induce a focused immune response. In certain aspects, a protein, peptide or mRNA coding for a protein or peptide to enhance development of an antigen-specific immune response is operably linked to the NP.

[0055] In certain aspects, NP that target pDCs via BDCA2 and contain TLR agonists are combined with protein antigens (e.g., flu vaccines), mRNA vaccines (e.g., COVID 19 vaccines), live vaccines with attenuated microorganisms (e.g., MMR or zoster vaccines) or virus-like particles (VLP) to improve the efficacy of vaccination or reduce the amount of vaccine needed to induce an effective immune response.

[0056] Nanoparticles (NP) containing TLR agonists.

[0057] NP composed of various polymers, and / or virus-like particles (VLP), that contain various agents have been prepared. For example, NPs comprising TLR agonists have been designed and tested. Incorporation of TLR agonists into NP overcomes some of the barriers to effective therapy with naked TLR agonists by protecting the TLR agonist from degradation and enhancing delivery to key immune system cells, most notably pDC.

[0058] For various positively charged dendrimers or polymers, with or without chemical modifications, the optimal N / P ratio for use with NP containing TLR agonists is defined during formulation optimization. The optimal N / P ratio exists in a range in which the TLR agonists are successfully entrapped within the NPs at minimal amounts of the positively charged dendrimers or polymers. In certain embodiments, the N / P ratio range is 3 : 1 to 10: 1.

[0059] Immunostimulatory Compositions

[0060] In certain aspects, provided herein is an immunostimulatory composition comprising: (a) a nanoparticle (NP), VHPM 17023.303WO1 / UIRF 25010

[0061] (b) an activating agent consisting of an TLR agonist operably linked to the NP, and

[0062] (c) a blood dendritic cell antigen 2 (BDCA2) ligand operably linked to the NP.

[0063] In certain aspects, the NP is a dendrimer.

[0064] In certain aspects, the dendrimer is polyamidoamine (PAMAM).

[0065] In certain aspects, the PAMAM is a generation 1, 2, 3, 4 or 5 architecture.

[0066] In certain aspects, the PAMAM is a generation 5 architecture.

[0067] In certain aspects, the PAMAM has a nitrogen / phosphate ratio is from 1 :3 to 1 : 10.

[0068] In certain aspects, the NP comprises polyethyleneimine, polypropyleneimine, poly-L-lysine, poly-D-lysine, poly-L-arginine, poly-D-arginine, poly-L-histidine, poly- D-histidine, and / or chitosan.

[0069] In certain aspects, the immunostimulatory composition further comprises a secondary polymer conjugated to the NP.

[0070] In certain aspects, the secondary polymer is polyethylene glycol (PEG).

[0071] In certain aspects, the secondary polymer is dextran.

[0072] In certain aspects, the secondary polymer is present at a molar ratio to PAMAM of 0 to 64.

[0073] In certain aspects, the particle is a liposome.

[0074] In certain aspects, the particle consists of a virus like particle.

[0075] In certain aspects, the activating agent is a Toll Like Receptor (TLR) agonist.

[0076] In certain aspects, the TLR agonist is a TLR7 agonist or TLR8 agonist

[0077] In certain aspects, the TLR agonist is a TLR9 agonist.

[0078] In certain aspects, the TLR agonist is a class A CpG ODN TLR9 agonist.

[0079] In certain aspects, the TLR agonist is TLR9 agonist G10.

[0080] In certain aspects, the BDCA2 ligand is heparin or a heparin-like molecule.

[0081] In certain aspects, the BDCA2 ligand is keratan sulfate or chondroitin sulfate.

[0082] In certain aspects, the BDCA2 ligand is an IgG or a glycan.

[0083] In certain aspects, the glycan is a galactose-terminated biantennary N-linked glycan.

[0084] In certain aspects, the immunostimulatory composition further comprises an additional agent operably linked to the NP. VHPM 17023.303WO1 / UIRF 25010

[0085] In certain aspects, the additional agent is a protein, peptide or mRNA coding for a protein or peptide.

[0086] In certain aspects, the additional agent is a vaccine including a protein, peptide, mRNA, attenuated live organism of VLP.

[0087] In certain aspects, the vaccine is a protein, peptide, mRNA, or attenuated live organism of VLP.

[0088] Formulations of Immunostimulatory Compositions

[0089] The invention also provides a pharmaceutical composition comprising an immunostimulatory composition or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0090] The pharmaceutical compositions of the invention can comprise one or more excipients. When used in combination with the pharmaceutical compositions of the invention the term “excipients” refers generally to an additional ingredient that is combined with the compound of formula (I) or the pharmaceutically acceptable salt thereof to provide a corresponding composition. For example, when used in combination with the pharmaceutical compositions of the invention the term “excipients” includes, but is not limited to: carriers, binders, disintegrating agents, lubricants, sweetening agents, flavoring agents, coatings, preservatives, and dyes.

[0091] In cases where compounds are sufficiently basic or acidic, a salt of a compound of formula I can be useful as an intermediate for isolating or purifying a compound of formula I. Additionally, administration of a compound of formula I as a pharmaceutically acceptable acid or base salt may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, a- ketoglutarate, and a-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.

[0092] Salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

[0093] The compounds of formula I can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes. VHPM 17023.303WO1 / UIRF 25010

[0094] Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0095] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

[0096] The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0097] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or VHPM 17023.303WO1 / UIRF 25010 dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0098] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0099] For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

[0100] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0101] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0102] Examples of useful dermatological compositions which can be used to deliver the compounds of formula I to the skin are known to the art; for example, see Jacquet et al. (U.S. VHPM 17023.303WO1 / UIRF 25010

[0103] Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

[0104] Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

[0105] The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

[0106] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

[0107] Methods of Making Immunostimulatory Compositions

[0108] PAMAM dendrimer chemical modification (i.e., synthesis of PEG conjugated PAMAM dendrimers (PAMAM-PEG)). The COOH-PEG-OH (average molecular weight of 2,000 Da; 20 mg) was dissolved in 10 ml 2-morpholin-4-ylethanesulfonic acid (MES) buffer (pH 6). A solution of l-ethyl-3 -(3 -dimethylaminopropyl) (EDC; 19 mg) and N-hydroxysulfosuccinimide (Sulfo-NHS; 54 mg) in 1 ml of the MES buffer was added dropwise to the COOH-PEG-OH solution while stirring. The resulting mixture was stirred for 15 min followed by a pH adjustment to the pH values of 7 to 8 using dilute NaOH solution. 5% (w / v) PAMAM (generation 5) methanolic solution (706.4 pL) was added dropwise to the pH-adjusted mixture and stirred for 24 h. The resulting mixture, which contains PAMAM-PEG, was purified using a 10-kDa MWCO cellulosic dialysis membrane against deionized water.

[0109] Preparation of heparin-coated dendrimer-TLR9 agonists NPs: To prepare dendrimer- TLR9 agonists NPs, G10 was dissolved in water at a concentration of 100 pg / ml, and PAMAM- PEG solution was also prepared. Same volumes of G10 and PAMAM-PEG were mixed at an N / P ratio of 4. The N / P ratio can be 3 to 10, and does not need to be limited to 4. The mixture was vortexed for 30 seconds and sat at room temperature for 30 min. To prepare heparin-coated NPs, heparin was dissolved in deionized water at a concentration of 1 mg / ml, and added to previously prepared dendrimer-TLR9 agonists NPs. The mass ratio of heparin / GlO VHPM 17023.303WO1 / UIRF 25010 ranges from 1 / 1 to 5 / 1. The mixture of heparin and dendrimer- TLR9 agonists NPs was vortexed for 10 seconds and sat at room temperature for 10 min.

[0110] Methods of Use of Immunostimulatory Compositions

[0111] The invention also provides a method for activating plasmacytoid dendritic cells (pDCs) in an animal (e.g., a mammal such as a human) comprising administering an immunostimulatory composition or a pharmaceutical composition to the animal.

[0112] The invention provides for a method of in situ immunization, where the agent is delivered directly into the tumor to enhance the local immune response against a tumor based on the unique antigen profile of the tumor.

[0113] The invention also provides a method of enhancing an immune response against cancer in an animal (e.g., a mammal such as a human) comprising administering an immunostimulatory composition or a pharmaceutical composition to the animal either through injection directly into the tumor or systemic treatment such as subcutaneous or intramuscular injection.

[0114] The invention also provides a method of enhancing an immune response to a vaccine in an animal (e.g., a mammal such as a human) comprising administering an immunostimulatory composition or a pharmaceutical composition to the animal. In such a case, a protein, peptide or mRNA is incorporated into the NP and the NP delivered systemically such as into the skin, subcutaneous tissue or muscle.

[0115] The invention also provides an immunostimulatory composition or a pharmaceutically acceptable salt thereof for use in medical therapy.

[0116] In certain aspects, the immunostimulatory compositions of the present invention can be used to prevent or treat a variety of conditions, such as cancers or enhancing the efficacy of vaccines. Figures 1A-1D. In certain aspects, the composition is used for in situ immunization in cancer or as an immune adjuvant in other vaccination settings.

[0117] Cancers

[0118] In certain aspects, systemic or intratumoral injection of NPs (e.g., heparin-coated NPs containing TLR9 agonists) are used to enhance the activation of pDCs within cancers, thereby enhancing the anti-tumor immune response more effectively than currently available agents.

[0119] Vaccines

[0120] Such NP also enhance the efficacy of vaccination for a variety of diseases by serving as an improved adjuvant for a broad range of vaccination approaches. In certain aspects, heparin- coated particles containing TLR9 agonists could serve as an improved adjuvant for a broad range of vaccination approaches. In certain aspects, combining vaccines based on protein antigens (e.g., flu vaccine) or mRNA vaccines (e.g., COVID 19 vaccines) with heparin-coated VHPM 17023.303WO1 / UIRF 25010 particles containing TLR9 agonists activate pDCs at the site of vaccination and so improve the efficacy of vaccination or reduce the amount of vaccine needed to induce an effective immune response.

[0121] The invention will now be illustrated by the following non-limiting Examples.

[0122] EXAMPLE 1

[0123] Heparin-coated particles containing TLR9 agonists are potent activators of pDCs

[0124] Heparin is readily available and is among the most potent ligands for BDCA2. To further explore BDCA2-heparin interactions, it was explored whether nanoparticles (NP) containing TLR9 agonists that have heparin on their surface are immunosuppressive or immunostimulatory.

[0125] Polyamidoamine (PAMAM) dendrimers were mixed with different concentrations of G10 to create lipid nanoparticles (LNP) containing three different concentrations of G10 (0.2, 1.0 and 2.5 pg / ml). Particles ere left unconjugated (LNP no heparin) or conjugated with different amounts of heparin (LNP-H in pg / ml) to create heparin-conjugated particles. Particles ere incubated in duplicate with normal donor human peripheral blood mononuclear cells at a concentration of 1 million cells / ml for 20 hours. Analyses were run in duplicate. Dotted line indicates level of activation induced by soluble G10.

[0126] Results from experiments with these particles indicate BDCA2 plays a more complex role than suggested by the literature. Such heparin-coated particles containing TLR9 agonists were potent activators of pDCs, in contrast with the previously published role of BDCA2. Figures 2A-2B. Similar particles that contained TLR9 agonists but were not coated by heparin had little effect. This activation of pDCs was confirmed by both flow cytometry demonstrating induction of a pDC activation phenotype and ELISA assay showing pDCs were stimulated to produce IFNa. Thus, particles containing TLR agonists coated with BDCA2 ligands are highly immunostimulatory.

[0127] Although the foregoing specification and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto.

[0128] All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments VHPM 17023.303WO1 / UIRF 25010 and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

[0129] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms ( / .< ., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0130] Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. VHPM 17023.303WO1 / UIRF 25010WHAT IS CLAIMED IS:

1. An immunostimulatory composition comprising:(a) a nanoparticle (NP),(b) an immune activating agent operably linked to the NP, and(c) a blood dendritic cell antigen 2 (BDCA2) ligand operably linked to the NP.

2. The immunostimulatory composition of claim 1, wherein the NP is a dendrimer.

3. The immunostimulatory composition of claim 2, wherein the dendrimer is polyamidoamine (PAMAM).

4. The immunostimulatory composition of claim 3, wherein the PAMAM is a generation 1, 2, 3, 4 or 5 architecture.

5. The immunostimulatory composition of claim 3, wherein the PAMAM is a generation 5 architecture.

6. The immunostimulatory composition of any one of claims 1-5, wherein the PAMAM has a nitrogen / phosphate ratio is from 3:1 to 10:1.

7. The immunostimulatory composition of claim 1, wherein the NP comprises polyethyleneimine, polypropyleneimine, poly-L-lysine, poly-D-lysine, poly-L- arginine, poly-D-arginine, poly-L-histidine, poly-D-histidine, and / or chitosan.

8. The immunostimulatory composition of any one of claims 1-7, wherein a secondary polymer is conjugated to the NP.

9. The immunostimulatory composition of claim 8, wherein the secondary polymer is polyethylene glycol (PEG).

10. The immunostimulatory composition of claim 9, wherein the secondary polymer is dextran.

11. The immunostimulatory composition of claim 9, wherein the secondary polymer is present at a molar ratio to PAMAM of 0 to 64.VHPM 17023.303WO1 / UIRF 2501012. The immunostimulatory composition of claim 1 where the NP is a liposome.

13. The immunostimulatory composition of claim 1 where the NP is a virus-like particle.

14. The immunostimulatory composition of any one of claims 1-13, wherein the activating agent is a Toll Like Receptor (TLR) agonist.

15. The immunostimulatory composition of claim 14, wherein the TLR agonist is a TLR7 agonist, TLR8 agonist, or TLR9 agonist.

16. The immunostimulatory composition of claim 14, wherein the TLR agonist is a TLR9 agonist.

17. The immunostimulatory composition of claim 14, wherein the TLR agonist is a class A CpG ODN TLR9 agonist.

18. The immunostimulatory composition of claim 14, wherein the TLR agonist is TLR9 agonist GIO.

19. The immunostimulatory composition of any one of claims 1-18, wherein the BDCA2 ligand is heparin or a heparin-like molecule.

20. The immunostimulatory composition of any one of claims 1-18, wherein the BDCA2 ligand is keratan sulfate or chondroitin sulfate.

21. The immunostimulatory composition of any one of claims 1-18, wherein the BDCA2 ligand is an IgG or a glycan.

22. The immunostimulatory composition of any one of claims 1-18, wherein the glycan is a galactose-terminated biantennary N-linked glycan.

23. The immunostimulatory composition of any one of claims 1-22, further comprising an additional agent operably linked to the NP.VHPM 17023.303WO1 / UIRF 2501024. The immunostimulatory composition of claim 23, wherein the additional agent is a protein, peptide or mRNA coding for a protein or peptide.

25. The immunostimulatory composition of claim 23, wherein the additional agent is a vaccine.

26. A pharmaceutical composition comprising the immunostimulatory composition of any one of claims 1-25 and a pharmaceutically acceptable excipient.

27. A method of activating plasmacytoid dendritic cells (pDCs), comprising administering the immunostimulatory composition of any one of claims 1-25 or the pharmaceutical composition of claim 26 to a patient in need thereof.

28. A method of enhancing an immune response against cancer, comprising administering the immunostimulatory composition of any one of claims 1-25 or the pharmaceutical composition of claim 26 to a patient in need thereof.

29. A method of enhancing an immune response to a vaccine, comprising administering the immunostimulatory composition of any one of claims 1-25 or the pharmaceutical composition of claim 26 to a patient in need thereof.

30. An immunostimulatory composition of any one of claims 1-25 or a pharmaceutical composition of claim 26 to a patient in need thereof for use in medical therapy.

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