Compositions and methods for metal-containing formulations capable of modulating immune responses - Patents.com

Abstract

The present disclosure provides compositions and methods for stimulating an innate immune response in a subject with agents (e.g., damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs)) that are capable of stimulating an innate immune response in a subject upon administration to the subject. In particular, the present invention relates to compositions of DAMPs / PAMPs and metal ions, as well as systems and methods that utilize such nanoparticles (e.g., in diagnostic and / or therapeutic settings).

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This invention claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 250,359, filed September 30, 2021, the entire contents of which are incorporated by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under CA210273 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] Incorporation by reference of electronically submitted material The Sequence Listing filed concurrently with this application and identified as follows: One 527,000 byte file entitled "UM-39947-601," created on September 30, 2022; is hereby incorporated by reference in its entirety.

[0004] [Technical Field] The present disclosure provides compositions and methods for stimulating a subject's innate immune response with agents (e.g., damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs)) that can be administered to a subject to stimulate the subject's innate immune response. In particular, the disclosure relates to compositions of DAMPs / PAMPs and metal ions, as well as systems and methods that utilize such nanoparticles (e.g., in diagnostic and / or therapeutic settings).

[0005] [Background technology] The innate immune system is the first line of defense in humans, and activation of the innate immune system can induce proinflammatory cytokine secretion and modulate the adaptive immune system. DAMPs and PAMPs are two major innate immune stimulators. DAMPs are endogenous host biomolecules released upon tissue injury, including heat shock proteins and HMGB1 (High Mobility Group Box 1), ATP, uric acid, hyaluronan fragments, heparin sulfate, and tumor-derived DNA. PAMPs are conserved pathogen components recognized by various pathogen recognition receptors (PRRs) and induce anti-pathogen inflammation. PAMPs include Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), cytoplasmic DNA sensors (CDSs), stimulators of IFN genes (STING) agonists, purine-containing or purine-derivative factors, and C-type lectin receptors (CLRs).

[0006] DAMPs and PAMPs can induce the production of proinflammatory cytokines and the proinflammatory phenotype of immune cells, which are important for both cancer and autoimmune diseases. Meanwhile, the proinflammatory phenotype disrupts the immunosuppressive tumor microenvironment and modulates a "cold tumor" into a "hot tumor." Therefore, TLR-3, TLR4, TLR7, TLR9, NLRP3, and STING agonists are currently undergoing clinical trials for cancer immunotherapy. In particular, the tumor-derived DNA-cGAS-STING pathway has recently been shown to be crucial for monitoring tumor immunity, demonstrating dramatic efficacy in preclinical cancer immunotherapy, leading to numerous phase I clinical trials of STING agonists. Meanwhile, DAMPs and PAMPs are widely involved in the development and progression of autoimmune diseases. Inhibiting aberrant innate immune responses is becoming increasingly clear as an effective treatment for many incurable autoimmune diseases. Modulating DAMP- and PAMP-mediated immune responses may provide new therapeutic approaches to a variety of human diseases, including cancer and autoimmune disorders.

[0007] The present invention addresses this need.

[0008] Summary of the Invention Immune checkpoint blockade enables a patient's own immune system to fight cancer. However, the current average response rate to immune checkpoint blockade is only approximately 30%. This is thought to be due to the fact that some tumors have characteristics that make them less recognizable by the immune system, known as "cold tumors." These tumor characteristics include a low inflammatory response, a low mutational burden, and a lack of tumor infiltration by T cells and other pro-inflammatory immune cells. On the other hand, "hot tumors," which have more inflammatory characteristics that are recognizable by the immune system, exhibit a favorable therapeutic response rate to cancer immunotherapy. Therefore, it is important to understand how to transform "cold tumors" into "hot tumors."

[0009] Accumulating evidence indicates that tumor immunity, mediated by the innate immune system, recognizes tumors by sensing tumor cell-derived DNA via the STING pathway. Activation of the STING pathway triggers the innate immune cascade, including type I interferon responses and other proinflammatory phenotypic changes, which further induce adaptive antitumor responses. Therefore, STING has been implicated as a "trigger" for reversing a "cold tumor" to a "hot tumor." For example, intratumoral administration of STING agonists can induce antitumor immune responses against both local and metastatic tumors. Clinical studies have demonstrated that type I interferon responses, along with antigen-specific T cell infiltration, are hallmarks of better cancer treatment outcomes. Therefore, developing STING agonists with significant in vivo stability, favorable pharmacokinetic properties, and an acceptable safety profile is of great importance and has high translational value.

[0010] However, most current evaluation of human STING agonists is based on cyclic dinucleotides and their derivatives, whose low molecular weight, poor pharmacokinetic parameters, and severe side effects severely limit their systemic administration.

[0011] Experiments conducted in the course of developing embodiments of the present invention demonstrated that various metal ions (e.g., Zn 2+ , Mn 2+ We have demonstrated that Toll-like receptor (TLR) agonists and cyclic dinucleotides (CDNs) (e.g., cdi-AMP, cGAMP, and cGMP) each assemble into uniform nanoparticles in the presence of various metal ions (e.g., Zn). 2+ , Mn 2+ It was also shown that such TLR agonists and CDNs assembled into uniform nanoparticles in the presence of (e.g., ) were further stabilized with lipid vesicles.

[0012] Further experiments demonstrated that CDNs or TLR agonists can be formulated into nanoparticles in the presence of calcium phosphate and copolymers of cationic poly(ethyleneimine) (PEI) and polyethylene glycol (PEG). Such CDN-nanoparticle assemblies (e.g., CDN and PEI-PEG copolymers formulated into nanoparticles in the presence of calcium phosphate) (e.g., Zn 2+ and CDNs formulated into nanoparticles in the presence of liposomes) were further shown to increase cancer cell uptake and provide more precise targeting to the tumor microenvironment (e.g., TME), thereby enabling increased STING agonist delivery efficacy and reduced STING agonist toxicity.

[0013] For TLR agonist or CDN / metal ion embodiments, these results demonstrate the following unique features compared to conventional drug delivery systems: 1) reversible assembly suitable for sustained drug release without loss of bioactivity, 2) high loading efficiency and capacity, 3) increased cellular uptake, 4) pH-sensitive release at low pH, 5) good biocompatibility, 6) flexible surface chemistry suitable for surface modification and functionalization, and 7) low cost and ease of scale-up.

[0014] For the CDN@CaP / PEI-PEG embodiment, these results demonstrate the following unique features compared to conventional drug delivery systems: 1) increased cellular uptake, 2) high loading efficiency, 3) pH-sensitive release at low pH, 4) biocompatibility, and 5) low cost and ease of scale-up.

[0015] These results are of great clinical importance because these CDN-related nanoparticles can induce specific tumor-directed immune responses via systemic administration, thereby avoiding the need for direct local injections into the tumor.

[0016] Further experiments conducted during the course of developing embodiments of the present invention discovered that certain metal ions can significantly enhance STING activation and type I IFN responses of STING agonists. For example, in optimized conditions, Mn 2+ or Co 2+ It was shown that cGAMP enhances STING activity by more than 60-fold. 2+ or Co 2+ Administration of a STING agonist in combination with Coxib significantly improved therapeutic efficacy, characterized by elevated serum type I IFN concentrations, greater tumor eradication, and longer animal survival. Following treatment, 80% of tumor-bearing mice were eradicated, and the resulting tumors remained resistant to challenging secondary tumors 80 days later, demonstrating long-term immunity against tumor recurrence. Furthermore, this phenomenon was found to be generalizable to a variety of other innate immune pathways, including, but not limited to, TLR3 / 4 / 7 / 8 / 9 ligands, NOD1 / 2 ligands, TLR7 / 8 ligands, RIG-I & CDS agonists, and inflammasome inducers. For example, Coxib 3+ dramatically increased Poly IC-mediated production of IFNb, TNFa, IL6, and IL2 by dendritic cells, whereas Mn 2+ Mn increased PolyIC-mediated IFNb production. 2+ increased MPLA-mediated IFNb and TNFa production, and Ni 2+ Mn increased MPLA-mediated TNFα production.2+ increased R848-mediated IFNb and TNFa production, and 2+ increased R848-mediated TNFα production. 2+ and Mn 2+ increased CpG-mediated production of IFNb and TNFa.

[0017] Based on these results, several pharmaceutically acceptable formulations have been developed to precisely deliver metal-innate immune stimulator combinations to the desired target and promote immune activation. For example, liposome-coated nanoparticles, CDA-Mn-His11-DOPE@liposome (Mn-CDA / H11@lip), can be used for systemic delivery of STING agonists and eradicate 60% of the resulting CT26 colon tumors. Co-CDA / His33-PEG significantly prolonged IFNb production, which was detectable even 4 days after injection. Furthermore, we conducted experiments to test whether chelated intracellular metal ions inhibit the innate immune response. Through equivalent screening, several chelators were identified that could effectively inhibit DNA-induced cGAS-STING-type I IFN / NFkB responses and Poly(I)-induced TLR3-cGAS-STING-type I IFN, potentially useful for the treatment of autoimmune diseases. Overall, these results represent a simple yet effective approach to solving several unmet medical challenges, such as improving the efficacy of vaccine adjuvants, developing cancer immunotherapies, and controlling autoimmune diseases.

[0018] Thus, these results and embodiments represent a new class of drug delivery systems for both local and systemic delivery of agents that, upon administration to a subject, can stimulate an innate immune response in the subject.

[0019] Thus, the present disclosure provides compositions and methods for stimulating a subject's innate immune response upon administration to a subject by administering an agent capable of stimulating the subject's innate immune response. In particular, the present invention is directed to such compositions comprising an agent capable of stimulating a subject's innate immune response upon administration to a subject, methods for synthesizing such compositions, and systems and methods that utilize such compositions (e.g., in diagnostic and / or therapeutic settings).

[0020] Thus, in certain embodiments, the present invention provides a method for detecting a mutated or modified nucleotide sequence comprising one or more DAMPs or PAMPs and: a) calcium phosphate and copolymers of cationic poly(ethyleneimine) (PEI) with polyethylene glycol (PEG), poly(histidine)-polyethylene glycol (PH-PEG), lipid-polyhistidine, poly(lysine)-polyethylene glycol PEG (PK-PEG), or anionic poly(glutamic acid)-polyethylene glycol (PGA-PEG); and b) Zn 2+ , Mn 2+ , Ca 2+ , Fe 2+ , Fe 3+ , Cu 2+ , Ni 2+ , Co 2+ , Pb 2+ , Sn 2+ , Ru 2+ , Au 2+ , Mg 2+ ,VO 2+ , Al 3+ , Co 3+ , Cr 3+ , Ga 3+ , Tl 3+ , Ln 3+ , MoO 3+ , Cu + , Au + , Tl + , Ag + , Hg 2+ , Pt 2+ , Pb 2+ , Hg 2+ , Cd 2+ , Pd 2+ , Pt4+ , Na + , K. + and one or more cations selected from the group consisting of associated phosphates or carbonates; and one or both of:

[0021] In certain embodiments, one or more DAMPs or PAMPs; below: Zn 2+ , Mn 2+ , Fe 2+ , Fe 3+ , Cu 2+ , Ni 2+ , Co 2+ , Pb 2+ , Sn 2+ , Ru 2+ , Au 2+ , Mg 2+ ,VO 2+ , Al 3+ , Co 3+ , Cr 3+ , Ga 3+ , Tl 3+ , Ln 3+ , MoO 3+ , Cu + , Au + , Tl + , Ag + , Hg 2+ , Pt 2+ , Pb 2+ , Hg 2+ , Cd 2+ , Pd 2+ , Pt 4+ , Na + , K. + and one or more cations selected from the group consisting of: and related phosphates or carbonates; and Lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC) , dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), DSPE-PEG, monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearyl ... one or more lipid molecules (e.g., phospholipids) selected from aroyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0PA), or 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (18:1PA); and one or more of the following:

[0022] In some embodiments, the composition is capable of stimulating an innate immune response in a subject upon administration to the subject. In some embodiments, the subject has cancer or is at risk of having cancer. In some embodiments, the composition is used to elicit an immune response to a vaccine. In some embodiments, the subject has cancer, and the composition is capable of stimulating an innate immune response in at least one cancer cell upon administration to the subject. In some embodiments, stimulating an innate immune response comprises stimulating an innate cytokine response mediated via a cytokine, wherein the innate cytokine response is mediated via type 1 interferon.

[0023] Thus, in certain embodiments, the present invention provides methods for treating cancer in a subject, the methods comprising administering to the subject a pharmaceutically effective amount of a composition comprising an agent (e.g., a DAMP / PAMP) capable of stimulating an innate immune response in the subject upon administration to the subject. In some embodiments, the innate immune response is an innate cytokine response mediated via cytokines in the subject. In some embodiments, the innate cytokine response is mediated via type 1 interferon in the subject.

[0024] Such methods are not limited to a particular mode of administration. In some embodiments, administration is systemic. In some embodiments, administration is local.

[0025] In some embodiments, the composition is administered with a chemotherapeutic agent, such as aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alfa, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarucizumab, dacarbazine (DTIC), ... One or more of the following: rubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (TAXOL), pilocarpine, prochlorperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate.

[0026] Such compositions are not limited to specific DAMP or PAMP agonists.In some embodiments, DAMP and PAMP agonists are selected from STING agonists, purine-containing or purine-derived factors, Toll-like receptor (TLR) agonists, NOD-like receptor (NLR) agonists, RIG-I-like receptor (RLR) agonists, cytoplasmic DNA sensor (CDS) agonists, C-type lectin receptor (CLR) agonists, and inflammasome inducers.In some embodiments, DAMP and PAMP agonists are selected from TLR-3 agonists, TLR-4 agonists, TLR-5 agonists, TLR-7 agonists (e.g., imiquimod), TLR-8 agonists (e.g., resiquimod), TLR-9 agonists, and NLRP3 agonists.

[0027] Such compositions are not limited to specific purine-containing or purine-derivative factors. In some embodiments, the purine-containing or purine-derivative factor is 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluor (cAIMP Difluor), cAIM(PS)2, difluor (Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluoride (3'3'-cGAMP Fluorinated), c-di-AMP fluoride, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluoride, 2'3' -c-di-GMP, c-di-IMP, cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, 3'3'-cGAMP, cGAM(PS)2, 2'3'-cGAM(PS)2(Rp / Sp), 2'2'-cGAM(PS)2, 2'3'-cGAM(PS)2, cGAMP fluoride, 3'3'-cGAMP fluoride, 2'3'-cGAMP fluoride, 2'2'-cGAMP fluoride, c-di-AMP, 2'3'-cdAMP, 2'2'-cdAMP, 3'3'-cdAMP, c-di-AM(PS)2, 2'3'-c-di-AM(PS)2(Rp,Rp), 2'2'-c-di-AM(PS)2, 3'3'-c-di-AM(PS)2, c-di-AMP fluoride, 2'3'-cdAMP fluoride, 2'2'-cdAMP fluoride fluoride, 3'3'-cdAMP fluoride, cdGMP, 2'3'-cdGMP, 2'2'-cdGMP, 3'3'-cdGMP, c-di-GM(PS)2, 2'3'-c-di- GM(PS)2, 2'2'-c-di-GM(PS)2, 3'3'-c-di-GM(PS)2, cdGMP fluoride, 2'3'-cdGMP fluoride, 2'2'-cdGMP fluoride Fluorinated, 3'3'-cdGMP fluorinated, cAIMP, 2'3'-cAIMP, 2'2'-cAIMP, 3'3'-cAIMP, cAIMP difluoro (3'3'-cAIMP fluorinated) Fluorinated compound, 2'3'-cAIMP fluorinated compound, 2'2'-cAIMP fluorinated compound, cAIM(PS)2 difluoro, 3'3'-cAIM(PS)2 difluoro(Rp / Sp), 2'3 '-cAIM(PS)2 difluoro, 2'2'-cAIM(PS)2 difluoro, c-di-IMP, 2'3'-cdIMP, 2'2'-cdIMP, 3'3'-cdIMP, c-d i-IM(PS)2, 2'3'-c-di-IM(PS)2, 2'2'-c-di-IM(PS)2, 3'3'-c-di-IM(PS)2, c-di-IMP fluoride, 2'3'-cdIMP fluoride, 2'2'-cdIMP fluoride, 3'3'-cdIMP fluoride, imiquimod, resiquimod, 6-(4-amino-imidazoquinolyl)-norleucine, [ka] The inhibitor is selected from RNA, siRNA, microRNA, interfering RNA, mRNA, replicon mRNA, RNA analog, DNA, and a purine-based PI3K inhibitor.

[0028] Such compositions are not limited to a specific type or type of STING agonist. In some embodiments, the STING agonist is a small molecule agonist of STING. In some embodiments, the small molecule agonist of STING is a cyclic dinucleotide. For example, in some embodiments, the cyclic dinucleotide comprises cGAMP, cdiAMP, cdiGMP, and cAIMP. Further examples of cyclic purine dinucleotides are described in some detail in, for example, U.S. Patent Nos. 7,709,458 and 7,592,326; WO2007 / 054279; and Yan et al., Bioorg. Med. Chem Lett. 18: 5631 (2008). Each of these documents is incorporated herein by reference. In some embodiments, the additional STING agonist is selected from 5,6-dimethylxanthenone-4-acetic acid (DMXAA), methoxyflavone, 6,4'-dimethoxyflavone, 4'-methoxyflavone, 3',6'-dihydroxyflavone, 7,2'-dihydroxyflavone, daidzein, formononetin, and letucin 7-methyl ether, or any derivative thereof. In some embodiments, small molecule agonists of STING include but are not limited to 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluoro, cAIM(PS)2, difluoro(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluorinated, c-di-AMP fluorinated, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluorinated, 2'3'-c-di-GMP, c-di-IMP, SB11285, STING-agonist-C11, STING agonist-1, STING agonist G10, and gemcitabine.

[0029] In some embodiments, the small molecule agonist of STING is [ka] 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluoro, cAIM(PS)2, difluoro(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluoride, c-di-AMP fluoride, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluoride, 2'3'-c-di-GMP, c-di-IMP, cGAMP, 2' 3'-cGAMP, 2'2'-cGAMP, 3'3'-cGAMP, cGAM(PS)2, 2'3'-cGAM(PS)2(Rp / Sp), 2'2'-cGAM(PS)2, 2'3'-cGAM(PS)2, cGAMP fluoride, 3'3'-cGAMP fluoride, 2'3'-cGAMP fluoride, 2'2'-cGAMP fluoride, c-di-AMP, 2'3'-cdAMP, 2'2'-cdAMP, 3'3'-cdAMP, c-di-AM(PS)2, 2'3'-c-di-AM(PS)2(Rp,Rp), 2'2'-c-di-AM(PS)2, 3'3'-c-di-AM(PS)2, c-di-AMP fluoride, 2'3'-cdAMP fluoride, 2'2'-cdAMP fluoride fluoride, 3'3'-cdAMP fluoride, cdGMP, 2'3'-cdGMP, 2'2'-cdGMP, 3'3'-cdGMP, c-di-GM(PS)2, 2'3'-c-di- GM(PS)2, 2'2'-c-di-GM(PS)2, 3'3'-c-di-GM(PS)2, cdGMP fluoride, 2'3'-cdGMP fluoride, 2'2'-cdGMP fluoride Fluorinated, 3'3'-cdGMP Fluorinated, cAIMP, 2'3'-cAIMP, 2'2'-cAIMP, 3'3'-cAIMP, cAIMP Difluoro (3'3'-cAIMP Fluorinated) fluoride, 2'3'-cAIMP fluoride, 2'2'-cAIMP fluoride, cAIM(PS)2 difluoro, 3'3'-cAIM(PS)2 difluoro(Rp / Sp), 2'3' -cAIM(PS)2 difluoro, 2'2'-cAIM(PS)2 difluoro, c-di-IMP, 2'3'-cdIMP, 2'2'-cdIMP, 3'3'-cdIMP, c-di The compound is selected from fluorinated c-di-IM(PS)2, 2'3'-c-di-IM(PS)2, 2'2'-c-di-IM(PS)2, 3'3'-c-di-IM(PS)2, c-di-IMP, fluorinated 2'3'-cdIMP, fluorinated 2'2'-cdIMP, and fluorinated 3'3'-cdIMP, and amidobenzimidazole (ABZI)-based compounds.

[0030] As previously mentioned, the use of CDNs as cancer therapeutics has two significant limitations: 1) poor pharmacokinetics and severe, undesirable side effects. Regarding poor pharmacokinetics, when administered via intratumoral injection, CDNs readily diffuse due to their low molecular weight and high hydrophilicity. However, when administered via intravenous infusion, CDNs may exhibit low bioavailability in tumor tissue due to their in vivo instability, low lipophilicity, and rapid elimination. Regarding severe, undesirable side effects, as an immunological sensor against viral infections, STING is widely distributed throughout the body. Therefore, high doses of STING agonists or systemically administered STING agonists can nonspecifically activate the innate immune system and cause a cytokine storm. The present invention addresses these limitations by providing prodrugs of such small molecule agonists of DAMPs and / or PAMPs (including STING agonists).

[0031] Indeed, in some embodiments, the small molecule agonist of a DAMP and / or PAMP is a prodrug of the small molecule agonist of a DAMP and / or PAMP. For example, in some embodiments, the prodrug of the small molecule agonist of a DAMP and / or PAMP is a prodrug of any of the small molecule agonists of a DAMP and / or PAMP listed herein. In some embodiments, the prodrug of the small molecule agonist of a DAMP and / or PAMP is conjugated to a hydrophobic moiety that supports nanoparticle loading and / or tissue retention.

[0032] In some embodiments, CDNs are modified with a cleavable lipid moiety to generate CDN prodrugs. For example, three synthetic routes for lipid-CDN prodrugs are contemplated, as shown in the following schemes. Each is activated by a different mechanism: esterase-based activation for Route 1, phosphoramidase-based activation for Route 2, and reduced environmentally sensitive activation for Route 3.

[0033] [ka]

[0034] [ka]

[0035] [ka]

[0036] After modification, the lipid-CDN prodrug can be administered either in free or liposomal form. Such an embodiment significantly improves pharmacokinetics and reduces CDN side effects. For example, an injected lipid-CDN prodrug is intended to be retained at the injection site and slowly release CDN into the tumor, conferring high bioavailability and reducing side effects on normal tissues. For example, liposomal lipid-CDN prodrugs can be administered either intravenously or locally. Such liposomal lipid-CDNs can significantly prolong drug circulation in the blood, increasing tumor accumulation and lymph node drainage. More importantly, CDNs are inactive after lipid modification and can only be reactivated when cleaved by esterase. Furthermore, previous studies have shown that metastatic lymph nodes can be distinguished from tumor-free lymph nodes by their high esterase levels, allowing for selective activation of lipid-CDN prodrugs at tumor sites.

[0037] In some embodiments, STING-activating compounds are provided (see, e.g., WO2017011920, WO2017027646, WO2017011622, U.S. Patent Application Publication No. 20160287623, WO2016100261, U.S. Patent Application Publication No. 20160074507, and WO2015161762).

[0038] In some embodiments, cGAS modulating compounds are provided (see, e.g., WO2014179335).

[0039] In some embodiments, STING inhibitory compounds are provided (see, e.g., U.S. Patent Application Publication No. 20170037400).

[0040] In some embodiments, compounds are provided that can kill STING-deficient and / or cGAS-deficient cancer cells (see, e.g., WO2016201450).

[0041] In some embodiments, a STING pathway agonist is provided in combination with a pharmaceutically active ingredient (see, e.g., STING activation / chemotherapy (WO2016096577), STING activation / selected vaccine formulations that stimulate immune responses (see U.S. Patent Application Publication Nos. 20150056224 and 20140205653), and STING activation / cytokine production (WO2013185052)).

[0042] In some embodiments, such compositions comprising agents (e.g., DAMPs / PAMPs) that can stimulate an innate immune response in a subject upon administration to the subject are associated with (e.g., by complexing, binding, encapsulation, absorption, adsorption, or mixing with) nanoparticles.

[0043] In some embodiments, such compositions associated with nanoparticles are further associated (e.g., by complexing, binding, encapsulating, absorbing, adsorbing, or mixing) with calcium phosphate and copolymers of PEI / PEG, PH-PEG, PK-PEG, or PGA-PEG. Indeed, in some embodiments, the association of nanoparticles with agents capable of stimulating an innate immune response in a subject is in the presence of calcium phosphate and copolymers of PEI / PEG, PH-PEG, PK-PEG, or PGA-PEG. In some embodiments, such compositions associated with nanoparticles are further associated (e.g., by complexing, binding, encapsulating, absorbing, adsorbing, or mixing) with calcium phosphate and copolymers of PEI / PEG, PH-PEG, PK-PEG, or PGA-PEG. 2+ , Mn 2+ , Ca 2+ , Fe 2+ , Fe 3+ , Cu 2+ , Ni 2+ , Co2+ , Pb 2+ , Sn 2+ , Ru 2+ , Au 2+ , Mg 2+ ,VO 2+ , Al 3+ , Co 3+ , Cr 3+ , Ga 3+ , Tl 3+ , Ln 3+ , MoO 3+ , Cu + , Au + , Tl + , Ag + , Hg 2+ , Pt 2+ , Pb 2+ , Hg 2+ , Cd 2+ , Pd 2+ , Pt 4+ , Na + , K. + and associated phosphates or carbonates. Indeed, in some embodiments, the association of the nanoparticle with an agent capable of stimulating an innate immune response in a subject is further associated (e.g., by complexing, binding, encapsulating, absorbing, adsorbing, or mixing) with one or more cations selected from the group consisting of Zn, ... 2+ , Co 2+ , or Mn 2+ In some embodiments, the cation is Mn 2+ In some embodiments, the cation is Zn 2+ is.

[0044] In some embodiments, nanoparticles and one or more cations (e.g., Zn 2+ , Co 2+ , or Mn 2+ ) or such compositions associated with calcium phosphate are further associated with (eg, by complexing, binding, encapsulating, absorbing, adsorbing, or mixing with) hydrophobic molecules.

[0045] In some embodiments, the hydrophobic molecule is a lipid molecule. In some embodiments, the lipid molecule is a membrane-forming lipid molecule. In some embodiments, the lipid molecule is a non-membrane-forming lipid molecule.

[0046] Examples of lipid molecules applicable to embodiments of the present invention include lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, lipid-polyhistidine (e.g., DOPE-H11), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine ... palmitoyloleoyl-phosphatidylethanolamine (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylethanolamine (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylethanolamine (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidyl Phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, 1,2-dimyristoyl-sn-glycero-3-phosphate; Avanti Polar Lipids Catalog No.: 830845 (14:0 PA), 1,2-dioleoyl-sn-glycero-3-phosphate; Avanti Polar Lipids Catalog No.: 840875 (18:1 PA) (DOPA), 1,2-distearoyl-sn-glycero-3-phosphate (sodium salt); Avanti Polar Lipids Catalog No.: 830865 (18:0 PA), and mixtures thereof.Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used, in which the acyl groups are preferably derived from fatty acids having C10 to C24 carbon chains, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0047] Other non-limiting examples of lipid molecules include sterols such as cholesterol, their derivatives such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

[0048] Other examples of lipid molecules suitable for use in the present invention include non-phosphorus-containing lipids such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkylaryl sulfate polyethyloxylate fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, sphingomyelin, and the like.

[0049] Other examples of lipid molecules suitable for use in the present invention include fatty acids and their derivatives or analogs, including oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilauric acid, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, and their C 1-10These include alkyl esters (e.g., methyl, isopropyl, and t-butyl) and their mono- and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0050] Other examples of lipid molecules suitable for use in the present invention include lipid molecules modified with PEG (PEG-lipid).Examples of PEG-lipid include, but are not limited to, PEG (PEG-DAA) linked to dialkyloxypropyl, for example, as described in PCT Publication No. WO05 / 026372; PEG (PEG-DAG) linked to diacylglycerol, for example, as described in US Patent Publication Nos. 20030077829 and 2005008689; PEG (PEG-PE) linked to phospholipids such as phosphatidylethanolamine; PEG linked to ceramide, for example, as described in US Patent No. 5,885,613; PEG linked to cholesterol or its derivatives, and mixtures thereof.The disclosures of these patent documents are incorporated herein by reference in their entirety for all purposes.Additional PEG-lipids include, but are not limited to, PEG-C-DOMG, 2KPEG-DMG, and mixtures thereof.

[0051] PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups.PEG is classified according to its molecular weight, for example, PEG2000 has an average molecular weight of about 2,000 daltons, and PEG5000 has an average molecular weight of about 5,000 daltons.PEG is commercially available from Sigma Chemical Co. and other companies, and includes, for example, monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH2), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), and monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM).Other PEGs (e.g., mPEG (20KDa) amine) as described in U.S. Patent Nos. 6,774,180 and 7,053,150 are also useful for preparing the PEG-lipid conjugates of the present invention. The disclosures of these patents are incorporated herein by reference in their entireties for all purposes. Additionally, monomethoxypolyethylene glycol acetic acid (MePEG-CH2COOH) is particularly effective for preparing PEG-lipid conjugates, including, for example, PEG-DAA conjugates.

[0052] The PEG moiety of the PEG-lipid conjugates described herein can comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain examples, the PEG moiety has an average molecular weight of about 750 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0053] In certain instances, PEG may be substituted with an alkyl group, an alkoxy group, an acyl group, or an aryl group.PEG can be directly bound to lipid, or can be linked to lipid via a linker moiety.Any linker moiety suitable for linking PEG to lipid can be used, including, for example, non-ester-containing linker moieties and ester-containing linker moieties.In a preferred embodiment, the linker moiety is a non-ester-containing linker moiety.As used herein, the term "non-ester-containing linker moiety" refers to a linker moiety that does not contain a carboxylic acid ester bond (-OC(O)-). Suitable non-ester containing linker moieties include, but are not limited to, amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, disulfide, and combinations thereof (such as linkers containing both carbamate and amide linker moieties). In a preferred embodiment, a carbamate linker is used to attach PEG to the lipid.

[0054] In other embodiments, an ester-containing linker moiety is used to attach PEG to the lipid. Suitable ester-containing linker moieties include, for example, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), sulfonate ester, and combinations thereof.

[0055] Phosphatidylethanolamines with various acyl chain groups of various chain lengths and saturations can be bound to PEG to form lipid conjugates. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. 10 ~C 20Phosphatidylethanolamines containing saturated or unsaturated fatty acids with a carbon chain length of 1 to 1000 are preferred. Phosphatidylethanolamines containing mono- or di-unsaturated fatty acids, as well as mixtures of saturated and unsaturated fatty acids, can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0056] In some embodiments, nanoparticles associated with such compositions comprising agents (e.g., DAMPs / PAMPs) that can stimulate an innate immune response in a subject upon administration to the subject are further associated (e.g., by complexing, binding, encapsulation, absorption, adsorption, mixing) with one or more agents configured to target cancer cells.

[0057] In some embodiments, the agent configured to target cancer cells is selected from the group consisting of α-actinin-4, Bcr-Abl fusion protein, Casp-8, β-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, Mum-2, and Mum-3, neo-PAP, myosin class I, OS-9, pml-R ARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Bage-1, Gage3, Gage4, Gage5, Gage6, Gage7, GnTV, Herv-K-mel, Lage-1, Mage-A1, Mage-A2, Mage-A3, Mage-A4, Mage-A6, Mage-A10, Mage-A12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100 (Pmel17), Tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGS), SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB 2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA19-9, CA-72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA125, CA15-3 (CA27.29\BCAA), CA195, CA242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250Ga733 (EpCAM), human EGFR protein or human EGFR protein fragments such as human EGFR residues 306-325 (SCVRACGADSYEMEEDGVRK (SEQ ID NO: 374)) and residues 897-915 (VWSYGVTVWELMTFGSKPY (SEQ ID NO: 375)), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein \cyclophilin C-related protein), TAAL6, TAG72, TLP, TPS, WT1 (and WT1-derived peptide sequences: WT1 126-134 (RMFP NAPYL (SEQ ID NO: 376)), WT1 122-140 (SGQARMFPNAPYLPSCLES (SEQ ID NO: 377)), and WT1 122-144 (SGQARMFPNAPYLPSCLESQPTI (SEQ ID NO: 378)), MUC1 (and MUC1-derived peptides and glycopeptides such as RPAPGS (SEQ ID NO: 379), PPAHGVT (SEQ ID NO: 380), and PDTRP (SEQ ID NO: 381)), LMP2, EGFRvIII, idiotype, GD2, Ras mutants, p53 mutants, proteinase 3 (PR1), survivin, hTERT, sarcoma translocation breakpoints, EphA2, EphA4, LMW-PTP, PAP, ML-IAP, AFP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl-GM1, mesothelin, sLe (animal), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AK AP-4, XAGE1, B7H3, legumain, Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-α, PDGFR-β, MAD-CT-2, Fos-related antigen 1, ERBB2, folate receptor 1 (FOLR1 or FBP), IDH1, IDO, LY6K, fms-related tyrosine kinase 1 (FLT1, best known as VEGFR1), KDR, PADRE, TA-CIN (recombinant HPV16 L2E7E6),The tumor antigen is selected from the group consisting of SOX2, and aldehyde dehydrogenase, and any derivative thereof.

[0058] In some embodiments, one or more agents configured to target cancer cells are attached to the exterior surface of the nanoparticle, hi some embodiments, one or more agents configured to target cancer cells are encapsulated within the nanoparticle.

[0059] In some embodiments, nanoparticles associated with such compositions comprising agents (e.g., DAMPs / PAMPs) that can stimulate an innate immune response in a subject upon administration to the subject are further associated (e.g., by complexing, binding, encapsulation, absorption, adsorption, mixing) with an adjuvant.

[0060] In some embodiments, the adjuvant is CPG, Poly IC, Poly ICLC, 1018 ISS, aluminum salts (e.g., aluminum hydroxide, aluminum phosphate), Amplivax, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, cytokines (such as GM-CSF, IL-2, IFN-α, Flt-3L), IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, Monophosphoryl Lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel™, Vector Systems, PLGA Microparticles, Imiquimod, Resiquimod, Gardiquimod, 3M-052, SRL172, Virosomes and Other Virus-Like Particles, YF-17D, VEGF Trap, β-Glucan, Pam3Cys, Aquila's QS21 Stimulon, Vadimesan, AsA404 (DMXAA), 3M MEDI9197, glucopyranosyl lipid adjuvant (GLA), GLA-SE, CD1d ligands (C20:2, OCH, AH04-2, α-galatosylceramide, α-C-galatosylceramide, α-mannosylceramide, α-fructosylceramide, β-galatosylceramide, β-mannosylceramide, etc.), STING agonists (e.g., cyclic [G(3',5')pA(3',5')p], cyclic [G(2',5')pA(3',5')p], cyclic [G(2',5')pA(2',5')p], cyclic diadenylate monophosphate, cyclic diglycerides and cyclic dinucleotides containing anilate monophosphate), CL401, CL413, CL429, flagellin, RC529, E6020, imidazoquinoline-based small molecules TLR-7 / 8a (including lipidated analogs thereof), virosomes, AS01, AS02, AS03, AS04, AS15, IC31, CAF01, ISCOM, cytokines (such as GM-CSF, IL-2, IFN-α, Flt-3L), and bacterial toxins (such as CT and LT), any derivative of an adjuvant, and any combination of adjuvants.In some embodiments, the adjuvant is any derivative of an adjuvant (e.g., cholesterol-modified CpG) or any combination thereof. In some embodiments, the adjuvant is a dendritic cell targeting molecule.

[0061] Such compositions comprising agents (e.g., DAMPs / PAMPs) capable of stimulating an innate immune response in a subject upon administration to the subject and associated with nanoparticles are not limited to a specific type of nanoparticle.

[0062] In some embodiments, the nanoparticles are sHDL nanoparticles. In some embodiments, the nanoparticles are sHDL nanoparticles, fullerenes, endohedral metallofullerene buckyballs, trimetal nitride templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitrate nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, any nanostructure, microstructure, or layer-by-layer process, self-assembly process. or derivatives thereof formed using polyelectrolytes, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold nanoparticles, silver nanoparticles, carbon nanoparticles, iron nanoparticles, modified micelles, metal-polyhistidine-DOPE@liposomes, metal-polyhistidine-PEG, 4-arm-PEG-polyhistidine-metal hydrogels, sHDL-polyhistidine, and metal-organic framework (MOF) coordination polymers (CPs).

[0063] In some embodiments, the average size of the nanoparticles is about 6 nm to about 500 nm, e.g., about 20 nm to about 500 nm, e.g., about 20, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, or about 500 nm, about 30 nm to about 500 nm, about 40 nm to about 500 nm, about 50 nm to about 500 nm, or about 75 nm to about 250 nm, e.g., about 75 nm, about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm, about 225 nm, or about 250 nm. In some embodiments, the nanoparticles are sHDL nanoparticles. In some embodiments, the sHDL nanoparticles comprise a mixture of at least one phospholipid and at least one HDL apolipoprotein or apolipoprotein mimetic. In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein AI (apoA-I), apolipoprotein A-II (apoA-II), apolipoprotein A4 (apoA4), apolipoprotein Cs (apoCs), and apolipoprotein E (apoE).In some embodiments, the phospholipid is dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N- [4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof. In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic.

[0064] In some embodiments, the ApoA-I mimetic is selected from the group consisting of SEQ ID NOs: 1-336, WDRVKDLATVYVDVLKDSGRDYVSQF (SEQ ID NO: 341), LKLLDNWDSVTSTFSKLREOL (SEQ ID NO: 342), PVTOEFWDNLEKETEGLROEMS (SEQ ID NO: 343), KDLEEVKAKVQ (SEQ ID NO: 344), KDLEEVKAKVO (SEQ ID NO: 345), PYLDDFQKKWQEEMELYRQKVE (SEQ ID NO: 346), PLRAELQEGARQKLHELOEKLS (SEQ ID NO: 347), PLG EEMRDRARAHVDALRTHLA (SEQ ID NO: 348), PYSDELRQRLAARLEALKENGG (SEQ ID NO: 349), ARLAEYHAKATEHLSTLSEKAK (SEQ ID NO: 350), PALEDLROGLL (SEQ ID NO: 351), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), PVLESFVSFLSALEEYTKKLN (SEQ ID NO: 353), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 354), QTVTDYGKDLME (SEQ ID NO: 355), KVKSPELOAEAKSYFEKSKE (SEQ ID NO: 356), VLTLALVAVAGARAEVSADOVATV (SEQ ID NO: 357), NNAKEAVEHLOKSELTOOLNAL (SEQ ID NO: 358), LPVLVWLSIVLEGPAPAOGTPDVSS (SEQ ID NO: 359), LPVLVVVLSIVLEGPAPAQGTPDVSS (SEQ ID NO: 360), ALDKLKEFGNTLEDKARELIS (SEQ ID NO: 361), VVALLALLASARASEAEDASLL( SEQ ID NO: 362), HLRKLRKRLLRDADDLQKRLAVYOA (SEQ ID NO: 363), AQAWGERLRARMEEMGSRTRDR (SEQ ID NO: 364), LDEVKEQVAEVRAKLEEQAQ (SEQ ID NO: 365), DWLKAFYDKVAEKLKEAF (SEQ ID NO: 236), DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA (SEQ ID NO: 366), PVLDLFRELLNELLEALKQKL (SEQ ID NO: 367), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 368),It is represented by any of PVLDLFRELLNELLEALKQKLK (SEQ ID NO: 4), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 369), PVLDLFRELLNELLEALKKLLK (SEQ ID NO: 370), PVLDLFRELLNELLEALKKLLA (SEQ ID NO: 371), PLLDLFRELLNELLEALKKLLA (SEQ ID NO: 372), and EVRSKLEEWFAAFREFAEEFLARLKS (SEQ ID NO: 373).

[0065] In some embodiments, the average particle size of the sHDL nanoparticles is between 6 and 70 nm.

[0066] In some embodiments, nanoparticles associated with such compositions comprising agents (e.g., DAMPs / PAMPs) capable of stimulating an innate immune response in a subject upon administration to the subject are further associated (e.g., by complexing, binding, encapsulation, absorption, adsorption, mixing) with one or more neo-antigenic peptides, wherein each of the one or more neo-antigenic peptides is specific for a neo-antigenic mutation identified from a tumorigenic biological sample obtained from the subject. In some embodiments, the subject is a human.

[0067] In some embodiments, the one or more neo-antigenic peptides range from about 5 to about 50 amino acids in length. In some embodiments, the one or more neo-antigenic peptides range from about 15 to about 35 amino acids in length. In some embodiments, the one or more neo-antigenic peptides range from about 18 to about 30 amino acids in length. In some embodiments, the one or more neo-antigenic peptides range from about 6 to about 15 amino acids in length.

[0068] In some embodiments, nanoparticles associated with such compositions comprising agents (e.g., DAMPs / PAMPs) that can stimulate an innate immune response in a subject upon administration to the subject are further associated (e.g., by complexing, binding, encapsulation, absorption, adsorption, mixing) with one or more biopolymeric agents.

[0069] Such compositions are not limited to any particular biopolymeric agent.

[0070] In some embodiments, the biopolymer agent is a nucleic acid. Such embodiments encompass any type of nucleic acid molecule, including, but not limited to, RNA, siRNA, microRNA, interfering RNA, mRNA, replicon mRNA, RNA analogs, and DNA.

[0071] In some embodiments, the biopolymer agent is a peptide.

[0072] In some embodiments, the peptide is selected from the group consisting of adrenocorticotropic hormone (ACTH), growth hormone peptides, melanocyte-stimulating hormone (MSH), oxytocin, vasopressin, corticotropin-releasing factor (CRF), CRF-related peptides, gonadotropin-releasing-related peptides (GAP), growth hormone-releasing factor (GRF), luteinizing hormone-releasing hormone (LH-RH), orexin, prolactin-releasing peptide (PRP), somatostatin, thyrotropin-releasing hormone (THR), THR analogs, calcitonin (CT), CT precursor peptide, calcitonin (CT), CT-related ... Cytokinin gene-related peptide (CGRP), parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), amylin, glucagon, insulin, insulin-like peptide, neuropeptide Y (NPY), pancreatic polypeptide (PP), peptide YY (PYY), cholecystokinin (CCK), CCK-related peptide, gastrin-releasing peptide (GRP), gastrin, gastrin-related peptide, gastrin-inhibitory peptide, motilin, secretin, vasoactive intestinal peptide (VIP), VIP-related peptide, atrial natriuretic peptide (ANP), Brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), tachykinin, angiotensin, renin substrate, renin inhibitor, endothelin, endothelin-related peptide, opioid peptide, thymic peptide, adrenomedullin peptide, alostatin peptide, amyloid beta protein fragment, antimicrobial peptide, antioxidant peptide, apoptosis-related peptide, capsule cell peptide (BCP), bombesin, bone Gla protein peptide, cocaine- and amphetamine-related transcript (CART) peptide, cell adhesion peptide, chemotactic peptide Tides, complement inhibitors, cortistatin peptides, fibronectin fragments, fibrin-related peptides, FMRF, FMRFamide-related peptides (FaRP), galanin, galanin-related peptides, growth factors, growth factor-related peptides, G therapeutic peptide-binding protein fragments, guarilin, uroguarilin, inhibin peptides, interleukins (IL), interleukin receptor proteins, laminin fragments, leptin fragment peptides, leukokinins, pituitary adenylate cyclase-activating polypeptides (PAPCAP), pancreastatin, polypeptide repeat chains,Signal transduction factors, thrombin inhibitors, toxins, trypsin inhibitors, virus-related peptides, adjuvant peptide analogs, alpha mating factor, antiarrhythmic peptides, anorexigenic peptides, alpha-1 antitrypsin, bovine pineal antireproductive peptide, brucine, C3 peptide P16, cadherin peptides, chromogranin A fragments, contraceptive tetrapeptides, conantokin G, conantokin T, crustacean cardioactive peptides, C-telopeptides, cytochrome b588 peptides, decorsin, delicious peptides peptide), delta sleep-inducing peptide, diazepam binding inhibitor fragment, nitric oxide synthase inhibitor peptide, OVA peptide, platelet calpain inhibitor (P1), plasminogen activator inhibitor 1, rigin, schizophrenia-related peptide, sodium potassium A therapeutic peptidase inhibitor-1, speract, sperm-activating peptide, systemin, thrombin receptor agonist, tuftsin, lipid mobilizing hormone, uremic pentapeptide, antifreeze polypeptide, tumor necrosis factor (TNF), Leech [Des Asp10]Decorsin, L-Ornityltaurine Hydrochloride, P-Aminophenylacetyltuftsin, Ac-Glu-Glu-Val-Val-Ala-Cys-pNA, Ac-Ser-Asp-Lys-Pro, Ac-rfwink-NH2, Cys-Gly-Tyr-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-Gly, D-Ala-Leu, DDDDD, DDDDDD, NPNANPNA, VAITVLVK, VGVRVR, VIHS, VPDPR , Val-Thr-Cys-Gly, RSR, sea urchin sperm activating peptide, SHU-9119 antagonist, MC3-R antagonist, MC4-R antagonist, Glaspimod, HP-228, α2-plasmin inhibitor, APC tumor suppressor, early pregnancy factor, gamma interferon, glandular kallikrein N-1, placental ribonuclease inhibitor, sarcolecin-binding protein, surfactant protein D, Wilms tumor suppressor, GABAB 1b receptor peptide, prion-related peptide (iPRP13), choline-binding protein fragment, telomerase inhibitor,Cardiostatin peptides, endostatin-derived peptides, prion inhibitory peptides, N-methyl D-aspartate receptor antagonists, and C-peptide analogs.

[0073] In some embodiments, the peptide is selected from the group consisting of 177Lu-DOTA0-Tyr3-octreotate, Abarelix acetate, ADH-1, Afamelanotidec, Melanotan-1, CUV1647, Albiglutide, Aprotinin, Argipressin, Atosiban acetate, Bacitracin, Bentiromide, BH3 domain, Bivalirudin, Bivalirudin trifluoroacetate hydrate. hydrate), blissibimod, bortezomib, buserelin, buserelin acetate, calcitonin, carbetocin, carbetocin acetate, cecropin A and B, ceruletide, ceruletide diethylamine, cetrorelix, cetrorelix acetate, cyclosporine, cilengitide (Cilengitidec), EMD121974, corticorelin acetate injection, hCRF, corticorelin sheep triflutate, corticorelin trifluoroacetate, corticotropin, cosyntropin, ACTH 1-24, tetracosactide hexaacetate, dalbavancin, daptomycin, degarelix acetate, deptreotide trifluoroacetate (with sodium pertechnetate), desmopressin acetate, desmopressin DDAVP, dulaglutide, ecallantide, edotreotide (with yttrium-90), elcatonin acetate, enalapril maleate (or 2-butanedioate), enfuvirtide, epfihibatide, exenatide, ganirelix acetate, glatiramer acetate, glutathione, gonadorelin, Gonadorelin acetate, GnRH, LHRH, goserelin, goserelin acetate, gramicidin, histrelin acetate, human calcitonin, icatibant, icatibant acetate, IM862, oglufanide disodium, KLAKLAK, lanreotide acetate, lepirudin, leuprolide, leuprolide acetate, leuprorelin, liraglutide, lisinopril, lixisenatide, lypressin, magenin 2, MALP-2Sc, macrophage-activating lipopeptide-2synthetic), nafarelin acetate, nesiritide, NGR-hTNF, octreotide acetate, oritavancin, oxytocin, pasireotide, peginesatide, pentagastrin, pentetreotide (with indium-111), phenypressin, pleurocidin, pramlintide, protirelin, thyroliberin, TRH, TRF, salmon calcitonin, saralasin acetate, secretin (human), secretin (porcine), semaglutide, seractide acetate, ACTH, corticotropin, sermorelin acetate, GRF 1-29, sinapultide, KL4 in lucinactant, sincalide, somatorelin acetate, GHRH, GHRF, GRF, somatostatin acetate, Spaglumat magnesium (or sodium) salt salt), substance P, taltirelin hydrate, teduglutide, teicoplanin, telavancin, teriparatide, terlipressin acetate, tetracosactide, thymalfasin, thymosin al, thymopentin, trebananib, triptorelin, triptorelin pamoate, tyroseruleutide, ularitide, vancomycin, vapreotide acetate, vasoactive intestinal peptide acetate, Vx-001c, TERT572Y, ziconotide acetate, α5-α6Bax peptide, and β-defensin.

[0074] In some embodiments, the peptide is any peptide that helps achieve a desired purpose using the composition, for example, in some embodiments, the peptide is any peptide that facilitates the treatment of any type of disease and / or disorder.

[0075] In some embodiments, the peptide is an antigen.

[0076] In some embodiments, the antigen is selected from the group consisting of a peptide-based antigen, a protein-based antigen, a polysaccharide-based antigen, a saccharide-based antigen, a lipid-based antigen, a glycolipid-based antigen, a nucleic acid-based antigen, an inactive organism-based antigen, an attenuated organism-based antigen, a viral antigen, a bacterial antigen, a parasitic antigen, an antigen derived from an allergen, and a tumor antigen.

[0077] In some embodiments, the antigen is a tumor antigen described herein.

[0078] In some embodiments, the antigen is any kind of viral, bacterial, or autoantigen, such as FimH for urinary tract infections; soluble F protein from respiratory syncytial virus (RSV); NEF, GAG, and ENV proteins from HIV; Streptococcus pneumoniae proteins; HMGB1 proteins; hemagglutinin and neuroamidase proteins for influenza; viral antigens from HPV types 16 and 18; gL2, ICP4, gD2ΔTMR, gD2ΔTMR, or ICP4.2 from HSV-2; pneumolysoid, choline-binding protein A (CbpA), or pneumococcal surface protein A (PspA), antigens from S. pneumoniae such as SP1912, SP1912, SP1912L, SP0148 with or without a signal sequence, SP2108 with or without a signal sequence; Chlamydia such as CT209 polypeptide antigen, CT253 polypeptide antigen, CT425 polypeptide antigen, CT497 polypeptide antigen, and CT843 polypeptide antigen. trachomatis; amyloid-beta peptide.

[0079] In some embodiments, the antigen is bound to the exterior surface of the nanoparticle, hi some embodiments, the antigen is encapsulated within the nanoparticle.

[0080] In certain embodiments, the present invention provides compositions capable of inhibiting cGAS-STING activation and type I IFN responses, comprising one or more cell-permeable chelators or derivatives thereof, which make intracellular metal ions unavailable for cGAS-STING-type I IFN activation.

[0081] In certain embodiments, the present invention provides compositions capable of modulating innate immune activation, the compositions comprising one or more cell-permeable chelators (e.g., metal ion chelators) that make intracellular metal ions unavailable to innate immune pathways.

[0082] In some embodiments, such cell-permeable chelators (e.g., metal ion chelators) include, but are not limited to, polyphenolic chelators such as (-)-epigallocatechin gallate (EGCG), punicalagin, (-)-catechin gallate, (-)-catechin, tannic acid, tannin, punicalin, vescalagin, procyanidin C1, geraniin, theaflavin 3,3'-digallate, lipid-modified NTA, porphyrin, EDTA, NOTA, DOTA, TPEN, and crofelemer.

[0083] In some embodiments, such compositions capable of inhibiting cGAS-STING activation and type I IFN responses are used in the treatment of subjects suffering from or at risk of suffering from an autoimmune disorder.

[0084] Thus, the present invention provides methods for treating autoimmune disorders by administering to a subject (e.g., a human subject) a composition capable of modulating innate immune activation, comprising one or more cell-permeable chelators (e.g., metal ion chelators), to render intracellular metal ions unavailable to innate immune pathways. In such embodiments, such cell-permeable chelators (e.g., metal ion chelators) include, but are not limited to, polyphenolic chelators such as (-)-epigallocatechin gallate (EGCG), punicalagin, (-)-catechin gallate, (-)-catechin, tannic acid, tannin, punicalin, vescalagin, procyanidin C1, geraniin, theaflavin 3,3'-digallate, lipid-modified NTA, porphyrin, EDTA, NOTA, DOTA, TPEN, and crofelemer.

[0085] Examples of autoimmune disorders include, but are not limited to, systemic lupus erythematosus, Aicardi-Goutières syndrome, acute pancreatitis, age-related macular degeneration, alcoholic liver disease, liver fibrosis, metastasis, myocardial infarction, nonalcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, inflammatory bowel disease, and multiple sclerosis.

[0086] In some embodiments, additional therapeutic agents are administered with such compositions. Examples of such therapeutic agents include, but are not limited to, disease-modifying antirheumatic drugs (e.g., leflunomide, methotrexate, sulfasalazine, hydroxychloroquine), biologics (e.g., rituximab, infliximab, etanercept, adalimumab, golimumab), nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, celecoxib, ketoprofen, naproxen, piroxicam, diclofenac), analgesics (e.g., acetaminophen, tramadol), immunomodulators (e.g., anakinra, abatacept), glucocorticoids (e.g., prednisone, methylprednisone), TNF-α inhibitors (e.g., adalimumab, certolizumab pegol, etanercept, golimumab, infliximab), IL-1 inhibitors, and metalloprotease inhibitors. In some embodiments, therapeutic agents include but are not limited to infliximab, adalimumab, etanercept, parenteral gold, or oral gold.

[0087] In certain embodiments, the present invention provides methods for treating cancer in a subject, comprising administering to the subject a composition described herein (e.g., a composition comprising one or more DAMPs and / or PAMPs) and one or more of an adjuvant (as described herein), a chemotherapeutic agent, an anti-immunosuppressant, an immunostimulatory agent, and an antigen (as described herein). In some embodiments, the subject is a human subject.

[0088] In some embodiments, the immune stimulatory agent is selected from an anti-CTLA-4 antibody, anti-PD-1, anti-PD-L1, anti-TIM-3, anti-BTLA, anti-VISTA, anti-LAG3, anti-CD25, anti-CD27, anti-CD28, anti-CD137, anti-OX40, anti-GITR, anti-ICOS, anti-TIGIT, and an inhibitor of IDO.

[0089] In some embodiments, the chemotherapeutic agent is aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alfa, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, Selected from idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (TAXOL), pilocarpine, prochlorperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate.

[0090] In some embodiments, the cancer is one or more selected from bladder cancer, brain cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, abdominal cancer, head and neck cancer, testicular cancer, melanoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, B-cell lymphoma, and uterine cancer.

[0091] In certain embodiments, the present invention provides a method for treating a cancer cell comprising: a) one or more DAMPs or PAMPs; b) Zn 2+ , Mn 2+ , Ca 2+ , Fe 2+ , Fe 3+ , Cu 2+ , Ni 2+ , Co 2+ , Pb 2+ , Sn 2+ , Ru 2+ , Au 2+ , Mg 2+ ,VO 2+ , Al3+ , Co 3+ , Cr 3+ , Ga 3+ , Tl 3+ , Ln 3+ , MoO 3+ , Cu + , Au + , Tl + , Ag + , Hg 2+ , Pt 2+ , Pb 2+ , Hg 2+ , Cd 2+ , Pd 2+ , Pt 4+ , Na + , K. + and one or more cations selected from the group consisting of related phosphates or carbonates; c) cholesterol; and d) Lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DSPC) Dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane one or more lipid molecules (e.g., phospholipids) selected from 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0PA), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), lipid-polyhistidine (e.g., DOPE-H11), and mixtures thereof, wherein in some embodiments, the lipid molecule is pegylated; A composition comprising:

[0092] In some embodiments, the composition is capable of stimulating an innate immune response in a subject upon administration to the subject.

[0093] In some embodiments, the subject has or is at risk of having cancer.

[0094] In some embodiments, the compositions are used to elicit an immune response to a vaccine application.

[0095] In some embodiments, the composition is capable of stimulating an innate immune response in at least one cancer cell upon administration to a subject, wherein the subject is afflicted with cancer. In some embodiments, stimulating the innate immune response comprises stimulating an innate cytokine response mediated via a cytokine, wherein the innate cytokine response is mediated via type 1 interferon.

[0096] In some embodiments, the one or more DAMPs or PAMPs are selected from a STING agonist, a purine-containing or purine-derivative agent, a Toll-like receptor (TLR) agonist, a NOD-like receptor (NLR) agonist, a RIG-I-like receptor (RLR) agonist, a cytoplasmic DNA sensor (CDS) agonist, a C-type lectin receptor (CLR) agonist, and an inflammasome inducer.

[0097] In some embodiments, the one or more STING agonists or prodrugs thereof (e.g., conjugated to a hydrophobic moiety) are selected from the group consisting of cGAMP, cdiAMP, cdiGMP, cAIMP, 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP difluoro, cAIM(PS)2, difluoro(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluorinated, c-di-AMP fluorinated, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluorinated, 2'3'-c-di-GMP, c-di-IMP, [ka] 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluoro, cAIM(PS)2, difluoro(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluoride, c-di-AMP fluoride, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluoride, 2'3'-c-di-GMP, c-di-IMP, cGAMP, 2' 3'-cGAMP, 2'2'-cGAMP, 3'3'-cGAMP, cGAM(PS)2, 2'3'-cGAM(PS)2(Rp / Sp), 2'2'-cGAM(PS)2, 2'3'-cGAM(PS)2, cGAMP fluoride, 3'3'-cGAMP fluoride, 2'3'-cGAMP fluoride, 2'2'-cGAMP fluoride, c-di-AMP, 2'3'-cdAMP, 2'2'-cdAMP, 3'3'-cdAMP, c-di-AM(PS)2, 2'3'-c-di-AM(PS)2(Rp,Rp), 2'2'-c-di-AM(PS)2, 3'3'-c-di-AM(PS)2, c-di-AMP fluoride, 2'3'-cdAMP fluoride, 2'2'-cdAMP fluoride fluoride, 3'3'-cdAMP fluoride, cdGMP, 2'3'-cdGMP, 2'2'-cdGMP, 3'3'-cdGMP, c-di-GM(PS)2, 2'3'-c-di- GM(PS)2, 2'2'-c-di-GM(PS)2, 3'3'-c-di-GM(PS)2, cdGMP fluoride, 2'3'-cdGMP fluoride, 2'2'-cdGMP fluoride Fluorinated, 3'3'-cdGMP Fluorinated, cAIMP, 2'3'-cAIMP, 2'2'-cAIMP, 3'3'-cAIMP, cAIMP Difluoro (3'3'-cAIMP Fluorinated) fluoride, 2'3'-cAIMP fluoride, 2'2'-cAIMP fluoride, cAIM(PS)2 difluoro, 3'3'-cAIM(PS)2 difluoro(Rp / Sp), 2'3' -cAIM(PS)2 difluoro, 2'2'-cAIM(PS)2 difluoro, c-di-IMP, 2'3'-cdIMP, 2'2'-cdIMP, 3'3'-cdIMP, c-di- The compound is selected from the group consisting of IM(PS)2, 2'3'-c-di-IM(PS)2, 2'2'-c-di-IM(PS)2, 3'3'-c-di-IM(PS)2, c-di-IMP fluorinated, 2'3'-cdIMP fluorinated, 2'2'-cdIMP fluorinated, 3'3'-cdIMP fluorinated, and amidobenzimidazole (ABZI)-based compounds.

[0098] In some embodiments, the TLR agonist is selected from a TLR-3 agonist, a TLR-4 agonist, a TLR-5 agonist, a TLR-7 agonist (eg, imiquimod), a TLR-8 agonist (eg, resiquimod), a TLR-9 agonist.

[0099] In some embodiments, the NLR agonist is an NLRP3 agonist.

[0100] In some embodiments, the purine-containing or purine-derivative agent is 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluoro, cAIM(PS)2, difluoro(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluorinated, c-di-AMP fluorinated, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluorinated, 2'3'-c-di-GMP, c-di-IMP, cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, 3'3'-cGAMP, cGAM(PS)2, 2'3'-cGAM(PS)2(Rp / Sp), 2'2'-cGAM(PS)2, 2'3'-cGAM(PS)2, cGAMP fluoride, 3'3'-cGAMP fluoride, 2'3'-cGAMP fluoride, 2'2'-cGAMP fluoride, c-di-AMP, 2'3'-cdAMP, 2'2'-cdAMP, 3'3'-cdAMP, c-di-AM(PS)2, 2'3'-c-di-AM(PS)2(Rp,Rp), 2'2'-c-di-AM(PS)2, 3'3'-c-di-AM(PS)2, c-di-AMP fluoride, 2'3'-cdAMP fluoride, 2'2'-cdAMP fluoride fluoride, 3'3'-cdAMP fluoride, cdGMP, 2'3'-cdGMP, 2'2'-cdGMP, 3'3'-cdGMP, c-di-GM(PS)2, 2'3'-c-di- GM(PS)2, 2'2'-c-di-GM(PS)2, 3'3'-c-di-GM(PS)2, cdGMP fluoride, 2'3'-cdGMP fluoride, 2'2'-cdGMP fluoride Fluorinated, 3'3'-cdGMP fluorinated, cAIMP, 2'3'-cAIMP, 2'2'-cAIMP, 3'3'-cAIMP, cAIMP difluoro (3'3'-cAIMP fluorinated) Fluorinated compound, 2'3'-cAIMP fluorinated compound, 2'2'-cAIMP fluorinated compound, cAIM(PS)2 difluoro, 3'3'-cAIM(PS)2 difluoro(Rp / Sp), 2'3 '-cAIM(PS)2 difluoro, 2'2'-cAIM(PS)2 difluoro, c-di-IMP, 2'3'-cdIMP, 2'2'-cdIMP, 3'3'-cdIMP, c-d i-IM(PS)2, 2'3'-c-di-IM(PS)2, 2'2'-c-di-IM(PS)2, 3'3'-c-di-IM(PS)2, c-di-IMP fluoride, 2'3'-cdIMP fluoride, 2'2'-cdIMP fluoride, 3'3'-cdIMP fluoride, imiquimod, resiquimod, 6-(4-amino-imidazoquinolyl)-norleucine, [ka] The inhibitor is selected from RNA, siRNA, microRNA, interfering RNA, mRNA, replicon mRNA, RNA analog, DNA, and a purine-based PI3K inhibitor.

[0101] In some embodiments, the nanoparticle is further associated with an antigen, where associated is selected from complexed, bound, encapsulated, absorbed, adsorbed, and mixed.

[0102] In some embodiments, the antigen is α-actinin-4, Bcr-Abl fusion protein, Casp-8, β-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, Mum-2, and Mum-3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK , K-ras, N-ras, triose phosphate isomerase, Bage-1, Gage3, Gage4, Gage5, Gage6, Gage7, GnTV, Herv-K-mel, Lage-1, Mage-A1, Mage-A2, Mage-A3, Mage-A4, Mage-A6, Mage-A10, Mage-A12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TR P-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO(LAGS), SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c -met, nm-23H1, PSA, TAG-72-4, CA19-9, CA-72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA125, CA15-3 (CA27.29\BCAA), CA195, CA242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM),Human EGFR protein or human EGFR protein fragments such as human EGFR residues 306-325 (SCVRACGADSYEMEEDGVRK (SEQ ID NO: 374)) and residues 897-915 (VWSYGVTVWELMTFGSKPY (SEQ ID NO: 375)), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein \cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, WT1 (and WT1-derived peptide sequences: WT1 126-134 (RMFP NAPYL (SEQ ID NO: 376)), WT1 122-140 (SGQARMFPNAPYLPSCLES (SEQ ID NO: 377)), and WT1 122-144 (SGQARMFPNAPYLPSCLESQPTI (SEQ ID NO: 378)), MUC1 (and MUC1-derived peptides and glycopeptides such as RPAPGS (SEQ ID NO: 379), PPAHGVT (SEQ ID NO: 380), and PDTRP (SEQ ID NO: 381)), LMP2, EGFRvIII, idiotype, GD2, Ras mutants, p53 mutants, proteinase 3 (PR1), survivin, hTERT, sarcoma translocation breakpoints, EphA2, EphA4, LMW-PTP, PAP, ML-IAP, AFP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl-GM1, mesothelin, sLe (animal), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AK AP-4, XAGE1, B7H3, legumain, Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-α, PDGFR-β, MAD-CT-2, Fos-related antigen 1, ERBB2, folate receptor 1 (FOLR1 or FBP), IDH1, IDO, LY6K, fms-related tyrosine kinase 1 (FLT1, best known as VEGFR1), KDR, PADRE, TA-CIN (recombinant HPV16 L2E7E6), SOX2, neoantigens,and aldehyde dehydrogenase.

[0103] In some embodiments, the antigen is derived from an autoantigen.

[0104] In some embodiments, the antigen is bound to the outer surface of the nanoparticle.

[0105] In some embodiments, the composition is in association with an adjuvant, where in association is selected from complexed, bound, encapsulated, absorbed, adsorbed, and mixed.

[0106] In some embodiments, the adjuvant is CPG, Poly IC, Poly ICLC, 1018 ISS, aluminum salts (e.g., aluminum hydroxide, aluminum phosphate), Amplivax, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, cytokines (such as GM-CSF, IL-2, IFN-α, Flt-3L), IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, Monophosphoryl Lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel™, Vector Systems, PLGA Microparticles, Imiquimod, Resiquimod, Gardiquimod, 3M-052, SRL172, Virosomes and Other Virus-Like Particles, YF-17D, VEGF Trap, β-Glucan, Pam3Cys, Aquila's QS21 Stimulon, Vadimesan, AsA404 (DMXAA), 3M MEDI9197, glucopyranosyl lipid adjuvant (GLA), GLA-SE, CD1d ligands (C20:2, OCH, AH04-2, α-galatosylceramide, α-C-galatosylceramide, α-mannosylceramide, α-fructosylceramide, β-galatosylceramide, β-mannosylceramide, etc.), STING agonists (e.g., cyclic [G(3',5')pA(3',5')p], cyclic [G(2',5')pA(3',5')p], cyclic [G(2',5')pA(2',5')p], cyclic diadenylate monophosphate, cyclic diguanylate monophosphate-containing cyclic dinucleotides), CL401, CL413, CL429, flagellin, RC529, E6020, imidazoquinoline-based small molecules TLR-7 / 8a (including lipidated analogs thereof), virosomes, AS01, AS02, AS03, AS04, AS15, IC31, CAF01, ISCOM, cytokines (such as GM-CSF, IL-2, IFN-α, Flt-3L), bacterial toxins (such as CT, and LT), any derivative of an adjuvant, and any combination of adjuvants.

[0107] In some embodiments, the nanoparticles are associated with an adjuvant, where associated is selected from complexed, bound, encapsulated, absorbed, adsorbed, and mixed.

[0108] In some embodiments, the adjuvant is CPG, Poly IC, Poly ICLC, 1018 ISS, aluminum salts (e.g., aluminum hydroxide, aluminum phosphate), Amplivax, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, cytokines (such as GM-CSF, IL-2, IFN-α, Flt-3L), IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, Monophosphoryl Lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel™, Vector Systems, PLGA Microparticles, Imiquimod, Resiquimod, Gardiquimod, 3M-052, SRL172, Virosomes and Other Virus-Like Particles, YF-17D, VEGF Trap, β-Glucan, Pam3Cys, Aquila's QS21 Stimulon, Vadimesan, AsA404 (DMXAA), 3M MEDI9197, glucopyranosyl lipid adjuvant (GLA), GLA-SE, CD1d ligands (C20:2, OCH, AH04-2, α-galatosylceramide, α-C-galatosylceramide, α-mannosylceramide, α-fructosylceramide, β-galatosylceramide, β-mannosylceramide, etc.), STING agonists (e.g., cyclic [G(3',5')pA(3',5')p], cyclic [G(2',5')pA(3',5')p], cyclic [G(2',5')pA(2',5')p], cyclic diadenylate monophosphate, cyclic diguanylate monophosphate-containing cyclic dinucleotides), CL401, CL413, CL429, flagellin, RC529, E6020, imidazoquinoline-based small molecules TLR-7 / 8a (including lipidated analogs thereof), virosomes, AS01, AS02, AS03, AS04, AS15, IC31, CAF01, ISCOM, cytokines (such as GM-CSF, IL-2, IFN-α, Flt-3L), bacterial toxins (such as CT, and LT), any derivative of an adjuvant, and any combination of adjuvants.

[0109] In some embodiments, the average particle size of the nanoparticles is 6 nm to 500 nm, e.g., about 20 nm to about 500 nm, e.g., about 20, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, or about 500 nm, about 30 nm to about 500 nm, about 40 nm to about 500 nm, about 50 nm to about 500 nm, or about 75 nm to about 250 nm, e.g., about 75 nm, about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm, about 225 nm, or about 250 nm.

[0110] Additional embodiments will be apparent to those skilled in the relevant art(s) based on the teachings contained herein.

[0111] BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1B are schematic diagrams of the synthesis of CDN-Zn, CDN-Zn@liposomes, and CDN@CaP / PEI-PEG. (Figure 1A) Zn 2+ The coordination bridge between the CDN and the CaP allows the assembly of CaP-ZnNPs, which are then further modified with liposomes (Figure 1B). The charge interaction between the CDN and the PEI-PEG backbone allows the CDN to be packed into the CaP / PEI-PEG NPs during synthesis.

[0112] Figures 2A–2E: Characterization of CDN-Zn, CDN-Zn@liposomes, and CDN@CaP / PEI-PEG. TEM images (upper panel), size (middle panel), and zeta potential (lower panel) of cdAMP-Zn (Figure 2A), cdGMP-Zn (Figure 2B), cGAMP-Zn (Figure 2C), CDN-Zn@liposomes (Figure 2D), and CDN@CaP / PEI-PEG (Figure 2E).

[0113] Figures 3A-3D: Release graphs of different CDN formulations and in vitro STING activation. (Figure 3A) Effect of CDN loading on the formulation. The red line shows the CDN absorbance before loading, while the blue line shows the absorbance of unloaded free CDN in the supernatant after loading. (Figure 3B) Release kinetics of CDN from the nanoformulation. (Figure 3C) Representative THP1 activation evaluation with different concentrations of free CDN and CDN-Zn. The CDN used was cdAMP. (Figure 3D) Representative THP1 activation with free CDN and CDN@CaP / PEI-PEG. The CDN used was cdAMP(ps)2.

[0114] Figures 4A-4F: Therapeutic effects of CDN formulations in the CT26 tumor model. (Figures 4A-4C) 1.5 x 10 5 CT26 tumor cells were inoculated into the tumor-bearing mice. On days 10 and 15, tumor-bearing mice were treated intratumorally with the indicated formulations containing 25 μg / dose of adAMP(ps)2. (Figure 4A) Average tumor growth curves of tumor-bearing mice are shown. (Figure 4B) Survival of mice after different treatments is shown. (Figure 4C) Tumor growth curves of individual mice from different groups are shown. (Figures 4D-4E) Seven days after the second dose of CDN treatment, PBMCs were collected for (Figure 4D) tetramer staining and (Figure 4E) ELISPOT analysis with AH1 peptide. (Figure 4F) Seven days after the first dose of CDN treatment, PBMCs were collected for ELISPOT analysis with AH1 peptide.

[0115] Figures 5A-5C: Enhancement of cGAS-STING-Type-I IFN activation by metal ions in vitro. (Figures 5A-5C) Bone marrow-derived dendritic cells (BMDCs) (Figures 5A-5B) and the human monocytic cell line THP1 (Figure 5C) were incubated with various concentrations of metal ions with or without a STING agonist. STING activation was quantified by interferon-β (IFN-β) release in the cell culture medium.

[0116] Figures 6A-6D: Co 2+ and Mn 2+Enhanced STING activation and cancer therapeutic efficacy in vivo. (Figure 6A) Individual tumor growth curves after three intratumoral injections of the indicated formulations on days 9, 12, and 15 after tumor inoculation. (Figure 6B) Serum IFN-β concentrations 8 hours after the first administration of the indicated formulation. (Figures 6C-6D) Tumor growth (Figure 6C) and survival (Figure 6D) of individual tumor-bearing mice after treatment with the indicated formulations.

[0117] Figures 7A-7E: Co 2+ and Mn 2+ Enhanced STING activation by IFN-γ resulted in improved antigen-specific immune responses after in vivo administration. (Figure 7A) Percentage of AH1-specific CD8+ T cells in PBMCs on day 16. (Figure 7B) Number of IFN-γ-secreting cells per 5E4 PBMCs after stimulation with AH1 peptide on day 22. (Figures 7C-7E) Timeline (Figure 7C), tumor growth curve (Figure 7D), and percentage of AH1-specific CD8+ T cells among splenic CD8+ T cells (Figure 7E) in a tumor rechallenge study starting on day 81.

[0118] Figures 8A-8J: Changes in cytokine profiles in vitro by metal ions of representative PAMPs. (Figures 8A-8D) Bone marrow-derived dendritic cells (BMDCs) were incubated with various concentrations of metal ions with or without the TLR3 agonist PolyIC. (Figures 8E-8F) BMDCs were incubated with various concentrations of metal ions with or without the TLR4 agonist MPLA. (Figures 8G-8H) BMDCs were incubated with various concentrations of metal ions with or without the TLR7 / 8 agonist R848. (Figures 8I-8J) BMDCs were incubated with various concentrations of metal ions with or without the TLR9 agonist CpG. Cytokine levels in the cell culture medium were quantified by ELISA assay.

[0119] Figures 9A-9L: Metal ions modulated immune responses of representative NOD-like receptor (NLR) ligands in vitro. (Figures 9A-9F) Bone marrow-derived dendritic cells (BMDCs) were incubated with various concentrations of metal ions with or without the NOD1 agonist C12-iE-DAP. (Figures 9G-9L) BMDCs were incubated with various concentrations of metal ions with or without the NOD2 agonist C18-MDP. Cytokine levels in cell culture medium were quantified by ELISA assay. Control: relative PAMP in saline.

[0120] Figures 10A-10F: Metal ions modulated immune responses to representative RIG-I-like receptor (RLR) ligands in vitro. (Figures 10A-10F) Bone marrow-derived dendritic cells (BMDCs) were transfected with the RLR ligand poly(dA:dT) / LyoVec. TM Cells were incubated with different concentrations of metal ions with or without (Invivogen). Cytokine levels in the cell culture medium were quantified by ELISA assay. Control: relative PAMP in saline.

[0121] Figures 11A-11K: Metal ions modulated the immune response to typical inflammatory inducers in vitro. (Figures 11A-11F) Bone marrow-derived dendritic cells (BMDCs) were pretreated with 300 ng / ml phorbol 12-myristate 13-acetate (PMA) for 3 hours, washed twice, and then treated with 10-200 mg / ml alum crystals. NLRP3 inflammasome formation was characterized by IL-1b secretion. (Figures 11G-11K) BMDCs were incubated with noncanonical inflammasome inducers in E. coli outer membrane vesicles and different concentrations of various metal ions. Cytokine levels in the cell culture medium were quantified by ELISA assay. Control: relative PAMP in saline.

[0122] Figures 12A-12F: Immunologic effects of metal ions alone in vitro. (Figures 12A-12F) Bone marrow-derived dendritic cells (BMDCs) were treated with different concentrations of metal ions. Cytokine levels in the cell culture medium were quantified by ELISA assay. Control: Relative PAMP of saline.

[0123] Figures 13A-13J: Representative formulation 1 composed of innate immune stimulators and metal ions. (Figure 13A) Overview of the composition of metal ion-polyHis-DOPE@liposome nanoparticles. (Figure 13B) TEM image of manganese-CDA-H11-DOPE@liposome nanoparticles (Mn-CDA / H11@liposomes). (Figures 13C-13E) Tumor growth curves of the CT26 colon tumor model treated with the indicated formulations, and the number of tumor-free treated mice out of five mice: (Figure 13C) Free CDA / Mn 2+ 5 μg of free CDA or 5 μg of Mn-CDA / H11@liposomes containing CDA were administered three times (Fig. 13D). 2+ 1 μg of free CDA or 1 μg of Mn-CDA / H11@liposomes containing CDA was administered three times by intratumoral (IT) injection on days 9, 12, and 15 after tumor inoculation; (Fig. 13E) Free CDA / Mn 2+ Mn-CDA / H11@liposomes containing 20 μg of CDA or 20 μg of CDA were administered three times by intravenous (IV) injection on days 9, 12, and 15 after tumor inoculation. (Figure 13F) Percentage of AH-1 antigen-specific T cells in PBMCs 7 days after the first administration. (Figure 13G) ELISPOT counts per 100,000 PBMCs 14 days after the first administration. (Figures 13H-J) Serum IFN-β, IP10, and TNF-α levels 4 hours after injection of the indicated formulations.

[0124] Figures 14A-14H: Representative formulation 2 composed of innate immune stimulators and metal ions. (Figure 14A) Overview of metal ion-polyHis-PEG nanoparticle configuration. (Figure 14B) TEM image of Co-CDA / H33-PEG nanoparticles. (Figure 14C) In vitro STING activation of BMDCs treated with the indicated formulations. (Figure 14D) Serum IFN-β after a single intratumoral injection of the indicated formulations in a B16F10 melanoma model. (Figures 14E-14F) Tumor growth (Figure 14E) and individual tumor growth (Figure 14F) in mice treated with the indicated formulations. Five micrograms of free CDA / Mn2+ or 5 micrograms of CDA containing Mn-CDA-H33-PEG were administered three times via IT injection into CT16 tumors on days 9, 12, and 15 after tumor inoculation. (FIGS. 14G to 14H) The ratio of AH-1 antigen-specific T cells in PBMCs 7 days after the first administration (FIG. 14G) and ELISPOT measurements per 100,000 PBMCs 14 days after the first administration.

[0125] Figures 15A-15F: Representative formulation 3 composed of innate immune stimulators and metal ions. (Figure 15A) Schematic composition of metal ion-4arm-PEG-polyHis coordination hydrogel. CDA@Co 2+ Figure 15B shows the retention of injectable Trypan Blue@4aH11-Co hydrogel at the injection site 6 hours after injection. Figures 15C-15E show the growth of individual tumors in mice treated with the indicated formulations. 20 μg of free CDA / Mn 2+ Or 20 μg of CDA-containing hydrogel was injected intratumorally (IT) three times on days 9, 12, and 15 after tumor inoculation. (Figure 15F) Representative tumor images after treatment with CDA@4aH11-Co hydrogel.

[0126] Figures 16A-16E: Several other representative formulations can be used to deliver metal ions and PAMPs. (Figure 16A) Self-assembly of metal ions and CDNs. (Figure 16B) Liposome-coated CDN-metal ion coordination nanoparticles. (Figure 16C) Polyhistidine-coated nanoparticles. (Figures 16D-16E) Polymer-stabilized metal-CDN coordination nanoparticles or metal mineral nanoparticles. Copolymers of poly(histidine) polyethylene glycol: PH-PEG or pHis-PEG, poly(ethyleneimine)-polyethylene glycol: PEI-PEG, poly(lysine) polyethylene glycol PEG: PK-PEG, anionic poly(glutamic acid) polyethylene glycol: PGA-PEG.

[0127] Figures 17A-17G: Therapeutic effects of selected formulations from Figure 12 in a CT26 colon tumor model. (Figure 17A) Representative THP1 activation assessment by different concentrations of free CDN and CDN-Zn. The CDN used here is cdAMP. (Figure 17B) Representative THP1 activation by free CDN and CDN@CaP / PEI-PEG. The CDN used here is cdAMP(ps)2. (Figures 17B-17E) 6-7 week old Balb / c mice were injected with 1.5 x 10 5 CT26 tumor cells were inoculated into the tumor-bearing mice. On days 10 and 15, tumor-bearing mice were treated with the indicated formulations containing 25 μg / dose of adAMP(ps)2 intratumorally. (Figure 17C) Average tumor growth curves for tumor-bearing mice, (Figure 17D) survival of mice after different treatments, and (Figure 17E) tumor growth curves for individual mice in different groups are shown. (Figures 17F-17G) Tetramer staining (Figure 17F) 7 days after the first dose of treatment, and ELISPOT analysis (Figure 17G) 7 days after the second dose of treatment.

[0128] Figures 18A-18F: Chelated metal ions for inhibiting the cGAS-STING-type I IFN pathway. (Figure 18A) Molecular structures of representative chelators that can inhibit the cGAS-STING-type I IFN pathway. (Figures 18B-18C) Dose-inhibition curves of IFN-I responses. (Figure 18B) NF-kB inflammatory responses. (Figure 18C) DNA / Lipofectamine 2000 (ThermoFisher, 11668027)-treated THP1 dual KI-hSTING WT(R232) (Figure 18D) Cell viability of Figures 18B-18C with the indicated compounds in reporter cells (Invivogen, thpd-r232). (Figure 18E) DNA / Lipofectamine 2000 (ThermoFisher, 11668027)-treated THP1-ISG hSTING HAQ Dose-inhibition curves of IFN-I responses by the indicated compounds in reporter cells (Invivogen, thp-isg). (Figure 18F) cGAMP-treated THP1 dual KI-hSTING WT(R232) Dose inhibition curves of IFN-I responses by the indicated compounds in reporter cells (Invivogen, thpd-r232).

[0129] Figure 19: Chelated metal ions to inhibit the TLR3-type I IFN pathway. Dose inhibition curves of IFN-I responses by the indicated compounds in Poly IC / Lipofectamine 2000 (ThermoFisher)-treated THP1 double STING KO reporter cells (Invivogen).

[0130] Figure 20: Molecular structures of other representative potent polyphenol chelating agents.

[0131] Figures 21A-21G: Amplification of STING activation by metal-containing lipid nanoparticles, CDN-manganese particles (CMPs), for cancer metalloimmunotherapy. (Figure 21A) CMPs are a cyclic dinucleotide (CDN)-manganese ion (Mn 2+ ), phospholipid-histidine 11 (DOPE-H11), and a PEG-lipid layer (DOPC:cholesterol:DSPE-PEG5000). 2+CDN and DOPE-H11 self-assembled into CDN-Mn@DOPE, which was subsequently PEGylated with a PEG-lipid layer to form the CMP (Figure 21B). CDA TEM image showing the CMP. Scale bar = 100 nm. CDA (Figure 21C) Dynamic light scattering and (Figure 21D) Zeta potential analysis of (Figure 21E) CMP CDA increased the cellular uptake of STING agonists. BMDCs were treated with free CDG-Dy547 or CDG-Dy547@CMP. CDA After incubation with CMP for 6, 12, or 24 hours, the cells were analyzed by flow cytometry (Figures 21F-21G). CDA BMDCs were treated with free forms of CDA and / or Mn, which increased STING activation and cytokine production. 2+ , blank nanoparticles without CDA (Mn-H11NP), or CMP CDA After 24 hours of treatment with IFN-β (Figure 21G) and TNF-α (Figure 21H), secretion was quantified by ELISA. Data represent the mean ± SEM from a representative experiment of two independent experiments with n = 3 (Figure 21E, Figures 21F-21G). Data were analyzed by one-way analysis of variance (Figures 21F-21G) or two-way analysis of variance with Bonferroni's multiple comparison test (Figure 21E).

[0132] Figures 22A-22I: CMP CDA Systemic IV administration of CMP on CT26 tumors after IV administration resulted in the disappearance of established tumors (Figures 22A-22I). CDA (Fig. 22A) CT26 tumor-bearing BALB / c mice were treated with CDA+Mn on days 9, 12, and 15. 2+ or CMP CDA(Figure 22B) Serum cytokines were measured by ELISA 6 hours after the second dose. (Figure 22C) Antigen-specific T cell responses were analyzed on day 21 by restimulating PBMCs with AH1 peptide followed by IFN-γ ELISPOT assay. (Figures 22D-22F) Tumor growth (Figures 22D-22E) and animal survival (Figure 22F) were monitored over time. (Figure 22G) Survivors re-challenged with CT26 tumor cells on day 145 were monitored for tumor growth and survival. (Figures 22H-22I) CMP against B16F10 tumors after IV administration CDA B16F10 tumor-bearing C57BL / 6 mice were treated with 20 μg CDA and 10 μg Mn on days 6, 9, and 13. 2+ Contains CDA+Mn 2+ or CMP CDA Mice were treated IV with 100 mg / kg / day (Figure 22H), and tumor growth was monitored over time (Figure 22I). Data represent the mean ± SEM from a representative experiment with n = 5 (Figures 22B-22C, Figure 22G) and n = 5-7 (Figures 22H, Figure 22I) from two independent experiments. Data were analyzed by one-way analysis of variance (Figures 22B, Figure 22C), or by two-way analysis of variance with Bonferroni's multiple comparison test (Figures 22F, Figure 22G, Figure 22I), or by log-rank (Mantel-Cox) test (Figure 22G).

[0133] Figures 23A-23P: CMP in multiple tumor models CDA (Figures 23A-23J) In the resulting B16F10 tumor model, CMP CDA The therapeutic effects of CMP were compared with other CDA formulations and other STING agonists (Figures 23A-23E). Tumor-bearing C57BL / 6 mice were treated with CMP at the indicated time points. CDA , CDA-Zn particles (CZP CDA), CDA liposomes, ADU-S100, or diABZI (all administered 5 μg of STING agonist intravenously) (Figure 23A). (Figure 23B) Individual tumor growth, (Figure 23C) representative photographs of tumors, (Figure 23D) mean tumor growth, and (Figure 23E) survival are shown. (Figures 23F-23J) Tumor-bearing C57BL / 6 mice were treated with the indicated regimens (all administered 20 μg intravenously) (Figure 23F). (Figure 23G) Individual tumor growth, (Figure 23H) representative photographs of tumors, (Figure 23I) mean tumor growth, and (Figure 23J) survival are shown. (Figures 23K-23P) CMP in an immune checkpoint inhibitor (ICB)-resistant tobacco-associated tumor model (NOOC1). CDA Therapeutic effects of 4NQO. (Fig. 23K) NOOC1 single-cell clones were isolated from visible oral squamous cell carcinoma lesions in C57BL / 6J mice treated with 4NQO-containing drinking water for 16 weeks. (Fig. 23L) Mutational signatures show NOOC1 tumors with high fidelity to human cancer. (Fig. 23M) Mutational profile of NOOC1 compared with other 4NQO-induced mouse squamous cell carcinoma cell lines (4MOSC). (Figs. 23N-23P) NOOC1 tumor-bearing C57BL / 6 mice were treated with CMP via the IT route (5 μg administration) or the IV route (20 μg administration) on days 9, 12, 16, and 20 after tumor inoculation. CDA The mice were treated with either CDA or the free form of CDA. (Figure 23N) Individual tumor growth, (Figure 23O) representative photographs of tumors, and (Figure 23P) mean tumor growth and survival are shown. Data represent the mean ± SEM from a representative experiment with n = 4–10 (Figures 23D–23E, Figures 23I–23J) and n = 7–8 (Figures 23N–23P) from two independent experiments. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparisons post-hoc test (Figures 23D, 23I, 23P). Survival in (Figures 23E, 23J, 23P) was analyzed by Kaplan-Meier survival analysis using the log-rank (Mantel-Cox) test. In (Figure 23P), *p and #p indicate statistical significance relative to the untreated and CDA groups, respectively.

[0134] Figures 24A-24E: Scalable synthesis of metal ion-containing lipid nanoparticles using the solvent dilution method. (Figure 24A) CDN-Mn@DOPE in a lipid mixture in ethanol is rapidly mixed with an aqueous buffer solution at a fixed ratio. CMPs are obtained by dialysis against a 10% sucrose solution. (Figure 24B) TEM image showing homogeneous CMPs formed using the solvent dilution method. Scale bar = 100 nm. (Figure 24C) Dynamic light scattering and (Figure 24E) zeta potential analysis of CMPs and CMPs with different compartmentalized lipids. (Figure 24D) CMPs increased STING activation and cytokine production. BMDCs were treated with CDN in free form or CMPs for 24 hours, followed by ELISA assays.

[0135] Figures 25A-25H: Robust therapeutic effect of CMP synthesized by solvent dilution in the MMTV-PyMT spontaneous tumor model. (Figures 25A-25H) MMTV-PyMT mice were treated with CMP on the indicated days. CDA Mice were treated with either CMP or free CDA via the intravenous route (20 μg). (Figures 25B-25E) CMP inhibited MMTV-PyMT tumor growth. (Figure 25B) Representative photographs of tumors at 94 days of age, (Figure 25C) total tumor volume for each mouse, (Figure 25D) average tumor volume for each group, and (Figure 25E) tumor number for each mouse at 14 weeks are shown. (Figures 25F-25G) CMP reduced lung metastasis of spontaneous tumors. (Figure 25H) CMP significantly prolonged the survival time of MMTV-PyMT mice.

[0136] Figures 26A-26B: CMPs modified with sorting lipids alter the biodistribution of CMPs. (Figure 26A) Addition of the ionic lipids 14-PA or 18-PA, or DOPE, altered the distribution of CMPs. Such lipids are called sorting lipids. (Figure 26B) Addition of different amounts of 14-PA altered the absolute drug concentrations and the relative amounts of drug in different organs.

[0137] Figures 27A-27C: CMP-14:0PA alleviates the side effects of original CMP. (Figure 27A) Representative images of the anal region of Balb / c mice 24 hours after IV treatment with 10 μg of CMP, CMP-14:0PA, or CMP-18:0PA. (Figure 27B) Incidence of diarrhea among Balb / c mice after treatment with 10 μg of CMP (n=10) and CMP-14:0PA (n=15). (Figure 27C) Body weight changes of mice on days 1, 2, and 3 after the first dose of 10 μg of CMP treatment.

[0138] Figure 28: CMP-14:0PA abolished hepatotoxicity in mice. B16F10 tumor-bearing C57BL / 6 mice were administered 10 μg of CMP or CMP-14:0PA IV on days 0, 4, and 7. Serum was collected on day 6, and aspartate transaminase (AST) and alanine transaminase (ALT) levels were measured as indicators of liver function.

[0139] Figures 29A-29C: CMP-14:0PA abrogated acute toxicity and exerted robust efficacy in B16F10 tumor-bearing mice. (Figure 29A) 10 μg of CMP or CMP-14:0PA was administered IV to B16F10 tumor-bearing mice on days 0, 5, and 10. (Figure 29B) Mean tumor growth curves and (Figure 29C) survival rates are shown.

[0140] Figures 30A-30C: CMP-14:0PA demonstrates robust efficacy in a mouse model of orthotopic pancreatic cancer. (Figure 30A) 20K Pan65671 was injected into the pancreas on day 0. 5 μg of CMP, CMP-14:0PA, or CDA was administered IV to tumor-bearing FVB / NJ mice on days 3, 7, and 10. (Figure 30B) Representative pancreatic images and (Figure 30C) pancreatic weights on day 14 are shown.

[0141] Figures 31A-31C: CMP-14:0PA is safe in rabbits. (Figure 31A) VX2 tumor tissue was cut into fragments and implanted intramuscularly into both hind limbs at week -3.5. Rabbits were treated IV with 0.5 mg of CMP-14:0PA or 1.5 mg of diABZi at weeks 0 and 1. (Figure 31B) Serum was collected at week 2, and AST and ALT levels were measured as indicators of liver function. (Figure 31C) Body weight changes in VX2 tumor-bearing rabbits were monitored over two cycles of treatment.

[0142] Figures 32A-32E: CMP-14:0PA demonstrates robust efficacy in a rabbit VX2 squamous cell carcinoma model. (Figure 32A) VX2 tumor tissue was cut into fragments and implanted intramuscularly into both hind limbs at week -3.5. Rabbits were treated IV with 0.5 mg of CMP-14:0PA or 1.5 mg of diABZi at weeks 0 and 1. (Figure 32B) Total weight of primary tumors detached from hind limb muscles is shown. (Figure 32C) Total volume of primary tumors detached from hind limb muscles. (Figure 32D) Percent tumor area measured based on H&E-stained slides of lung sections. (Figure 32E) Representative images of lungs.

[0143] Figures 33A-33G: Mn 2+ Improves the anticancer effects of LMW-PolyIC, HMW-PolyIC, MPLA, R848, CpG1826, and cyclic di-AMP (CDA) in the CT26 tumor model (Figure 33A). CT26 tumor-bearing BALB / c mice were treated with either TLR agonists (10 μg) or Mn on days 10, 13, 16, and 19. 2+ (2 μg) of TLR agonists were administered IT. (Figures 33B-33G) 2+ Average tumor growth in CT26 tumor-bearing mice treated with each of the TLR agonists in combination with .

[0144] Figures 34A-34D: TLR agonist / Mn 2+Systemic delivery of lipid nanoparticles delayed tumor growth in the CT26 mouse tumor model without apparent toxicity (Figure 34A). CT26 tumor-bearing BALB / c mice were treated with Mn 2+ (10 μg) together with TLR agonist (50 μg), or TLR agonist / Mn 2+ Treatment with lipid nanoparticles (50 μg) by IV administration. (Figure 34B) Mean tumor growth of CT26 tumor-bearing mice. (Figure 34C) Individual tumor growth curves of CT26 tumor-bearing mice. (Figure 34D) Body weight changes of CT26 tumor-bearing mice treated with different formulations.

[0145] [Definition] To facilitate understanding of the present invention, several terms and expressions are defined below.

[0146] As used herein, the term "lipid" or "lipid molecule" refers to a water-insoluble fatty substance, including fats, oils, waxes, and related compounds. They can be produced in the blood (endogenous) or ingested in the diet (exogenous). Lipids are essential for normal bodily function, and whether produced from exogenous or endogenous sources, they must be transported to and then released for use by cells. The production, transport, and release of lipids for use by cells is called lipid metabolism. There are several classes of lipids, but the two major classes are cholesterol and triglycerides. Cholesterol can be ingested through the diet and manufactured by cells in most organs and tissues in the body, primarily the liver. Cholesterol can be found in a free form or, more frequently, bound to a fatty acid, called cholesterol esters. As used herein, "lipid" or "lipid molecule" refers to any fat-soluble compound. Non-limiting examples of lipid compounds include fatty acids, cholesterol, phospholipids, complex lipids, and their derivatives or analogs, which are generally divided into at least three classes: (1) "simple lipids," which include fats and oils, and waxes; (2) "lipid compounds," which include phospholipids and glycolipids; and (3) "lipid derivatives," such as steroids. Lipids or lipid molecules suitable for use in the present invention include both membrane-forming and non-membrane-forming lipids.

[0147] As used herein, the term "lipoprotein" refers to a spherical compound structured so that water-insoluble lipids are partially contained within a water-soluble outer shell. Depending on the type of lipoprotein, the contents contain varying amounts of free and esterified cholesterol, triglycerides, and apoproteins, or apolipoproteins. There are five major types of lipoproteins, which differ in function and lipid and apoprotein content and are classified according to increasing density: (i) chylomicrons and chylomicron remnants, (ii) very low-density lipoproteins ("VLDL"), (iii) intermediate-density lipoproteins ("IDL"), (iv) low-density lipoproteins ("LDL"), and (v) high-density lipoproteins ("HDL"). Cholesterol circulates in the bloodstream as particles associated with lipoproteins.

[0148] As used herein, the term "HDL" or "high-density lipoprotein" refers to high-density lipoprotein. HDL contains approximately equal amounts of lipid and protein complexes and functions as a transporter of cholesterol in the blood. HDL is primarily synthesized in and secreted from epithelial cells in the liver and small intestine. Immediately after secretion, HDL is in the form of discoidal particles containing apolipoprotein AI (also known as apoA-I) and phospholipids as its major components, also known as nascent HDL. This nascent HDL receives free cholesterol from the cell membranes of peripheral cells in the blood or is produced during the hydrolysis of other lipoproteins. It forms mature spherical HDL by retaining cholesterol esters converted from the cholesterol in its hydrophobic center through the action of LCAT (lecithin cholesterol acyltransferase). HDL plays a crucial role in the lipid metabolic process known as "reverse cholesterol transport," transporting cholesterol from peripheral tissues into the blood and transporting it to the liver. Reverse cholesterol transport is thought to be one of the main mechanisms of HDL's protective effect against atherosclerosis, and therefore high levels of HDL are associated with a reduced risk of atherosclerosis and coronary heart disease (CHD).

[0149] As used herein, the terms "synthetic HDL," "sHDL," "reconstituted HDL," or "rHDL" refer to particles structurally similar to native HDL, composed of one or more lipids, preferably ApoA-I or a mimetic thereof, in association with at least one protein of HDL. Typically, the components of sHDL are derived from blood or may be produced by recombinant technology.

[0150] As used herein, the term "complexation" as used herein relates to the non-covalent interaction of a biopolymer drug (e.g., an antigen, an adjuvant, etc.) with a nanoparticle and / or microparticle.

[0151] As used herein, the term "conjugated" as used herein refers to a covalent association between a biopolymer agent (e.g., an antigen, an adjuvant, etc.) and a nanoparticle and / or microparticle.

[0152] As used herein, the term "encapsulation" refers to the placement of a biopolymer agent (eg, antigen, adjuvant, etc.) wrapped around or completely contained within the nanoparticles and / or microparticles.

[0153] As used herein, the term "absorbed" refers to the incorporation and stable retention of a biopolymer agent (eg, antigen, adjuvant, etc.) within the interior, i.e., outer surface, of the nanoparticles and / or microparticles.

[0154] As used herein, the term "adsorption" refers to the attachment of a biopolymeric agent (e.g., an antigen, an adjuvant, etc.) to the outer surface of a nanoparticle and / or microparticle. Such adsorption preferably occurs through electrostatic attraction. Electrostatic attraction is the attraction or bond generated between two or more oppositely charged or ionic chemical groups. In general, adsorption is typically reversible.

[0155] As used herein, the term "mixing" refers to a biopolymeric agent (e.g., an antigen, an adjuvant, etc.) dissolved, dispersed, or suspended in nanoparticles and / or microparticles. In some cases, the biopolymeric agent may be homogeneously mixed in the nanoparticles and / or microparticles.

[0156] As used herein, the term "biological biopolymer" or "biopolymer" or "biopolymer agent" refers to a molecule having a molecular weight of greater than 1 kDa that can be isolated from an organism or cell culture, e.g., a eukaryotic (e.g., mammalian) cell culture or a prokaryotic (e.g., bacterial) cell culture. In some embodiments, the term refers to polymers, e.g., nucleic acids (including, but not limited to, RNA, siRNA, microRNA, interfering RNA, mRNA, replicon mRNA, RNA analogs, DNA, etc.), polypeptides (such as proteins), carbohydrates, and lipids. In some embodiments, the term "biopolymer" refers to proteins. In some embodiments, the term "biopolymer" refers to recombinant proteins or fusion proteins. In some embodiments, the proteins are soluble. In some embodiments, the biopolymer is an antibody, e.g., a monoclonal antibody. In some embodiments, the biopolymer is an adjuvant, antigen, therapeutic agent, imaging agent, etc.

[0157] The term "antigen" as used herein is defined herein as a molecule containing one or more epitopes that stimulates the host immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response. Antigens can be peptides, proteins, polysaccharides, sugars, lipids, nucleic acids, and combinations thereof. Antigens can be derived from viruses, bacteria, parasites, plants, protozoa, fungi, tissues, or transformed cells such as cancer or leukemia cells, and can be whole cells or immunogenic components thereof, such as cell wall components. Antigens can be oligonucleotides or polynucleotides that express the antigen. Antigens can be natural or synthetic antigens, such as haptens, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens (see, e.g., Bergmann, et al., Eur. J.Immunol., 23:2777-2781 (1993); Bergmann, et al., J. Immunol., 157:3242-3249 (1996); Suhrbier, Immunol. and Cell Biol., 75:402-408 (1997)).

[0158] As used herein, the term "neoantigen" or "neoantigenic" refers to a class of tumor antigens that arise from tumor-specific mutation(s) that alter the amino acid sequence of a genome-encoded protein.

[0159] As used herein, a "tumor-specific antigen" is defined herein as an antigen that is unique to tumor cells and does not occur in or on other cells of the body.

[0160] As used herein, the term "tumor-associated antigen" is defined herein as an antigen that is not native to tumor cells and occurs within or on normal cells under conditions that are incapable of inducing an immune response to the antigen.

[0161] As used herein, the term "adjuvant" is defined herein as a substance that, when administered together with other antigens, increases the immune response to the other antigens. Adjuvants are also referred to herein as "immune enhancing agents" and "immunomodulating agents."

[0162] The term "antigen-presenting cell" as used herein is defined as a highly specialized cell that can process antigens and present peptide fragments on the cell surface together with molecules required for lymphocyte activation.The main antigen-presenting cells for T cells are dendritic cells, macrophages, and B cells.The main antigen-presenting cells for B cells are follicular dendritic cells.

[0163] The term "cross-presentation" used herein is defined as the ability of antigen-presenting cells to take in extracellular antigens, process them, and present them to CD8 T cells (cytotoxic T cells) with MHC class I molecules.This processing induces cellular immunity against most tumors and viruses that do not infect antigen-presenting cells.Cross-presentation is also necessary for the induction of cytotoxic immunity in vaccination with protein antigens, for example, in tumor vaccination.

[0164] As used herein, the terms "immune," "immunological," or "immune" response is the development of a humoral and / or cellular response to an antigen.

[0165] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of the sHDL nanoparticles described herein (e.g., compositions comprising sHDL nanoparticles encapsulating siRNA) (e.g., compositions comprising sHDL nanoparticles configured to activate an immune response), such delivery systems include systems that allow for the storage, transportation, or delivery of such compositions and / or auxiliary components (e.g., written instructions for using the materials, etc.) from one location to another. For example, a kit may include one or more enclosures (e.g., boxes) containing the necessary agents and / or auxiliary components. As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers, each containing a portion of the overall kit components. The containers can be delivered to the intended recipient together or separately. For example, a first container may contain a composition comprising sHDL nanoparticles or components necessary to synthesize such sHDL nanoparticles, while a second container contains a second agent (e.g., siRNA, antigen, adjuvant) (e.g., antibiotic or spray applicator). Indeed, any delivery system containing two or more separate containers, each containing a portion of the overall kit components, is included in the term "fragmentation kit." In contrast, a "combination kit" refers to a delivery system containing all of the components necessary to synthesize and utilize any of the sHDL nanoparticles as described (e.g., in a single box containing each of the desired components). The term "kit" includes both fragmentation kits and combination kits.

[0166] As used herein, the term "subject" refers to any animal (e.g., mammal) that is to be the recipient of a particular treatment, including, but not limited to, humans, non-human primates, rodents, etc. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0167] The term "sample" as used herein is used in the broadest sense. In one sense, it is meant to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma and serum. Environmental samples include environmental materials such as surface matter, soil, water, quartz, and industrial samples. However, these examples should not be construed as limiting the types of samples applicable to the present invention.

[0168] As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell cultures. The term "in vivo" refers to a natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

[0169] As used herein, the term "drug" or "therapeutic agent" is meant to include any molecule, molecular complex, or substance that is administered to a living organism for diagnostic or therapeutic purposes, including medical imaging, monitoring, contraceptive, cosmetic, nutraceutical, pharmaceutical, and prophylactic uses. The term "drug" is further meant to include any such molecule, molecular complex, or substance that is chemically modified and / or operably linked to a biological or biocompatible structure.

[0170] The term "solvent" as used herein refers to the medium in which a reaction is carried out. A solvent may be a liquid, but is not limited to liquid form. Categories of solvents include, but are not limited to, non-polar, polar, protic, and aprotic.

[0171] Detailed Description of the Invention The CDNs cyclic di-AMP (produced by Listeria monocytogenes) and its analog, cyclic di-GMP (produced by Legionella pneumophila) are recognized by host cells as PAMPs (Pathogen Associated Molecular Pattern Modulators), which bind to a PRR (pathogen recognition receptor) known as STING. STING is an adaptor protein in the cytoplasm of host mammalian cells that activates the TANK-binding kinase (TBK1)-IRF3 signaling axis, resulting in the induction of IFN-β and other IRF-3-dependent gene products, which potently activate innate immunity. It is now recognized that STING is a component of the host cytosolic surveillance pathway, sensing infection by intracellular pathogens and inducing the production of IFN-β in response, leading to the development of an adaptive, protective pathogen-specific immune response consisting of both antigen-specific CD4 and CD8 T cells and pathogen-specific antibodies.

[0172] Immunotherapy is advancing cancer treatment in multiple areas. Recently, it has been discovered that activation of the innate immune system via cyclic GAM-AMP (cGAMP), which activates the stimulator of IFN genes (STING) pathway, can initiate a strong antitumor immune response. Besides cGAMP, various other cyclic dinucleotides (CDNs), such as cdiAMP, cdiGMP, and cAIMP, can activate the STING pathway, which is recognized as an essential immune defense mechanism against tumors and exogenous pathogens. However, due to their low molecular weight, poor pharmacokinetic properties, and severe toxicity to non-target cells, STING agonists require direct local injection into tumors. Experiments conducted during the development of embodiments for the present invention demonstrated that TLR agonists and CDNs each have the following properties: (1) a metal (e.g., Mn 2+ , Zn 2+We found that the formulations could assemble into uniform nanoparticles in the presence of either (1) calcium phosphate and PEI-PEG, or (2) calcium phosphate and PEI-PEG. Based on these results, two categories of drug delivery systems were developed for the delivery of TLR agonists and CDNs. In a subcutaneous CT26 tumor model, the formulations were shown to significantly inhibit tumor growth, achieving complete regression rates of 40% and 60%. Thus, these formulations represent a new class of drug delivery systems for both local and systemic delivery of STING agonists.

[0173] These results are of great clinical importance because these CDN-related nanoparticles can induce specific immune responses against tumors via systemic administration, thereby avoiding the need for direct local injection into the tumor.

[0174] Further experiments conducted in the course of developing embodiments for the present invention revealed that Mn 2+ and Co 2+ It was discovered that certain metal ions, such as Mn, can enhance STING activation and type I IFN responses induced by STING agonists. In a murine CT26 colon tumor model, Mn 2+ / Co 2+The combination of IFN-STING agonists was shown to increase serum type I IFN levels, resulting in greater tumor eradication and longer survival in tumor-bearing mice. After 80 days, 80% of the mice were cured and resistant to difficult secondary tumors. Furthermore, this phenomenon was found to be generalized to various other innate immune pathways, including, but not limited to, Toll-like receptor (TLR) 3 / 4 / 7 / 8 / 9 ligands, NOD1 / 2 ligands, TLR7 / 8 ligands, RIG-I & CDS agonists, and inflammasome inducers. Based on this discovery, several pharmaceutically acceptable formulations, such as metal salts of DAMPs / PAMPs, coordination complexes, and other metal-loaded formulations (e.g., hydroxide / carbonate / phosphate minerals, liposomes, self-assembled nanoparticles, PLGA, hydrogels, emulsions, etc.), have been developed to precisely deliver metal innate immune stimulator combinations to the desired targets and release them in an ideal manner. Finally, we found that several chelators could effectively inhibit DNA-induced cGAS-STING-type I IFN / NFkB responses and Poly IC-induced TLR3-cGAS-STING-type I IFN.

[0175] Thus, such results and embodiments represent a new class of drug delivery systems for both local and systemic delivery of agents that, upon administration to a subject, can stimulate an innate immune response in the subject.

[0176] Thus, the present disclosure provides compositions and methods for stimulating an innate immune response in a subject by administering an agent capable of stimulating an innate immune response in a subject upon administration to the subject. In particular, the present invention is directed to such compositions comprising an agent capable of stimulating an innate immune response in a subject upon administration to the subject, methods for synthesizing such compositions, and systems and methods that utilize such compositions (e.g., in diagnostic and / or therapeutic settings).

[0177] Thus, in certain embodiments, the present invention provides a method for detecting a mutated or modified nucleotide sequence comprising one or more DAMPs and / or PAMPs in combination with one or more of the following: a) calcium phosphate and copolymers of cationic poly(ethyleneimine) (PEI) with polyethylene glycol (PEG), poly(histidine)polyethylene glycol (PH-PEG), lipid poly-histidine, poly(lysine)polyethylene glycol PEG (PK-PEG), or anionic poly(glutamic acid)polyethylene glycol (PGA-PEG); and b) Zn 2+ , Mn 2+ , Ca 2 +, Fe 2+ , Fe 3+ , Cu 2+ , Ni 2+ , Co 2+ , Pb 2+ , Sn 2+ , Ru 2+ , Au 2+ , Mg 2+ ,VO 2+ , Al 3+ , Co 3+ , Cr 3+ , Ga 3+ , Tl 3+ , Ln 3+ , MoO 3+ , Cu + , Au + , Tl + , Ag + , Hg 2+ , Pt 2+ , Pb 2+ , Hg 2+ , Cd 2+ , Pd 2+ , Pt 4+ , Na + , K. + and one or more cations selected from the group consisting of related phosphates or carbonates; and one or both of:

[0178] In certain embodiments, a nanoparticle composition (e.g., nanoparticles comprising a particle size in the range of 20-500 nm) comprises one or more DAMPs or PAMPs and one or more of the following: Zn 2+ , Mn 2+ , Fe 2+ , Fe 3+, Cu 2+ , Ni 2+ , Co 2+ , Pb 2+ , Sn 2+ , Ru 2+ , Au 2+ , Mg 2+ ,VO 2+ , Al 3+ , Co 3+ , Cr 3+ , Ga 3+ , Tl 3+ , Ln 3+ , MoO 3+ , Cu + , Au + , Tl + , Ag + , Hg 2+ , Pt 2+ , Pb 2+ , Hg 2+ , Cd 2+ , Pd 2+ , Pt 4+ , Na + , K. + and one or more cations selected from the group consisting of: and related phosphates or carbonates; and Lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC) , dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), DSPE-PEG, monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearyl ... one or more lipid molecules (e.g., phospholipids) selected from aroyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0PA), or 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (18:1PA); and one or more of:

[0179] Such compositions are not limited to a particular DAMP or PAMP agonist.

[0180] In some embodiments, the DAMP and PAMP agonists are selected from STING agonists, purine-containing or purine-derived factors, Toll-like receptor (TLR) agonists, NOD-like receptor (NLR) agonists, RIG-I-like receptor (RLR) agonists, cytoplasmic DNA sensor (CDS) agonists, C-type lectin receptor (CLR) agonists, and inflammasome inducers.

[0181] In some embodiments, the DAMP and PAMP agonists are selected from a TLR-3 agonist, a TLR-4 agonist, a TLR-5 agonist, a TLR-7 agonist (e.g., imiquimod), a TLR-8 agonist (e.g., resiquimod), a TLR-9 agonist, and an NLRP3 agonist.

[0182] Such compositions are not limited to specific purine-containing or purine-derivative factors. In some embodiments, the purine-containing or purine-derivative factors include 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluoro, cAIM(PS)2, difluoro(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluoride, c-di-AMP fluoride, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluoride, 2'3'-c-di-GMP, c-di-IMP, cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, 3'3'-cGAMP, cGAM(PS)2, 2'3'-cGAM(PS)2(Rp / Sp), 2'2'-cGAM(PS)2, 2'3'-cGAM(PS)2, cGAMP fluoride, 3'3'-cGAMP fluoride, 2'3'-cGAMP fluoride, 2'2'-cGAMP fluoride, c-di-AMP, 2'3'-cdAMP, 2'2'-cdAMP, 3'3'-cdAMP, c-di-AM(PS)2, 2'3'-c-di-AM(PS)2(Rp,Rp), 2'2'-c-di-AM(PS)2, 3'3'-c-di-AM(PS)2, c-di-AMP fluoride, 2'3'-cdAMP fluoride, 2'2'-cdAMP fluoride fluoride, 3'3'-cdAMP fluoride, cdGMP, 2'3'-cdGMP, 2'2'-cdGMP, 3'3'-cdGMP, c-di-GM(PS)2, 2'3'-c-di- GM(PS)2, 2'2'-c-di-GM(PS)2, 3'3'-c-di-GM(PS)2, cdGMP fluoride, 2'3'-cdGMP fluoride, 2'2'-cdGMP fluoride Fluorinated, 3'3'-cdGMP fluorinated, cAIMP, 2'3'-cAIMP, 2'2'-cAIMP, 3'3'-cAIMP, cAIMP difluoro (3'3'-cAIMP fluorinated) Fluorinated compound, 2'3'-cAIMP fluorinated compound, 2'2'-cAIMP fluorinated compound, cAIM(PS)2 difluoro, 3'3'-cAIM(PS)2 difluoro(Rp / Sp), 2'3 '-cAIM(PS)2 difluoro, 2'2'-cAIM(PS)2 difluoro, c-di-IMP, 2'3'-cdIMP, 2'2'-cdIMP, 3'3'-cdIMP, c-d i-IM(PS)2, 2'3'-c-di-IM(PS)2, 2'2'-c-di-IM(PS)2, 3'3'-c-di-IM(PS)2, c-di-IMP fluoride, 2'3'-cdIMP fluoride, 2'2'-cdIMP fluoride, 3'3'-cdIMP fluoride, imiquimod, resiquimod, 6-(4-amino-imidazoquinolyl)-norleucine, [ka] and purine-based PI3K inhibitors.

[0183] Such compositions are not limited to specific STING agonist.In some embodiments, STING agonist is cyclic dinucleotide.For example, in some embodiments, cyclic dinucleotide is cdi-AMP, cGAMP or cGMP, or any of their derivatives. In some embodiments, small molecule agonists of STING include, but are not limited to, 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluoro, cAIM(PS)2, difluoro(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluorinated, c-di-AMP fluorinated, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluorinated, 2'3'-c-di-GMP, c-di-IMP, SB11285, STING-agonist-C11, STING agonist-1, STING agonist G10, gemcitabine, and additional STING agonists described herein.

[0184] Suitable STING agonists for use in the disclosed compositions and methods include, but are not limited to, cyclic dinucleotide molecules.For example, in some embodiments, the small molecule agonist of STING is a cyclic dinucleotide selected from cGAMP, cdiAMP, cdiGMP and cAIMP.More examples of cyclic purine dinucleotides are described in detail in, for example, U.S. Patent Nos. 7,709,458 and 7,592,326; WO2007 / 054279; and Yan et al., Bioorg.Med.Chem Lett.18:5631 (2008).Each of these is incorporated herein by reference.

[0185] Further suitable STING agonists for use in the disclosed methods include, but are not limited to, flavonoids. In some embodiments, the STING agonist may comprise a flavonoid. In other embodiments, the STING agonist may consist of a flavonoid. Suitable flavonoids include, but are not limited to, 10-(carboxymethyl)-9(10H)acridone (CMA), 5,6-dimethylxanthenone-4-acetic acid (DMXAA), methoxyflavone, 6,4'-dimethoxyflavone, 4'-methoxyflavone, 3',6'-dihydroxyflavone, 7,2'-dihydroxyflavone, daidzein, formononetin, lettuce 7-methyl ether, xanthone, or any combination thereof. In some embodiments, the STING agonist may be 10-(carboxymethyl)-9(10H)acridone (CMA). In some embodiments, the STING agonist may be 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In some embodiments, the STING agonist can be methoxyflavone. In some embodiments, the STING agonist can be 6,4'-dimethoxyflavone. In some embodiments, the STING agonist can be 4'-methoxyflavone. In some embodiments, the STING agonist can be 3',6'-dihydroxyflavone. In some embodiments, the STING agonist can be 7,2'-dihydroxyflavone. In some embodiments, the STING agonist can be daidzein. In some embodiments, the STING agonist can be formononetin. In some embodiments, the STING agonist can be letusin 7-methyl ether. In some embodiments, the STING agonist can be xanthone. In some embodiments, the STING agonist can be any combination of the above flavonoids. Thus, for example, in some embodiments, the flavonoid comprises DMXAA.

[0186] In some embodiments, small molecule agonists of STING include but are not limited to 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluoro, cAIM(PS)2, difluoro(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluorinated, c-di-AMP fluorinated, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluorinated, 2'3'-c-di-GMP, c-di-IMP, SB11285, STING-agonist-C11, STING agonist-1, STING agonist G10, and gemcitabine.

[0187] In certain embodiments, the present invention provides compositions capable of inhibiting cGAS-STING activation and type I IFN responses, comprising one or more cell-permeable chelators or derivatives thereof, which make intracellular metal ions unavailable for cGAS-STING-type I IFN activation.

[0188] In certain embodiments, the present invention provides compositions capable of modulating innate immune activation, the compositions comprising one or more cell-permeable chelators (e.g., metal ion chelators) that make intracellular metal ions unavailable to innate immune pathways.

[0189] In some embodiments, such cell-permeable chelators (e.g., metal ion chelators) include, but are not limited to, polyphenolic chelators such as (-)-epigallocatechin gallate (EGCG), punicalagin, (-)-catechin gallate, (-)-catechin, tannic acid, tannin, punicalin, vescalagin, procyanidin C1, geraniin, theaflavin 3,3'-digallate, lipid-modified NTA, porphyrin, EDTA, NOTA, DOTA, TPEN, and crofelemer.

[0190] In some embodiments, such compositions capable of inhibiting cGAS-STING activation and type I IFN responses are used in the treatment of subjects suffering from or at risk of suffering from an autoimmune disorder.

[0191] Thus, the present invention provides methods for treating autoimmune disorders by administering to a subject (e.g., a human subject) a composition capable of modulating innate immune activation, comprising one or more cell-permeable chelators (e.g., metal ion chelators), to render intracellular metal ions unavailable to innate immune pathways. In such embodiments, such cell-permeable chelators (e.g., metal ion chelators) include, but are not limited to, polyphenolic chelators such as (-)-epigallocatechin gallate (EGCG), punicalagin, (-)-catechin gallate, (-)-catechin, tannic acid, tannin, punicalin, vescalagin, procyanidin C1, geraniin, theaflavin 3,3'-digallate, lipid-modified NTA, porphyrin, EDTA, NOTA, DOTA, TPEN, and crofelemer.

[0192] Examples of autoimmune disorders include, but are not limited to, systemic lupus erythematosus, Aicardi-Goutières syndrome, acute pancreatitis, age-related macular degeneration, alcoholic liver disease, liver fibrosis, metastasis, myocardial infarction, nonalcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, inflammatory bowel disease, and multiple sclerosis.

[0193] In some embodiments, additional therapeutic agents are administered with such compositions, including, but not limited to, disease-modifying antirheumatic drugs (e.g., leflunomide, methotrexate, sulfasalazine, hydroxychloroquine), biologics (e.g., rituximab, infliximab, etanercept, adalimumab, golimumab), nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, celecoxib, ketoprofen, naproxen, piroxicam, diclofenac), analgesics (e.g., acetaminophen, tramadol), immunomodulators (e.g., anakinra, abatacept), glucocorticoids (e.g., prednisone, methylprednisone), TNF-α inhibitors (e.g., adalimumab, certolizumab pegol, etanercept, golimumab, infliximab), IL-1 inhibitors, and metalloprotease inhibitors. In some embodiments, the additional therapeutic agent includes but is not limited to infliximab, adalimumab, etanercept, parenteral gold, or oral gold.

[0194] In certain embodiments, a composition comprising an agent (e.g., a DAMP / PAMP) that can stimulate an innate immune response in a subject upon administration to the subject is associated with (e.g., by complexing, binding, encapsulation, absorption, adsorption, or mixing with) a nanoparticle.

[0195] In some embodiments, such compositions associated with nanoparticles are further associated (e.g., by complexing, binding, encapsulating, absorbing, adsorbing, or mixing) with calcium phosphate and copolymers of PEI / PEG, PH-PEG, PK-PEG, or PGA-PEG. Indeed, in some embodiments, the association of nanoparticles with agents capable of stimulating an innate immune response in a subject is in the presence of calcium phosphate and copolymers of PEI / PEG, PH-PEG, PK-PEG, or PGA-PEG.

[0196] In some embodiments, such compositions in association with nanoparticles include Zn 2+ , Mn 2+ , Ca 2+ , Fe2+ , Fe 3+ , Cu 2+ , Ni 2+ , Co 2+ , Pb 2+ , Sn 2+ , Ru 2+ , Au 2+ , Mg 2+ ,VO 2+ , Al 3+ , Co 3+ , Cr 3+ , Ga 3+ , Tl 3+ , Ln 3+ , MoO 3+ , Cu + , Au + , Tl + , Ag + , Hg 2+ , Pt 2+ , Pb 2+ , Hg 2+ , Cd 2+ , Pd 2+ , Pt 4+ , Na + , K. + and associated phosphates or carbonates. Indeed, in some embodiments, the association of the nanoparticle with an agent capable of stimulating an innate immune response in a subject is further associated (e.g., by complexing, binding, encapsulating, absorbing, adsorbing, or mixing) with one or more cations selected from the group consisting of Zn, ... 2+ , Co 2+ , or Mn 2+ ) in the presence of

[0197] It is known in the art that STING (stimulator of interferon genes) is an adaptor for multiple cytoplasmic DNA receptors and a pattern recognition receptor (PRR) that recognizes the bacterial second messengers cyclic di-adenosine monophosphate (c-di-AMP) and cyclic di-guanosine monophosphate (c-di-GMP). Cytoplasmic DNA binds to cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) to produce cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which then binds to and activates the adaptor protein STING, inducing IFN. STING contains five putative transmembrane domains and resides primarily in the endoplasmic reticulum. It activates both the NF-κB and IRF3 transcription pathways to induce the expression of type I interferons (IFN-α and IFN-β), which can exert a potent antiviral state after expression.

[0198] Thus, DAMPs and PAMPs (e.g., STING agonists) can stimulate an innate cytokine response in cancer cells. Thus, in some embodiments, DAMPs and PAMPs (e.g., STING agonists) can stimulate an innate cytokine response in cancer cells.

[0199] The innate cytokine response stimulated by a DAMP or PAMP is mediated through a cytokine, hi some embodiments, for example, the innate cytokine response can be mediated through type 1 interferon.

[0200] As discussed above, the present disclosure provides compositions and methods for stimulating the innate immune response of cancer cells (e.g., tumor cells) using agents (e.g., DAMPs / PAMPs) that, upon administration to a subject, can stimulate the subject's innate immune response to suppress and / or inhibit the proliferation of cancer cells (e.g., tumor cells). In particular, the present invention is directed to compositions comprising nanoparticles associated (e.g., by complexing, binding, encapsulation, absorption, adsorption, mixing) with agents (e.g., DAMPs / PAMPs) that, upon administration to a subject, can stimulate the subject's innate immune response, methods for synthesizing such nanoparticles, and systems and methods (e.g., in diagnostic and / or therapeutic settings) that utilize such nanoparticles.

[0201] Indeed, experiments conducted in the course of developing embodiments of the present invention have shown that Zn 2+ We demonstrated that CDNs containing cGAMP, cdiAMP, cdiGMP, and cAIMP assemble into uniform nanoparticles in the presence of Zn. 2+ It was also shown that such CDNs assembled into uniform nanoparticles in the presence of calcium phosphate and copolymers of cationic poly(ethyleneimine) (PEI) and polyethylene glycol (PEG) could be further stabilized with lipid vesicles. Further experiments demonstrated that CDNs could be formulated into nanoparticles in the presence of calcium phosphate and copolymers of cationic poly(ethyleneimine) (PEI) and polyethylene glycol (PEG). Such CDN-nanoparticle assemblies (e.g., CDN and PEI-PEG copolymers formulated into nanoparticles in the presence of calcium phosphate) (e.g., Zn) 2+ , Co 2+ or Mn 2+ , and CDNs formulated into nanoparticles in the presence of liposomes) have further been shown to provide increased cancer cell uptake and more precise targeting to the tumor microenvironment (e.g., TME), thereby allowing for increased STING agonist delivery efficacy and reduced STING agonist toxicity.

[0202] The present invention is not limited to a particular type or kind of nanoparticle that is associated (e.g., by complexing, binding, encapsulating, absorbing, adsorbing, mixing) with such a composition comprising an agent (e.g., DAMP / PAMP) that is capable of stimulating an innate immune response in a subject upon administration to the subject.

[0203] Examples of nanoparticles include, but are not limited to, metal-polyhistidine-DOPE@liposomes, metal-polyhistidine-PEG, 4-arm-PEG-polyhistidine-metal hydrogels, and sHDL-polyhistidine, fullerenes (mainly C). 60 , C 70 , C 76 , C 80 , C 84 Examples of nanoparticles include endohedral metallofullerene (EMI) buckyballs containing additional atoms, ions, or clusters within a fullerene cage, trimetal nitride-templated endohedral metallofullerenes (TNT EMIs, highly symmetrical four-atom molecular cluster inclusions formed in a trimetal nitride template within a carbon cage), single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitrate nanotubes, carbon nanotube peapods (nanotubes with internal metallofullerene and / or other internal chemical structures), carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, quantum dots, superparamagnetic nanoparticles, nanorods, and cellulose nanoparticles. Particle embodiments can also include microparticles capable of enhancing efficacy or selectivity. Other non-limiting nanoparticles include glass and polymeric microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold, silver, carbon, and iron nanoparticles.

[0204] In some embodiments, the nanoparticles are modified micelles. In these embodiments, the modified micelles comprise a polyol polymer modified to contain a hydrophobic polymer block. As used in this disclosure, the term "hydrophobic polymer block" refers to a segment of a polymer that is itself hydrophobic. As used herein, the term "micelle" refers to an aggregate of molecules dispersed in a liquid. Typical micelles in aqueous solution form aggregates with a hydrophilic "head" region in contact with the surrounding solvent, isolating a single hydrophobic tail region at the center of the micelle. In some embodiments, the head region can be, for example, a surface region of the polyol polymer, and the tail region can be, for example, a hydrophobic polymer block region of the polyol polymer.

[0205] The present invention further encompasses the use of micrometer-scale particles in addition to nanometer-scale particles. When microparticles are used, they are preferably relatively small, on the order of 1 to 50 micrometers. For ease of discussion, "nanoparticles" as used herein encompass true nanoparticles (sizes of about 1 nm to about 1000 nm), microparticles (e.g., about 1 micrometer to about 50 micrometers), or both.

[0206] Examples of nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers, dendrimers with covalently bound metal chelates, nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In some embodiments, the nanoparticles are metal nanoparticles (e.g., nanoparticles of gold, palladium, platinum, silver, copper, nickel, cobalt, iridium, or alloys of two or more thereof). The nanoparticles can comprise a core or a core and a shell, such as core-shell nanoparticles.

[0207] In some embodiments, the nanoparticles are sHDL nanoparticles. Generally, sHDL nanoparticles are composed of a mixture of HDL apolipoproteins and amphipathic lipids.

[0208] The present invention is not limited to the use of any particular type or variety of HDL apolipoproteins, including, for example, apolipoprotein AI (apo AI), apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), and apolipoprotein E (apo E). In some embodiments, the HDL apolipoprotein is selected from preproapolipoprotein, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, preproApoA-IV, proApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproapoA-I Milano, proApoA-I Milano, ApoA-I Milano, preproapoA-IParis, proapoA-IParis, and ApoA-IParis, as well as peptidomimetics of these protein mixtures. Preferably, the carrier particles are composed of ApoA-I or ApoA-II, although other lipoproteins, including apolipoprotein A4, apolipoprotein Cs, or apolipoprotein E, may be used alone or in combination to formulate carrier particle mixtures for delivery of therapeutic agents. In some embodiments, mimetics of such HDL apolipoproteins are used.

[0209] ApoA-I is synthesized by the liver and small intestine as a preproapolipoprotein, secreted as a proprotein that is rapidly cleaved to generate a mature polypeptide of 243 amino acid residues. ApoA-I primarily consists of six to eight distinct 22-amino acid repeats separated by linker moieties, often proline, and in some cases, stretches of several residues. ApoA-I forms three types of stable complexes with lipids: small lipid-poor complexes called pre-β-1 HDL; flattened, discoidal particles containing polar lipids (phospholipids and cholesterol) called pre-β-2 HDL; and spherical or mature HDL (HDL3 and HDL2), spherical particles containing both polar and nonpolar lipids. Most HDL in the circulating population contains both ApoA-I and ApoA-II (the second major HDL protein).

[0210] In some embodiments, ApoA-I agonists or mimetics are provided. In some embodiments, such ApoA-I mimetics can form an amphipathic α-helix that mimics the activity of ApoA-I and have specific activity that approaches or exceeds that of the native molecule. Sometimes, the ApoA-I mimetic is a peptide or peptide analog that forms an amphipathic helix (in the presence of lipids), binds to lipids, forms pre-β-like or HDL-like complexes, activates lecithin:cholesterol acyltransferase (LCAT), increases serum levels of the HDL fraction, and promotes cholesterol efflux.

[0211] The present invention is not limited to the use of a specific ApoA-I mimetic. In some embodiments, any of the ApoA-I mimetics described in Srinivasa, et al., 2014 Curr. Opinion Lipidology Vol. 25(4): 304-308 are utilized. In some embodiments, any of the ApoA-I mimetics described in U.S. Patent Application Publication Nos. 2011 / 0046056 and 2013 / 0231459 are used.

[0212] In some embodiments, the "22A" ApoA-I mimetic is used (PVLDLFRELLNELLEALKQKLK) (SEQ ID NO: 4) (see, e.g., U.S. Patent No. 7,566,695). In some embodiments, any of the following ApoA-I mimetics shown in Table 1, as described in U.S. Patent No. 7,566,695, are utilized.

[0213] [Table 1] TIFF2024536138000011.tif249121TIFF2024536138000012.tif249121TIFF2024536138000013.tif248121 TIFF2024536138000014.tif249121TIFF2024536138000015.tif249122TIFF2024536138000016.tif103162

[0214] * indicates an N-terminally acetylated and C-terminally amidated peptide; indicates an N-terminally dansylated peptide; sp indicates a peptide that showed solubility problems under the experimental conditions; X is Aib; Z is NaI; O is Orn; He(%) indicates percent helicity; mic indicates micelles; ~ indicates a missing amino acid.

[0215] In some embodiments, an ApoA-I mimetic is utilized having the following sequence: Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (SEQ ID NO: 255), as described in U.S. Patent No. 6,743,778.

[0216] In some embodiments, any of the following ApoA-I mimetics shown in Table 2, as described in US Patent Application Publication No. 2003 / 0171277, are utilized.

[0217] [Table 2] TIFF2024536138000018.tif250150TIFF2024536138000019.tif250154

[0218] In some embodiments, an ApoA-I mimetic having the following sequence as described in US Patent Application Publication No. 2006 / 0069030 is utilized: FAEKFKEAVKDYFAKFWD (SEQ ID NO: 333).

[0219] In some embodiments, ApoA-I mimetics having the following sequences as described in U.S. Patent Application Publication No. 2009 / 0081293 are utilized: DWFKAFYDKVAEKFKEAF (SEQ ID NO: 334); DWLKAFYDKVAEKLKEAF (SEQ ID NO: 335); PALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ (SEQ ID NO: 336).

[0220] In some embodiments, an ApoA-I mimetic having one of the following sequences is utilized: WDRVKDLATVYVDVLKDSGRDYVSQF (SEQ ID NO: 341), LKLLDNWDSVTSTFSKLREOL (SEQ ID NO: 342), PVTOEFWDNLEKETEGLROEMS (SEQ ID NO: 343), KDLEEVKAKVQ (SEQ ID NO: 344), KDLEEVKAKVO (SEQ ID NO: 345), PYLDDFQKKWQEEMELYRQKVE (SEQ ID NO: 346), PLRAELQEGARQKLHELOEKLS (SEQ ID NO: 347), 347), PLGEEMRDRARARAHVDALRTHLA (SEQ ID NO: 348), PYSDELRQRLAARLEALKENGG (SEQ ID NO: 349), ARLAEYHAKATEHLSTLSEKAK (SEQ ID NO: 350), PALEDLROGLL (SEQ ID NO: 351), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), PVLESFVSFLSALEEYTKKLN (SEQ ID NO: 353), PVLESFKVSFLSALEEYTKKLN (SEQ ID NO: 352), TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 354). 354), QTVTDYGKDLME (SEQ ID NO: 355), KVKSPELOAEAKSYFEKSKE (SEQ ID NO: 356), VLTLALVAVAGARAEVSADOVATV (SEQ ID NO: 357), NNAKEAVEHLOKSELTOOLNAL (SEQ ID NO: 358), LPVLVWLSIVLEGPAPAOGTPDVSS (SEQ ID NO: 359), LPVLVVVLSIVLEGPAPAQGTPDVSS (SEQ ID NO: 360), ALDKLKEFGNTLEDKARELIS (SEQ ID NO: 361), VVALLALLASARASEAEDASL L (SEQ ID NO: 362), HLRKLRKRLLRDADDLQKRLAVYOA (SEQ ID NO: 363), AQAWGERLRARMEEMGSRTRDR (SEQ ID NO: 364), LDEVKEQVAEVRAKLEEQAQ (SEQ ID NO: 365), DWLKAFYDKVAEKLKEAF (SEQ ID NO: 236), DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA (SEQ ID NO: 366), PVLDLFRELLNELLEALKQKL (SEQ ID NO: 367), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 368),PVLDLFRELLNELLEALKQKLK (SEQ ID NO: 4), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 369), PVLDLFRELLNELLEALKKLLK (SEQ ID NO: 370), PVLDLFRELLNELLEALKKLLA (SEQ ID NO: 371), PLLDLFRELLNELLEALKKLLA (SEQ ID NO: 372), and EVRSKLEEWFAAFREFAEEFLARLKS (SEQ ID NO: 373).

[0221] Amphipathic lipids include, for example, any lipid molecule having both hydrophobic and hydrophilic portions. Examples include phospholipids or glycolipids. Examples of phospholipids that can be used in sHDL-TA nanoparticles include, but are not limited to, lipid-polyhistidine (e.g., DOPE-H11), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl ... phospholipids include 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof. In some embodiments, the phospholipid is conjugated with a contrast agent (e.g., rhodamine (Rhod)-labeled DOPE (DOPE-Rhod)). In some embodiments, the phospholipid is a thiol-reactive phospholipid such as, for example, dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dihexadecanoyl-sn-glycero-3-phosphothioethanol, or N-4-(p-maleimidophenyl)butyryl)dipalmitoylphosphatidylethanolamine (MPB-DPPE).

[0222] In some embodiments, exemplary phospholipids include small alkyl chain phospholipids, egg phosphatidylcholine, soy phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, Dioleoylphosphatidylcholine, Dioleoylphosphatidylethanolamine, Dilauroylphosphatidylglycerol, Phosphatidylcholine, Phosphatidylserine, Phosphatidylethanolamine, Phosphatidylinositol, Phosphatidylglycerol, Dimyristoylphosphatidylglycerol, Dipalmitoylphosphatidylglycerol, Dimyristoylphosphatidylglycerol, Dipalmitoylphosphatidylglycerol, Distearoylphosphatidylglycerol Glycerol, diphosphatidylglycerols such as dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, egg sphingomyelin, milk sphingomyelin, palmitoyl sphingomyelin, phytosphingomyelin These include, but are not limited to, sphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin, dipalmitoylphosphatidylglycerol salts, phosphatidic acid, galactocerebrosides, gangliosides, cerebrosides, dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3) diglyceride, aminophenylglycosides, 3-cholesteryl-6'-(glycosylthio)hexyl ether glycolipids, and cholesterol, and derivatives thereof.The phospholipid fraction containing SM and palmitoylsphingomyelin can optionally contain small amounts of any type of lipid, including, but not limited to, lysophospholipids, sphingomyelins other than palmitoylsphingomyelin, galactocerebrosides, gangliosides, cerebrosides, glycerides, triglycerides, and cholesterol, and derivatives thereof.

[0223] In some embodiments, the sHDL nanoparticles have a phospholipid / HDL apolipoprotein molar ratio of 2 to 250 (eg, 10 to 200, 20 to 100, 20 to 50, 30 to 40).

[0224] Generally, the sHDL nanoparticles so formed are spherical and have a diameter of about 5 nm to about 20 nm (e.g., 4-75 nm, 4-60 nm, 4-50 nm, 4-22 nm, 6-18 nm, 8-15 nm, 8-10 nm, etc.). In some embodiments, the sHDL nanoparticles are subjected to size exclusion chromatography to obtain a more homogeneous preparation.

[0225] Compared to other strategies, including traditional nanoparticle vehicles, sHDL nanoparticles possess attractive biocompatibility and cargo-loading capabilities. For example, their ultrasmall yet tunable size (e.g., 10–20 nm) allows sHDL nanoparticles to efficiently flow to lymph nodes and deliver cargo peptide antigens and nucleic acid-based adjuvants to lymph node-resident dendritic cells, thereby positioning them as an efficient platform for the co-delivery of STING agonists and adjuvants for tumor immunotherapy.

[0226] In certain embodiments, compositions are provided comprising nanoparticles associated with such compositions comprising one or more agents (e.g., DAMPs / PAMPs) capable of stimulating the subject's innate immune response upon administration to the subject, wherein any type of biopolymer agent (e.g., nucleic acid, peptide, glycolipid, etc.) is associated with the nanoparticles.

[0227] In some embodiments, the biopolymer agent is a peptide.

[0228] For example, in some embodiments, the peptide is an antigen.

[0229] In some embodiments, the antigen is a tumor antigen, including tumor-associated or tumor-specific antigens, such as α-actinin-4, Bcr-Abl fusion protein, Casp-8, β-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, Mum-2, and Mum-3, neo-PAP, myosin class I, OS-9, pm l-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Bage-1, Gage3, Gage4, Gage5, Gage6, Gage7, GnTV, Herv-K-mel, Lage-1, Mage-A1, Mage-A2, Mage-A3, Mage-A4, Mage-A6, Mage-A10, Mage-A12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100( Pmel17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGS), SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MA GE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA19-9, CA-72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA125, CA15-3 (CA27.29\BCAA), CA195, CA242, CA-50, CAM43, CD68\KP1,CO-029, FGF-5, G250, Ga733 (EpCAM), human EGFR protein or human EGFR protein fragments such as human EGFR residues 306-325 (SCVRACGADSYEMEEDGVRK (SEQ ID NO: 374)) and residues 897-915 (VWSYGVTVWELMTFGSKPY (SEQ ID NO: 375)), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein \cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, WT1 (and WT1-derived peptide sequence: WT1 126-134 (RMFP NAPYL (SEQ ID NO: 376)), WT1 122-140 (SGQARMFPNAPYLPSCLES (SEQ ID NO: 377)) and WT1 122-144 (SGQARMFPNAPYLPSCLESQPTI (SEQ ID NO: 378)), MUC1 (and MUC1-derived peptides and glycopeptides such as RPAPGS (SEQ ID NO: 379), PPAHGVT (SEQ ID NO: 380), and PDTRP (SEQ ID NO: 381))), LMP2, EGFRvIII, idiotype, GD2, Ras mutants, p53 mutants, proteinase 3 (PR1), survivin, hTERT, sarcoma translocation breakpoints, EphA2, EphA4, LMW-PTP, PAP, ML-IAP, AFP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl-GM1, mesothelin, sLe (animal), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HM WMAA, AKAP-4, XAGE1, B7H3, legumain, Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-α, PDGFR-β, MAD-CT-2, Fos-related antigen 1, ERBB2, folate receptor 1 (FOLR1 or FBP), IDH1, IDO, LY6K, fms-related tyrosine kinase 1 (FLT1, best known as VEGFR1), KDR, PADRE,These include, but are not limited to, TA-CIN (recombinant HPV16 L2E7E6), SOX2, and aldehyde dehydrogenase.

[0230] In some embodiments in which the biopolymer is an antigen, the composition further comprises an adjuvant (as described herein).

[0231] In some embodiments, the peptide is selected from the group consisting of adrenocorticotropic hormone (ACTH), growth hormone peptides, melanocyte-stimulating hormone (MSH), oxytocin, vasopressin, corticotropin-releasing factor (CRF), CRF-related peptides, gonadotropin-releasing-related peptides (GAP), growth hormone-releasing factor (GRF), luteinizing hormone-releasing hormone (LH-RH), orexin, prolactin-releasing peptide (PRP), somatostatin, thyrotropin-releasing hormone (THR), THR analogs, calcitonin (CT), CT precursor peptide, calcitonin (CT), CT-related ... Cytokinin gene-related peptide (CGRP), parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), amylin, glucagon, insulin, insulin-like peptide, neuropeptide Y (NPY), pancreatic polypeptide (PP), peptide YY (PYY), cholecystokinin (CCK), CCK-related peptide, gastrin-releasing peptide (GRP), gastrin, gastrin-related peptide, gastrin-inhibitory peptide, motilin, secretin, vasoactive intestinal peptide (VIP), VIP-related peptide, atrial natriuretic peptide (ANP), Brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), tachykinin, angiotensin, renin substrate, renin inhibitor, endothelin, endothelin-related peptide, opioid peptide, thymic peptide, adrenomedullin peptide, alostatin peptide, amyloid beta protein fragment, antimicrobial peptide, antioxidant peptide, apoptosis-related peptide, capsule cell peptide (BCP), bombesin, bone Gla protein peptide, cocaine- and amphetamine-related transcript (CART) peptide, cell adhesion peptide, chemotactic peptide Tides, complement inhibitors, cortistatin peptides, fibronectin fragments, fibrin-related peptides, FMRF, FMRFamide-related peptides (FaRP), galanin, galanin-related peptides, growth factors, growth factor-related peptides, G therapeutic peptide-binding protein fragments, guarilin, uroguarilin, inhibin peptides, interleukins (IL), interleukin receptor proteins, laminin fragments, leptin fragment peptides, leukokinins, pituitary adenylate cyclase-activating polypeptides (PAPCAP), pancreastatin, polypeptide repeat chains,Signal transduction factors, thrombin inhibitors, toxins, trypsin inhibitors, virus-related peptides, adjuvant peptide analogs, alpha mating factor, antiarrhythmic peptides, anorexigenic peptides, alpha-1 antitrypsin, bovine pineal antireproductive peptide, brucine, C3 peptide P16, cadherin peptides, chromogranin A fragments, contraceptive tetrapeptides, conantokin G, conantokin T, crustacean cardioactive peptides, C-telopeptides, cytochrome b588 peptides, decorsin, delicious peptides peptide), delta sleep-inducing peptide, diazepam binding inhibitor fragment, nitric oxide synthase inhibitor peptide, OVA peptide, platelet calpain inhibitor (P1), plasminogen activator inhibitor 1, rigin, schizophrenia-related peptide, sodium potassium A therapeutic peptidase inhibitor-1, speract, sperm-activating peptide, systemin, thrombin receptor agonist, tuftsin, lipid mobilizing hormone, uremic pentapeptide, antifreeze polypeptide, tumor necrosis factor (TNF), Leech [Des Asp10]Decorsin, L-Ornityltaurine Hydrochloride, P-Aminophenylacetyltuftsin, Ac-Glu-Glu-Val-Val-Ala-Cys-pNA, Ac-Ser-Asp-Lys-Pro, Ac-rfwink-NH2, Cys-Gly-Tyr-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-Gly, D-Ala-Leu, DDDDD, DDDDDD, NPNANPNA, VAITVLVK, VGVRVR, VIHS, VPDPR , Val-Thr-Cys-Gly, RSR, sea urchin sperm activating peptide, SHU-9119 antagonist, MC3-R antagonist, MC4-R antagonist, Glaspimod, HP-228, α2-plasmin inhibitor, APC tumor suppressor, early pregnancy factor, gamma interferon, glandular kallikrein N-1, placental ribonuclease inhibitor, sarcolecin-binding protein, surfactant protein D, Wilms tumor suppressor, GABAB 1b receptor peptide, prion-related peptide (iPRP13), choline-binding protein fragment, telomerase inhibitor,Cardiostatin peptides, endostatin-derived peptides, prion inhibitory peptides, N-methyl D-aspartate receptor antagonists, and C-peptide analogs.

[0232] In some embodiments, the peptide is selected from the group consisting of 177Lu-DOTA0-Tyr3-octreotate, Abarelix acetate, ADH-1, Afamelanotidec, Melanotan-1, CUV1647, Albiglutide, Aprotinin, Argipressin, Atosiban acetate, Bacitracin, Bentiromide, BH3 domain, Bivalirudin, Bivalirudin trifluoroacetate hydrate. hydrate), blissibimod, bortezomib, buserelin, buserelin acetate, calcitonin, carbetocin, carbetocin acetate, cecropin A and B, ceruletide, ceruletide diethylamine, cetrorelix, cetrorelix acetate, cyclosporine, cilengitide (Cilengitidec), EMD121974, corticorelin acetate injection, hCRF, corticorelin sheep triflutate, corticorelin trifluoroacetate, corticotropin, cosyntropin, ACTH 1-24, tetracosactide hexaacetate, dalbavancin, daptomycin, degarelix acetate, deptreotide trifluoroacetate (with sodium pertechnetate), desmopressin acetate, desmopressin DDAVP, dulaglutide, ecallantide, edotreotide (with yttrium-90), elcatonin acetate, enalapril maleate (or 2-butanedioate), enfuvirtide, epfihibatide, exenatide, ganirelix acetate, glatiramer acetate, glutathione, gonadorelin, Gonadorelin acetate, GnRH, LHRH, goserelin, goserelin acetate, gramicidin, histrelin acetate, human calcitonin, icatibant, icatibant acetate, IM862, oglufanide disodium, KLAKLAK, lanreotide acetate, lepirudin, leuprolide, leuprolide acetate, leuprorelin, liraglutide, lisinopril, lixisenatide, lypressin, magenin 2, MALP-2Sc, macrophage-activating lipopeptide-2synthetic), nafarelin acetate, nesiritide, NGR-hTNF, octreotide acetate, oritavancin, oxytocin, pasireotide, peginesatide, pentagastrin, pentetreotide (with indium-111), phenypressin, pleurocidin, pramlintide, protirelin, thyroliberin, TRH, TRF, salmon calcitonin, saralasin acetate, secretin (human), secretin (porcine), semaglutide, seractide acetate, ACTH, corticotropin, sermorelin acetate, GRF 1-29, sinapultide, KL4 in lucinactant, sincalide, somatorelin acetate, GHRH, GHRF, GRF, somatostatin acetate, Spaglumat magnesium (or sodium) salt salt), substance P, taltirelin hydrate, teduglutide, teicoplanin, telavancin, teriparatide, terlipressin acetate, tetracosactide, thymalfasin, thymosin al, thymopentin, trebananib, triptorelin, triptorelin pamoate, tyroseruleutide, ularitide, vancomycin, vapreotide acetate, vasoactive intestinal peptide acetate, Vx-001c, TERT572Y, ziconotide acetate, α5-α6Bax peptide, and β-defensin.

[0233] In some embodiments, the peptide is any peptide that helps achieve a desired purpose using the composition, for example, in some embodiments, the peptide is any peptide that facilitates the treatment of any type of disease and / or disorder.

[0234] In some embodiments, the biopolymer agent is a nucleic acid. Such embodiments encompass any type of nucleic acid molecule, including, but not limited to, RNA, siRNA, microRNA, interfering RNA, mRNA, replicon mRNA, RNA analogs, and DNA.

[0235] In certain embodiments, nanoparticles associated with such compositions comprising an antigen and an agent (e.g., DAMP / PAMP) capable of stimulating an innate immune response in a subject upon administration to the subject are used to elicit an immune response. In some embodiments, such nanoparticles are further associated (e.g., by complexing, binding, encapsulation, absorption, adsorption, or mixing) with an adjuvant (e.g., a dendritic cell targeting molecule (DC)). In some embodiments, the nanoparticles are administered with an adjuvant. In some embodiments, the antigen is further associated (e.g., by complexing, binding, encapsulation, absorption, adsorption, or mixing) with an adjuvant. In some embodiments, the antigen is not associated (e.g., by complexing, binding, encapsulation, absorption, adsorption, or mixing) with an adjuvant. In some embodiments, the antigen is associated with a hydrophobic molecule. In some embodiments, the adjuvant is associated with a hydrophobic molecule. In some embodiments, the average size of the nanoparticles is 6 nm to 500 nm, e.g., about 20 nm to about 500 nm, e.g., about 20, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, or about 500 nm, about 30 nm to about 500 nm, about 40 nm to about 500 nm, about 50 nm to about 500 nm, or about 75 nm to about 250 nm, e.g., about 75 nm, about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm, about 225 nm, or about 250 nm.

[0236] In some embodiments, the hydrophobic molecule is a lipid molecule. In some embodiments, the lipid molecule is a membrane-forming lipid molecule. In some embodiments, the lipid molecule is a non-membrane-forming lipid molecule.

[0237] Examples of lipid molecules applicable to embodiments of the present invention include lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, 1,2-dileoyl-sn-glycero-3-phosphate (DOPA, 14:0PA), 1,2-distearoyl-sn-glycero- 3-phosphate (18:0PA), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (18:1PA), distearoylphosphatidylcholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylcholine (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine (DPPG), dioleoylphosphatidylcholine (DOPG), dioleoylphosphatidylcholine (DPPG), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine (DPPG), dioleoylphosphatidylcholine (DOPG), dioleoylphosphatidylcholine (DPPG), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine (DPPC ... phosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), DSPE-PEG, monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used.The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10 to C24 carbon chains, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0238] Other non-limiting examples of lipid molecules include sterols such as cholesterol, their derivatives such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

[0239] Other examples of lipid molecules suitable for use in the present invention include non-phosphorus-containing lipids such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkylaryl sulfate polyethyloxylate fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, sphingomyelin, and the like.

[0240] Other examples of lipid molecules suitable for use in the present invention include fatty acids and their derivatives or analogs, including oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilauric acid, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, and their C 1-10These include alkyl esters (e.g., methyl, isopropyl, and t-butyl) and their mono- and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0241] Other examples of lipid molecules suitable for use in the present invention include lipid molecules modified with PEG (PEG-lipid).Examples of PEG-lipid include, but are not limited to, PEG (PEG-DAA) linked to dialkyloxypropyl, for example, as described in PCT Publication No. WO05 / 026372; PEG (PEG-DAG) linked to diacylglycerol, for example, as described in US Patent Publication Nos. 20030077829 and 2005008689; PEG (PEG-PE) linked to phospholipids such as phosphatidylethanolamine; PEG linked to ceramide, for example, as described in US Patent No. 5,885,613; PEG linked to cholesterol or its derivatives, and mixtures thereof.The disclosures of these patent documents are incorporated herein by reference in their entirety for all purposes.Additional PEG-lipids include, but are not limited to, PEG-C-DOMG, 2KPEG-DMG, and mixtures thereof.

[0242] PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups.PEG is classified according to its molecular weight, for example, PEG2000 has an average molecular weight of about 2,000 daltons, and PEG5000 has an average molecular weight of about 5,000 daltons.PEG is commercially available from Sigma Chemical Co. and other companies, and includes, for example, monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH2), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), and monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM).Other PEGs (e.g., mPEG (20KDa) amine) as described in U.S. Patent Nos. 6,774,180 and 7,053,150 are also useful for preparing the PEG-lipid conjugates of the present invention. The disclosures of these patents are incorporated herein by reference in their entireties for all purposes. Additionally, monomethoxypolyethylene glycol acetic acid (MePEG-CH2COOH) is particularly effective for preparing PEG-lipid conjugates, including, for example, PEG-DAA conjugates.

[0243] The PEG moiety of the PEG-lipid conjugates described herein can comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain examples, the PEG moiety has an average molecular weight of about 750 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0244] In certain instances, PEG may be substituted with an alkyl group, an alkoxy group, an acyl group, or an aryl group.PEG can be directly bound to lipid, or can be linked to lipid via a linker moiety.Any linker moiety suitable for linking PEG to lipid can be used, including, for example, non-ester-containing linker moieties and ester-containing linker moieties.In a preferred embodiment, the linker moiety is a non-ester-containing linker moiety.As used herein, the term "non-ester-containing linker moiety" refers to a linker moiety that does not contain a carboxylic acid ester bond (-OC(O)-). Suitable non-ester containing linker moieties include, but are not limited to, amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, disulfide, and combinations thereof (such as linkers containing both carbamate and amide linker moieties). In a preferred embodiment, a carbamate linker is used to attach PEG to the lipid.

[0245] In other embodiments, an ester-containing linker moiety is used to attach PEG to the lipid. Suitable ester-containing linker moieties include, for example, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), sulfonate ester, and combinations thereof.

[0246] Phosphatidylethanolamines with various acyl chain groups of various chain lengths and saturation degrees can be linked to PEG to form lipid conjugates.Such phosphatidylethanolamines can be commercially available or can be isolated or synthesized by using conventional techniques known to those skilled in the art.

[0247] C 10 ~C 20Phosphatidylethanolamines containing saturated or unsaturated fatty acids with a carbon chain length of 1 to 1000 are preferred. Phosphatidylethanolamines containing mono- or di-unsaturated fatty acids, as well as mixtures of saturated and unsaturated fatty acids, can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0248] Such embodiments are not limited to a particular antigen. Indeed, the antigen can be a peptide, protein, polysaccharide, sugar, lipid, glycolipid, nucleic acid, or a combination thereof. The antigen can be derived from any source, including, but not limited to, a virus, a bacterium, a parasite, a plant, a protozoan, a fungus, a tissue, or a transformed cell, such as a cancer or leukemia cell, and can be a whole cell or an immunogenic component thereof, such as a cell wall component or a molecular component thereof.

[0249] In some embodiments, antigens are known in the art and available from commercial, government, and scientific sources. In some embodiments, antigens are fully inactivated or attenuated organisms. These organisms may be infectious organisms such as viruses, parasites, and bacteria. These organisms may be tumor cells. Antigens may be purified or partially purified polypeptides derived from tumors or viral or bacterial sources. Criteria for identifying and selecting effective antigenic peptides (e.g., minimal peptide sequences capable of eliciting an immune response) can be found in the art. Antigens may be recombinant polypeptides produced by expressing DNA encoding the polypeptide antigen in a heterologous expression system. Antigens may be DNA encoding all or part of the antigen protein. The DNA may be in the form of vector DNA, such as plasmid DNA.

[0250] The antigens can be provided as single antigens or as combinations of antigens. The antigens can be provided as complex mixtures of polypeptides or nucleic acids.

[0251] In some embodiments, the antigen is an autoantigen. As used herein, the term "autoantigen" refers to an immunogenic antigen or epitope that is unique to a mammal and that may be involved in the pathogenesis of an autoimmune disease.

[0252] In some embodiments, the antigen is a viral antigen. Viral antigens can be isolated from any virus, including, but not limited to, viruses from any of the following viral families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., coronaviruses such as severe acute respiratory syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strains)), Flaviviridae (e.g., hepatitis C virus, dengue virus 1, dengue virus 2, dengue virus 3, and dengue virus 4), Hepadnaviridae, Herpesviridae (e.g., human herpesvirus, HIV-1, HIV-2, HIV-3, HIV-4, HIV-5, HIV-6, HIV-7, HIV-8, HIV-9, HIV-11, HIV-12, HIV-13, HIV-14, HIV-15, HIV-16, HIV-17, HIV-18, HIV-19, HIV-20, HIV-21, HIV-22, HIV-23, HIV-24, HIV-25, HIV-26, HIV-27, HIV-28, HIV-29, HIV-30, HIV-31, HIV-32, HIV-33, HIV-34, HIV-35, HIV-36, HIV-37, HIV-38, HIV-39, HIV-41, HIV-42 Herpesvirus 1, human herpesvirus 3, human herpesvirus 4, human herpesvirus 5, and human herpesvirus 6, and cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Liposthrixviridae, Microviridae, Orthomyxoviridae (e.g., influenza virus A, influenza virus B, and influenza virus C), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae Family: Mycoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentiviruses such as human immunodeficiency virus (HIV)-1 and HIV-2), Rhabdoviridae (e.g., rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (e.g., rubella virus, dengue virus, etc.), and Totiviridae.Suitable viral antigens also include all or part of dengue protein M, dengue protein E, dengue D1NS1, dengue D1NS2 and dengue D1NS3.

[0253] Viral antigens may be derived from specific strains of papillomavirus, herpesvirus, i.e., herpes simplex 1 and herpes simplex 2; hepatitis viruses, e.g., hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis delta virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV); tick-borne encephalitis viruses; parainfluenza, varicella-zoster, cytomegalovirus, Epstein-Barr, rotavirus, rhinovirus, adenovirus, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choriomyelitis.

[0254] In some embodiments, the antigen is a bacterial antigen, such as Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Haemophilus influenzae type B (HIB), Hyphomicrobium, Legionella, Leptospira, Listeria, Neisseria meningitidis A, Neisseria meningitidis B, and Neisseria meningitidis. The bacterial strains may be derived from any bacteria, including, but not limited to, Bacillus subtilis, Bacillus casei, Bacillus subtilis ...

[0255] In some embodiments, the antigen is a parasitic antigen, such as, but not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia rickettsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni. These include all or part of a sporozoite antigen, a plasmodian antigen, such as a circumsporozoite protein, a sporozoite surface protein, a liver stage antigen, an apical membrane associated protein, or a merozoite surface protein.

[0256] In some embodiments, the antigens are allergens and environmental antigens, such as, but not limited to, antigens derived from the following natural allergens: pollen allergens (tree pollen allergens, herb pollen allergens, weed pollen allergens, and grass pollen allergens), insect allergens (inhalant allergens, saliva allergens, and venom allergens), animal hair and dander allergens, and food allergens. Important pollen allergens from trees, grasses, and herbs are from the taxonomic orders of Fagales, Oleaceae, Pinales, and Platanales (including, inter alia, birch (Betula), alder (Alnus), hazel (Hazel), hornbeam (Carpinus), and olive (Olea), cedars (Cedar and Juniper), and plane trees (Platanus)), Poales (i.e., including grasses of the genera Lolium, Timothy, Poa, Cynodon, Orchardgrass, Picea, Reed grass, Rye, and Sorghum), Asterales, and Urticales (including, inter alia, herbs of the genera Ragweed, Artemisia, and Lycopersicon). Other allergen antigens that may be used include allergens from dust mites of the genera Dermatophagoides and Euroglyphus, allergens from dust mites (e.g., Lepidoglyphys, Glycyphagus, and Tyrophagus), allergens from cockroaches, horseflies, and fleas (e.g., Blattella germanica, Periplaneta americana, etc.), and allergens from insects such as mites, cockroaches, and fleas (e.g., Blattella germanica, Blattella americana, etc.). Venom allergens, including those from stinging insects such as those from the genera Periplaneta, Chironomus, and Ctenocepphalides, mammals such as cats, dogs, and horses, birds, and the Hymenoptera order, which includes bees (Apidae), hornets (Vespidae), and ants (Formicoidae). Still other allergen antigens that can be used include inhalant allergens from fungi such as Alternaria and Cladosporium.

[0257] In some embodiments, the antigen is a tumor antigen (as described herein).

[0258] One of the significant barriers to the development of therapeutic and tumor-specific immunotherapy is the identification and selection of highly specific and restricted tumor antigens to circumvent autoimmunity. Tumor neoantigens, which arise as a result of genetic alterations within malignant cells (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.), are the most tumor-specific antigen class.

[0259] In some embodiments, the antigen is a neoantigen. The term neoantigen is used herein to define any newly expressed antigenic determinant. Neoantigens can arise upon conformational changes in proteins as newly expressed determinants (especially on the surface of transformed or infected cells) as a result of complex formation of one or more molecules or as a result of molecular cleavage resulting in the presentation of new antigenic determinants. Thus, as used herein, the term neoantigen encompasses antigens expressed during infection (e.g., viral, protozoan, or bacterial infection), prion-mediated diseases, and cell transformation (cancer); in the latter case, neoantigens are sometimes referred to as tumor-associated antigens.

[0260] The present invention is not limited to a particular technique for identifying neoantigens. In some embodiments, identifying neoantigens involves identifying all or nearly all mutations in a neoplasm / tumor at the DNA level using whole genome sequencing, whole exome (e.g., captured exons only) sequencing, or RNA sequencing of tumors on matched germline samples from each patient. In some embodiments, identifying neoantigens involves analyzing the identified mutations with one or more peptide-MHC binding prediction algorithms to generate multiple candidate neoantigen T cell epitopes expressed in the neoplasm / tumor and capable of binding to patient HLA alleles. In some embodiments, identifying neoantigens involves synthesizing multiple candidate neoantigen peptides selected from a set of all neo open reading frame peptides and predicted binding peptides used in cancer vaccines.

[0261] Thus, the present invention is based, at least in part, on the ability to identify all or nearly all mutations within a neoplasm / tumor (e.g., translocations, inversions, large and small deletions, and insertions, missense mutations, splice site mutations, etc.). In particular, these mutations are present in the genome of a subject's neoplasm / tumor cells but are absent in normal tissues derived from the subject. Such mutations are of particular interest when they result in changes that result in proteins with altered amino acid sequences unique to the patient's neoplasm / tumor (e.g., neoantigens). For example, useful mutations may include: (1) nonsynonymous mutations resulting in different amino acids in the protein; (2) read-through mutations in which a stop codon is altered or deleted, resulting in translation of a longer protein with a novel tumor-specific sequence at the C-terminus; (3) splice site mutations that contain introns in the mature mRNA, thereby resulting in a unique tumor-specific protein sequence; (4) chromosomal rearrangements (i.e., gene fusions) that result in chimeric proteins with tumor-specific sequences at the junction of two proteins; (5) frameshift mutations or deletions that result in new open reading frames with novel tumor-specific protein sequences; and the like. For example, peptides with mutations or mutant polypeptides resulting from splice site, frameshift, readthrough, or gene fusion mutations in tumor cells can be identified by sequencing DNA, RNA, or protein of tumor versus normal cells.

[0262] Individualized neo-antigenic peptides derived from common tumor driver genes are also within the scope of the present invention and may further include previously identified tumor-specific mutations.

[0263] Preferably, any suitable sequencing-by-synthesis platform can be used to identify mutations. Four major sequencing-by-synthesis platforms are currently available: the Genome Sequencers available from Roche / 454 Life Sciences, the HiSeq Analyzer available from Illumina / Solexa, the SOLiD system available from Applied BioSystems, and the Heliscope system available from Helicos Biosciences. Sequencing-by-synthesis platforms have also been described by Pacific Biosciences and VisiGen Biotechnologies. Each of these platforms can be used in the methods of the present invention. In some embodiments, multiple nucleic acid molecules to be sequenced are attached to a support (e.g., a solid support). To immobilize the nucleic acid on the support, a capture sequence / universal priming site can be added to the 3' and / or 5' end of the template. The nucleic acid may be attached to the support by hybridizing a capture sequence to a complementary sequence covalently attached to the support. A capture sequence (also called a universal capture sequence) is a nucleic acid sequence complementary to a support-bound sequence that can double as a universal primer.

[0264] Instead of capture sequences, members of a binding pair (e.g., antibody / antigen, receptor / ligand, or avidin-biotin pair, as described in U.S. Patent Application Publication No. 2006 / 0252077) may be linked to each fragment to be captured on a surface coated with the second member of the binding pair. Following capture, the sequence may be analyzed, for example, by single-molecule detection / sequencing, including sequencing by template-dependent synthesis, as described in the Examples and U.S. Patent No. 7,283,337. In decoding-by-synthesis methods, surface-bound molecules are exposed to multiple labeled nucleotide triphosphates in the presence of a polymerase. The sequence of the template is determined by the order of labeled nucleotides incorporated at the 3' end of the growing strand. This can be done in real time or step-and-repeat mode. For real-time analysis, different optical labels for each nucleotide may be incorporated, and multiple lasers may be utilized for stimulation of the incorporated nucleotides.

[0265] Any cell type or tissue can be used to obtain the nucleic acid sample used in the sequencing method described herein.In some embodiments, DNA or RNA samples are obtained from neoplasms / tumors or body fluids such as blood or saliva obtained by known techniques (e.g., venipuncture).Alternatively, nucleic acid testing can be performed on dry samples (e.g., hair or skin).

[0266] Various methods are available for detecting the presence of specific mutations or alleles in an individual's DNA or RNA. Advances in this field have provided accurate, easy, and inexpensive large-scale SNP genotyping. More recently, several new techniques have been described, including dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, various DNA "chip" technologies such as the TaqMan system and the Affymetrix SNP chip. These methods require amplification of the target gene region, usually by PCR. Still other newly developed methods based on invasive cleavage followed by mass spectrometry or the generation of small signal molecules by immobilized padlock probes and rolling circle amplification may ultimately eliminate the need for PCR. Some of the methods known in the art for detecting specific single nucleotide polymorphisms are summarized below. It is understood that the method of the present invention includes all available methods.

[0267] PCR-based detection means may involve multiplex amplification of multiple markers simultaneously, for example, selecting PCR primers to generate PCR products that do not overlap in size and that can be analyzed simultaneously is well known in the art.

[0268] Alternatively, different markers can be amplified using primers that are differently labeled and therefore can be differentially detected. Naturally, hybridization-based detection means allow for differential detection of multiple PCR products in a sample. Other techniques that allow for multiplex analysis of multiple markers are known in the art.

[0269] Several methods have been developed to facilitate the analysis of single nucleotide polymorphisms in genomic DNA or cellular RNA. In one embodiment, single nucleotide polymorphisms can be detected by using specialized exonuclease-resistant nucleotides, as disclosed in U.S. Pat. No. 4,656,127, for example. According to this method, a primer complementary to the allelic sequence immediately 3' from the polymorphic site is allowed to hybridize to a target molecule from a specific animal or human. If the polymorphic site on the target molecule contains a nucleotide complementary to a specific exonuclease-resistant nucleotide derivative present, that derivative will be incorporated onto the end of the hybridized primer. This incorporation confers exonuclease resistance to the primer, thereby enabling its detection. Since the identity of the exonuclease-resistant derivative in the sample is known, the finding that the primer is exonuclease-resistant reveals that the nucleotide present at the polymorphic site in the target molecule is complementary to the nucleotide in the nucleotide derivative used in the reaction. This method has the advantage of not requiring the determination of large amounts of unrelated sequence data.

[0270] In another embodiment of the invention, a solution-based method is used to determine the identity of the nucleotide at a polymorphic site (see, e.g., French Patent No. 2,650,840; PCT Application No. WO 1991 / 02087). As in the method of U.S. Patent No. 4,656,127, a primer complementary to the allelic sequence immediately 3' to the polymorphic site may be used. This method uses a labeled dideoxynucleotide derivative that, if complementary to the nucleotide at the polymorphic site, becomes incorporated onto the end of the primer to determine the identity of the nucleotide at that site.

[0271] An alternative method known as Genetic Bit Analysis (GBA®) is described in International Patent Publication No. WO 1992 / 15712. GBA® uses a mixture of labeled terminators and primers complementary to the sequence 3' of the polymorphic site. The incorporated labeled terminators are therefore determined by and complementary to the nucleotide present at the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent No. 2,650,840; International Patent Application No. WO 1991 / 02087), the GBA® method is preferably a heterogeneous assay in which the primers or target molecule are immobilized on a solid phase. Recently, several primer-directed nucleotide incorporation procedures for assaying polymorphic sites in DNA have been described (e.g., Komher, J. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990); Kuppuswamy, M. et al., Proc. Natl. Acad. Sci. (USA) 88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. et al. (See, e.g., Syvanen, A.-C, et al., Anal. Biochem. 208:171-175 (1993)). These methods differ from GBA® in that they all rely on the incorporation of labeled deoxynucleotides to discriminate between bases at polymorphic sites. In such formats, signal is proportional to the number of incorporated deoxynucleotides, so polymorphisms occurring in runs of the same nucleotide can result in a signal proportional to the length of the run (see, e.g., Syvanen, A.-C, et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

[0272] An alternative method for identifying tumor-specific neoantigens is direct protein sequencing. Protein sequencing of enzymatic digests using multidimensional MS techniques (MSn), including tandem mass spectrometry (MS / MS), can also be used to identify neoantigens of the present invention. Such proteomic approaches allow for rapid and highly automated analysis (see, e.g., K. Gevaert and J. Vandekerckhove, Electrophoresis 21:1145-1154 (2000)). It is further contemplated within the scope of the present invention that high-throughput methods for de novo sequencing of unknown proteins can be used to analyze the proteome of a patient's tumor to identify expressed neoantigens. For example, meta-shotgun protein sequencing can be used to identify expressed neo-antigens (see, e.g., Guthals et al. (2012) Shotgun Protein Sequencing with Meta-contig Assembly, Molecular and Cellular Proteomics 11(10):1084-96).

[0273] Tumor-specific neoantigens may also be identified using MHC multimers to identify neoantigen-specific T cell responses. For example, high-throughput analysis of neoantigen-specific T cell responses in patient samples may be performed using MHC tetramer-based screening techniques (see, e.g., Hombrink et al. (2011) High-Throughput Identification of Potential Minor Histocompatibility Antigens by MHC Tetramer-Based Screening: Feasibility and Limitations 6(8):1-11; Hadrup et al. (2009) Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers, Nature Methods, 6(7):520-26; van Rooij et al. (2013) Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an ipilimumab-responsive melanoma, Journal of Clinical Oncology, 31:1-4; and Heemskerk et al. (2013) The cancer antigenome, EMBO Journal, 32(2):194-203). It is considered within the scope of the present invention that such tetramer-based screening techniques may be used for the initial identification of tumor-specific neoantigens or as a secondary screening protocol to determine which neoantigens a patient may have already been exposed to, thereby facilitating the selection of candidate neoantigens for the vaccines of the present invention.

[0274] The present invention further includes isolated peptides (e.g., neo-antigenic peptides containing tumor-specific mutations identified by the described methods, peptides containing known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the described methods). These peptides and polypeptides are referred to herein as "neo-antigenic peptides" or "neo-antigenic polypeptides." The polypeptides or peptides can be of various lengths and will minimally include a small region predicted to bind to a patient's HLA molecule (the "epitope") and additional flanking amino acids extending at both the N- and C-termini. The polypeptides or peptides can be in either neutral (uncharged) or salt form, and can either contain no or contain modifications such as glycosylation, side chain oxidation, or phosphorylation, provided that the modifications do not destroy the biological activity of the polypeptide as described herein.

[0275] In certain embodiments, the size of the at least one neo-antigenic peptide molecule may include, but is not limited to, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino acid residues, and any range derivable therein. In specific embodiments, the neo-antigenic peptide molecule is 50 amino acids or less. In preferred embodiments, the neo-antigenic peptide molecule is equal to about 20 to about 30 amino acids.

[0276] Thus, the present invention provides nanoparticles associated with such compositions comprising an agent (e.g., a DAMP / PAMP) capable of stimulating an innate immune response in a subject upon administration to the subject and one or more neo-antigenic peptides. In some embodiments, the nanoparticles are associated with two neo-antigenic peptides. In some embodiments, the nanoparticles are associated with at least five or more neo-antigenic peptides. In some embodiments, the nanoparticles are associated with at least about 6, about 8, about 10, about 12, about 14, about 16, about 18, or about 20 different peptides. In some embodiments, the nanoparticles are associated with at least 20 different peptides.

[0277] Neoantigenic peptides, polypeptides, and analogs can be further modified to contain additional chemical moieties not normally part of proteins. These derivatized moieties can improve solubility, biological half-life, protein absorption, or binding affinity. These moieties can also reduce or eliminate any desired side effects of proteins, etc. A summary of these moieties can be found in Remington's Pharmaceutical Sciences, 20 thed., Mack Publishing Co., Easton, PA (2000). For example, neo-antigenic peptides and polypeptides having desired activity may be modified as needed to provide certain desired attributes, e.g., improved pharmacological properties, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide in binding to the desired MHC molecule and activating the appropriate T cells. For example, neo-antigenic peptides and polypeptides may undergo various modifications, such as either conservative or non-conservative substitutions, provided that the modifications may provide certain advantages in use, such as improved MHC binding. Such conservative substitutions may involve replacing one amino acid residue with another that is biologically and / or chemically similar (e.g., one hydrophobic residue with another hydrophobic residue, or one polar residue with another polar residue). The effects of single amino acid substitutions may also be examined using D-amino acids. Such modifications can be carried out using well-known peptide synthesis procedures, as described, for example, in Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (NY, Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, III., Pierce), 2d Ed. (1984).

[0278] In some embodiments, neoantigenic peptides and polypeptides may be modified with a linking agent to facilitate association with nanoparticles (e.g., sHDL nanoparticles). The present invention is not limited to a particular type or kind of linking agent. In some embodiments, the linking agent is a cysteine-serine-serine (CSS) molecule.

[0279] In some embodiments in which the nanoparticles are sHDL and the neoantigenic peptide or polypeptide is modified with CSS, the sHDL is further modified with dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), and upon mixing, the DOPE-PDP and CSS associate, thereby resulting in complexation (linkage) of the CSS-Ag with the sHDL.

[0280] Neoantigenic peptides and polypeptides can be modified by extending or reducing the amino acid sequence of the compound (e.g., adding or deleting amino acids). Neoantigenic peptides, polypeptides, or analogs can also be modified by changing the order or composition of specific residues. Those skilled in the art will understand that certain amino acid residues essential for biological activity (e.g., residues at critical contact sites or conserved residues) generally cannot be altered without adversely affecting the biological activity. Non-essential amino acids need not be limited to amino acids naturally occurring in proteins, such as La-amino acids or their D-isomers, but may also include unnatural amino acids, such as β-γ-δ-amino acids, as well as many derivatives of La-amino acids.

[0281] Typically, neoantigen polypeptides or peptides may be optimized by using a series of peptides with single amino acid substitutions to determine the effects of electrostatic charge, hydrophobicity, and the like on MHC binding. For example, a series of positively (e.g., Lys or Arg) or negatively (e.g., Glu) amino acid substitutions can be made along the length of the peptide to reveal distinct patterns of sensitivity to various MHC molecules and T cell receptors. In addition, multiple substitutions using small, relatively neutral moieties such as Ala, Gly, or Pro, or multiple substitutions using similar residues, may be used. Substitutions may be homo- or hetero-oligomeric. The number and type of residues substituted or added depend on the spacing required between essential contact points and the specific functional attributes desired (e.g., hydrophobicity vs. hydrophilicity). Such substitutions can achieve increased binding affinity for MHC molecules or T cell receptors compared to the affinity of the parent peptide. In either case, such substitutions may use amino acid residues or other molecular fragments selected to avoid, for example, steric hindrance and charge interference that could disrupt binding. Amino acid substitutions are typically of single residues. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at a final peptide.

[0282] Those skilled in the art will understand that there are various methods for producing such tumor-specific neoantigens. Generally, such tumor-specific neoantigens can be produced either in vitro or in vivo. Tumor-specific neoantigens can be produced in vitro as peptides or polypeptides, which can then be formulated into personalized neoplasia vaccines and administered to subjects. Such in vitro production can occur by various methods known to those skilled in the art, such as, for example, peptide synthesis or expression of peptides / polypeptides from DNA or RNA molecules in any of a variety of bacterial, eukaryotic, or viral recombinant expression systems, followed by purification of the expressed peptides / polypeptides.

[0283] Alternatively, tumor-specific neoantigens may be produced in vivo by introducing a molecule (e.g., DNA, RNA, viral expression system, etc.) encoding the tumor-specific neoantigen into a subject, whereupon the encoded tumor-specific neoantigen is expressed.

[0284] Proteins or peptides can be produced by any technique known to those skilled in the art, including expressing proteins, polypeptides, or peptides using standard molecular biology techniques, isolating proteins or peptides from natural sources, or chemically synthesizing proteins or peptides. Nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have been previously disclosed and can be found in computer databases known to those skilled in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information at the National Institutes of Health website. The coding regions of known genes can be amplified and / or expressed using techniques disclosed herein or known to those skilled in the art. Alternatively, various commercially available preparations of proteins, polypeptides, and peptides are known to those skilled in the art.

[0285] Peptides can be readily synthesized chemically using reagents that are free from contaminating bacterial or animal materials (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-54, 1963).

[0286] A further aspect of the present invention provides nucleic acids (e.g., polynucleotides) encoding the neo-antigenic peptides of the present invention, which can be used to produce the neo-antigenic peptides in vitro. The polynucleotides can be, for example, single-stranded and / or double-stranded DNA, cDNA, PNA, CNA, RNA, or polynucleotides in their natural or stabilized forms (e.g., polynucleotides with phosphorothioate backbones), or combinations thereof. The polynucleotides may or may not contain introns, so long as they encode the peptide. A further aspect of the present invention provides expression vectors capable of expressing the polypeptides of the present invention. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, the DNA is inserted into an expression vector, such as a plasmid, in the appropriate orientation and correct reading frame for expression. If necessary, the DNA may be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria); such control is generally provided in the expression vector. The vector is then introduced into a host bacterium for cloning using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0287] The present invention further encompasses variants and equivalents that are substantially homologous to the tumor-specific neoantigens identified herein. These can contain, for example, conservative substitution mutations (i.e., replacing one or more amino acids with similar amino acids). For example, conservative substitution refers to replacing an amino acid with another amino acid in the same general class, such as replacing one acidic amino acid with another acidic amino acid, replacing one basic amino acid with another basic amino acid, or replacing one neutral amino acid with another neutral amino acid. The purpose of conservative amino acid substitution is well known in the art.

[0288] The present invention also includes expression vectors containing the isolated polynucleotides and host cells containing the expression vectors. It is also considered within the scope of the present invention that the neo-antigenic peptides may be provided in the form of RNA or cDNA molecules encoding the desired neo-antigenic peptides. The present invention also provides that one or more neo-antigenic peptides of the present invention may be encoded by a single expression vector. The present invention also provides that one or more neo-antigenic peptides of the present invention may be encoded and expressed in vivo using a virus-based system (e.g., an adenovirus system).

[0289] The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that contain only the coding sequence of a polypeptide, as well as polynucleotides that contain additional coding and / or non-coding sequences. The polynucleotides of the present invention can be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or non-coding (antisense) strand.

[0290] In embodiments, the polynucleotide may include, for example, a coding sequence for a tumor-specific neo-antigenic peptide fused in the same reading frame as the polynucleotide that aids in the expression and / or secretion of the polypeptide from a host cell (e.g., a leader sequence that functions as a secretory sequence to control export of the polypeptide from the cell). Polypeptides with leader sequences can be preproteins, with the leader sequence being cleaved by the host cell to form the mature form of the polypeptide.

[0291] In some embodiments, the polynucleotide can include a coding sequence for a tumor-specific neoantigenic peptide fused in the same reading frame to a marker sequence that allows for purification of the encoded polypeptide, e.g., a polypeptide that can subsequently be incorporated into a personalized neoplasia vaccine. For example, the marker sequence can be a hexa-histidine tag provided by the pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. Additional tags include, but are not limited to, calmodulin tag, FLAG tag, Myc tag, S tag, SBP tag, Softag1, Softag3, V5 tag, Xpress tag, Isopeptag, SpyTag, biotin carboxyl carrier protein (BCCP) tag, GST tag, fluorescent protein tag (e.g., green fluorescent protein tag), maltose-binding protein tag, Nus tag, Strep tag, thioredoxin tag, TC tag, Ty tag, etc. In embodiments, the polynucleotide may contain coding sequences for one or more of the tumor-specific neo-antigenic peptides fused in the same reading frame to create a single concatemerized neo-antigenic peptide construct capable of producing multiple neo-antigenic peptides.

[0292] In embodiments, the present invention provides isolated nucleic acid molecules having a nucleotide sequence that is at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 96%, 97%, 98%, or 99% identical to a polynucleotide encoding a tumor-specific neo-antigenic peptide of the present invention.

[0293] A polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence can contain up to 5 point mutations for every 100 nucleotides of the reference nucleotide sequence.In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with other nucleotides, or up to 5% of the total number of nucleotides in the reference sequence can be inserted into the reference sequence.These mutations of the reference sequence can occur at the amino-terminal or carboxy-terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, and can be distributed separately between the nucleotides in the reference sequence or in one or more adjacent groups within the reference sequence.

[0294] In practice, whether any particular nucleic acid molecule is at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments at least 95%, 96%, 97%, 98%, or 99% identical to a reference sequence can be determined conventionally using known computer programs, such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the invention, parameters are set such that the percentage of identity is calculated across the full-length reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0295] The isolated tumor-specific neo-antigenic peptides described herein can be produced in vitro (e.g., in the laboratory) by any suitable method known in the art. Such methods range from direct protein synthesis to constructing DNA sequences encoding the isolated polypeptide sequences and expressing those sequences in a suitable transformed host. In some embodiments, recombinant techniques are used to construct DNA sequences by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutagenized by site-directed mutagenesis to provide a functional analog thereof. See, e.g., Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066 (1984), and U.S. Pat. No. 4,588,585.

[0296] In an embodiment, a DNA sequence encoding a polypeptide of interest is constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and by selecting codons that are preferred in the host cell in which the recombinant polypeptide of interest is to be produced. Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest. For example, the complete amino acid sequence can be used to construct a reverse-translated gene. Furthermore, it is possible to synthesize a DNA oligomer containing a nucleotide sequence encoding a particular isolated polypeptide. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides usually contain 5' or 3' overhangs for complementary assembly.

[0297] Once assembled (e.g., by synthesis, site-directed mutagenesis, or another method), the polynucleotide sequence encoding the specific isolated polypeptide of interest is inserted into an expression vector and, optionally, operably linked to expression control sequences suitable for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to obtain high levels of expression of the transgene in the host, the gene can be operably linked to transcriptional and translational expression control sequences functional in the selected expression host. Recombinant expression vectors can be used to amplify and express DNA encoding tumor-specific neo-antigenic peptides. Recombinant expression vectors are replicable DNA constructs having synthetic or cDNA-derived DNA fragments encoding tumor-specific neo-antigenic peptides or biologically equivalent analogs operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit generally comprises an assembly of (1) genetic elements or elements that play a regulatory role in gene expression (e.g., transcriptional promoters or enhancers), (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcriptional and translational start and stop sequences, as described in detail below. Such regulatory elements may include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may also be incorporated. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor involved in the secretion of the polypeptide. A promoter is operably linked to a coding sequence if it controls the transcription of the sequence. Alternatively, a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation.Generally, operably linked means contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Structural elements intended for use in yeast expression systems will include a leader sequence enabling the translated protein to be secreted extracellularly by a host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue which can optionally be subsequently cleaved from the expressed recombinant protein to provide the final product.

[0298] The choice of expression control sequence and expression vector will depend on the choice of host. A wide variety of expression host / vector combinations can be used. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids such as Escherichia coli-derived plasmids, including pCR1, pBR322, pMB9, and their derivatives, and broad-host-range plasmids such as M13 and filamentous single-stranded DNA phages.

[0299] Suitable host cells for expressing polypeptides include prokaryotes, yeast, insect, or higher eukaryotic cells under the control of an appropriate promoter. Prokaryotes include gram-negative or gram-positive bacteria, such as E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems can also be used. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985).

[0300] Various mammalian or insect cell culture systems can also be advantageously used to express recombinant proteins. Expression of recombinant proteins in mammalian cells can be carried out because such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells described by Gluzman (Cell 23: 175, 1981), as well as other cell lines capable of expressing suitable vectors, including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Mammalian expression vectors can include nontranscribed elements (e.g., an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5'- or 3'-flanking nontranscribed sequences), 5'- or 3'-untranslated sequences (e.g., necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites), and transcription termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0301] Proteins produced by transformed hosts can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange chromatography, affinity chromatography, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Affinity tags (e.g., hexahistidine, maltose-binding domain, influenza coat sequence, glutathione-S-transferase, etc.) can be attached to the protein to allow for easy purification by passage through an appropriate affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0302] For example, supernatants from systems secreting recombinant proteins into culture media can be first concentrated using commercially available protein concentration filters (e.g., Amicon or Millipore Pellicon ultrafiltration units). Following the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin (e.g., a matrix or substrate with pendant diethylaminoethyl (DEAE) groups) can be used. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, one or more reverse-phase high-performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium (e.g., silica gel with pendant methyl or other aliphatic groups) can be used to further purify the cancer stem cell protein-Fc composition. Various combinations of some or all of the above purification steps can also be used to provide a homogeneous recombinant protein. For example, recombinant proteins produced in bacterial culture can be isolated by initial extraction from a cell pellet, followed by one or more concentration, salting out, aqueous ion exchange, or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be used for final purification steps. Microbial cells used to express recombinant proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents.

[0303] Thus, in certain embodiments, the present invention relates to a personalized strategy for the treatment of disorders (e.g., neoplasms), more particularly tumors, by administering to a subject (e.g., a mammal such as a human) a therapeutically effective amount of a composition (e.g., a vaccine composition capable of generating a specific T cell response) comprising an agent (e.g., a DAMP / PAMP) capable of stimulating an innate immune response in the subject upon administration to the subject (as described herein) and one or more neoplasm / tumor-specific neoantigens. In some embodiments, such compositions are further associated with nanoparticles. Indeed, in certain embodiments, whole genome / exome sequencing can be used to identify all or nearly all mutant neoantigens uniquely present in an individual patient's neoplasm / tumor, and this collection of mutant neoantigens can be analyzed to identify a specific, optimized subset of neoantigens to be used as a personalized cancer vaccine for the treatment of the patient's neoplasm / tumor. For example, in some embodiments, a population of neoplasm / tumor-specific neoantigens can be identified by sequencing each patient's neoplasm / tumor and normal DNA to identify tumor-specific mutations and determining the patient's HLA allotype. The population of neoplasm / tumor-specific neoantigens and their cognate natural antigens may then be subjected to bioinformatics analysis using validated algorithms that predict which tumor-specific mutations generate epitopes that will be able to bind to the patient's HLA allotype, particularly which tumor-specific mutations generate epitopes that can bind to the patient's HLA allotype more effectively than the cognate natural antigen. Based on this analysis, one or more peptides corresponding to a subset of these mutations may be designed and synthesized for each patient and pooled together for use as a cancer vaccine when immunizing the patient. The neoantigen peptides may be combined with other anti-neoplastic agents. In some embodiments, such neoantigens are expected to bypass central thymic tolerance (thereby enabling stronger anti-tumor T cell responses) while reducing the potential for autoimmunity (e.g., by avoiding targeting normal self-antigens).

[0304] The present invention further provides methods of treating and / or alleviating symptoms of cancer in a subject by inducing a neoplasm / tumor-specific immune response in the subject, vaccinating against a neoplasm / tumor, and administering to the subject a neoantigenic peptide or vaccine composition of the present invention.

[0305] According to the present invention, the cancer vaccine can be used in patients diagnosed with cancer or at risk of developing cancer. In one embodiment, the patient may have a solid tumor (e.g., breast, ovarian, prostate, lung, kidney, stomach, colon, testicular, head and neck, pancreas, brain, melanoma, and other tumors of tissues and organs) and a hematological tumor (e.g., lymphomas and leukemias, including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma).

[0306] The peptides or compositions of the present invention are administered in an amount sufficient to induce a CTL response. The neoantigenic peptides, polypeptides, or vaccine compositions of the present invention can be administered alone or in combination with other therapeutic agents. The therapeutic agent can be, for example, a chemotherapeutic or biological agent, radiation, or immunotherapy. Any suitable therapeutic treatment for the specific cancer can be administered. Examples of chemotherapeutic and biological agents include aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alfa, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ibuprofen, riboflavin ... Include but are not limited to phosphamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol®), pilocarpine, prochlorperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine and vinorelbine tartrate.For treating prostate cancer, the preferred chemotherapeutic agent that can be combined with anti-CTLA-4 is paclitaxel (Taxol®).

[0307] In addition, the subject may further be administered an anti-immunosuppressant or an immunostimulant. For example, the subject may further be administered an inhibitor of anti-CTLA-4 antibody, anti-PD-1, anti-PD-L1, anti-TIM-3, anti-BTLA, anti-VISTA, anti-LAG3, anti-CD25, anti-CD27, anti-CD28, anti-CD137, anti-OX40, anti-GITR, anti-ICOS, anti-TIGIT, and IDO. Blocking CTLA-4 or PD-1 / PD-L1 with an antibody can enhance the patient's immune response to cancerous cells. In particular, CTLA-4 blockade has been shown to be effective when used in vaccination protocols.

[0308] Those skilled in the art can determine the optimal amount and optimal dosing regimen of each peptide to be included in the vaccine composition without undue experimentation. For example, the peptide or its variants can be prepared and prepared for intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. Preferred methods of peptide injection include sc, id, ip, im, and iv. Preferred methods of DNA injection include id, im, sc, ip, and iv. For example, a dose of 1 to 500 mg, 50 μg to 1.5 mg, preferably 10 μg to 500 μg of peptide or DNA may be administered, depending on the amount of peptide or DNA. The use of this dose range has been successful in previous clinical trials (Brunsvig PF, et al., Cancer Immunol Immunother. 2006; 55(12): 1553-1564; M. Staehler, et al., ASCO meeting 2007; Abstract No. 3017). Other methods of administering vaccine compositions are known to those skilled in the art.

[0309] The vaccines of the present invention may be tailored so that the selection, number, and / or amount of peptides present in the composition are tissue-, cancer-, and / or patient-specific. For example, the exact selection of peptides can be guided by the expression pattern of the parent protein in a given tissue to avoid side effects. The selection may depend on the specific type of cancer, the state of the disease, the early treatment regimen, the patient's immune status, and, of course, the patient's HLA-haplotype. Furthermore, the vaccines of the present invention may contain individual components depending on the individual needs of a particular patient. Examples include varying the amount of peptide according to the expression of relevant neoantigens in a particular patient, unwanted side effects due to individual allergies or other treatments, and adjustments for secondary treatments after the first round or treatment regimen.

[0310] Such vaccines can be administered to individuals already suffering from cancer. In therapeutic applications, such vaccines are administered to patients in an amount sufficient to elicit an effective CTL response against tumor antigens and to cure or partially halt at least symptoms and / or complications. An amount sufficient to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend, for example, on the peptide composition, the mode of administration, the stage and severity of the disease being treated, the patient's weight and general health, and the judgment of the prescribing physician. Generally, effective amounts for this use range from about 1.0 μg to about 50,000 μg of peptide for an initial immunization (whether therapeutic or prophylactic) for a 70 kg patient, followed by about 1.0 μg to about 10,000 μg of peptide according to boosting dosages or boosting regimens over several weeks to months, depending on the patient's response and condition, and optionally by measuring specific CTL activity in the patient's blood. It should be noted that the peptides and compositions of the present invention can generally be used in severe disease states, i.e., life-threatening or potentially life-threatening situations, especially when the cancer has metastasized. For therapeutic use, administration should be initiated as soon as possible after tumor detection or surgical removal. This is followed by increasing the dose at least until symptoms are substantially alleviated and thereafter. Pharmaceutical compositions (e.g., vaccine compositions) for therapeutic treatment are intended for parenteral, topical, nasal, oral, or local administration. Pharmaceutical compositions are preferably administered parenterally, for example, intravenously, subcutaneously, intradermally, or intramuscularly. The composition may also be administered at the surgical resection site to induce a local immune response to the tumor.

[0311] Such embodiments are not limited to a particular type of adjuvant. Generally, an adjuvant is any substance whose incorporation into a vaccine composition increases or otherwise modifies the immune response to a variant peptide. The carrier is a scaffold, e.g., a polypeptide or polysaccharide, to which the antigenic peptide (e.g., neoantigenic peptide) can be associated. Optionally, the adjuvant is covalently or noncovalently bound to the peptide or polypeptide of the invention.

[0312] The ability of an adjuvant to increase the immune response to an antigen is usually manifested by a significant increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity is usually manifested by a significant increase in the titer of antibodies elicited against the antigen, and an increase in T cell activity is usually manifested by an increase in cell proliferation, cytotoxicity, or cytokine secretion. Adjuvants may also modify the immune response, for example, by shifting a predominantly humoral or Th2 response to a predominantly cellular or Th1 response.

[0313] Suitable adjuvants include 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA These include, but are not limited to, 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, vector systems, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF traps, R848, β-glucans, Pam3Cys, Aquila's QS21 Stimulon (Aquila Biotech, Worcester, MA, USA) derived from saponins, mycobacterial extracts and synthetic bacterial cell wall mimics, and other trademarked or patented adjuvants such as Ribi's Detox. Quil or Superfos. Some immunological adjuvants specific for dendritic cells (e.g., MF59) and their preparation have been previously described (Dupuis M, et al., Cell Immunol. 1998; 186(1): 18-27; Allison AC; Dev Biol Stand. 1998; 92:3-11). Cytokines may also be used. Some cytokines have been directly implicated in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), accelerating dendritic cell maturation into effective antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (see, inter alia, U.S. Pat. No. 5,849,589, incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI, et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418).Toll-like receptors (TLRs), which may also be used as adjuvants, are important members of a family of pattern recognition receptors (PRRs) that recognize conserved motifs shared by many microorganisms, called "pathogen-associated molecular patterns" (PAMPS).

[0314] Recognition of these "danger signals" activates multiple components of the innate and adaptive immune systems. TLRs are expressed by cells of the innate and adaptive immune systems, such as dendritic cells (DCs), macrophages, T and B cells, mast cells, and granulocytes, and are localized in different cellular compartments, such as the plasma membrane, lysosomes, endosomes, and endolysosomes. Different TLRs recognize distinct PAMPs. For example, TLR4 is activated by LPS contained in bacterial cell walls, TLR9 is activated by unmethylated bacterial or viral CpG DNA, and TLR3 is activated by double-stranded RNA. TLR ligand binding leads to the activation of one or more intracellular signaling pathways, ultimately resulting in the production of many important molecules related to inflammation and immunity, particularly the transcription factor NF-κB and type I interferons. TLR-mediated DC activation leads to DC activation, phagocytosis, activation, and upregulation of costimulatory markers such as CD80, CD83, and CD86, enhanced expression of CCR7, which enables DC migration to draining lymph nodes and promotes antigen presentation to T cells, and increased secretion of cytokines such as type I interferon, IL-12, and IL-6. All of these downstream events are important for the induction of adaptive immune responses.

[0315] Other receptors that can be targeted include Toll-like receptors (TLRs). TLRs recognize and bind to pathogen-associated molecular patterns (PAMPs). PAMPs target TLRs on the surface of dendritic cells and signal internally, potentially increasing DC antigen uptake, maturation, and T cell stimulatory capacity. PAMPs bound or co-encapsulated on particle surfaces include unmethylated CpG DNA (bacterial), double-stranded RNA (viral), lipopolysaccharide (bacterial), peptidoglycan (bacterial), lipoarabinomannan (bacterial), zymosan (yeast), mycoplasma lipoproteins such as MALP-2 (bacterial), flagellin (bacterial), poly(inosinic-cytidylic) acid (bacterial), lipoteichoic acid (bacterial), or imidazoquinoline (synthetic).

[0316] Among the most promising cancer vaccine adjuvants currently in clinical development are the TLR9 agonist CpG and the synthetic double-stranded RNA (dsRNA) TLR3 ligand poly-ICLC. In preclinical studies, poly-ICLC appears to be the most potent TLR adjuvant compared with LPS and CpG due to its lack of induction of proinflammatory cytokines and stimulation of IL-10 in DCs and the maintenance of high levels of costimulatory molecules. Furthermore, poly-ICLC was recently directly compared with CpG in non-human primates (rhesus macaques) as an adjuvant for a protein vaccine consisting of human papillomavirus (HPV) 16 capsomeres (Stahl-Hennig C, Eisenblatter M, Jasny E, et al. Synthetic double-stranded RNAs are adjuvants for the induction of T helper 1 and humoral immune responses to human papillomavirus in rhesus macaques. PLoS pathogens. Apr 2009;5(4)).

[0317] In some embodiments, the adjuvant is a dendritic cell (DC) targeting molecule. DCs are potent and involved in initiating antigen-specific immune responses. One biological characteristic of DCs is their ability to sense antigen-encounter conditions and initiate the process of "DC maturation." Using receptors for various microorganisms and inflammatory products, DCs respond to antigen exposure in different ways depending on the nature of the pathogen (virus, bacteria, protozoan) they encounter. This information is conveyed to T cells by altering the cytokine release pattern upon antigen presentation in lymph nodes, altering the type of T cell response elicited. Therefore, targeting DCs not only generally enhances antigen delivery and antigen responses quantitatively, but also provides an opportunity to qualitatively control the nature of the immune response depending on the desired vaccination outcome.

[0318] Dendritic cells express multiple cell surface receptors that can mediate endocytosis of bound antigens. Targeting exogenous antigens to internalizing surface molecules on systemically distributed antigen-presenting cells facilitates antigen uptake, thereby overcoming a major rate-limiting step in immunization and, therefore, vaccination.

[0319] Dendritic cell targeting molecules include monoclonal or polyclonal antibodies or fragments thereof that recognize and bind to epitopes displayed on the surface of dendritic cells. Dendritic cell targeting molecules also include ligands that bind to cell surface receptors on dendritic cells. One such receptor, the lectin DEC-205, has been used in vitro and in mice to increase both humoral (antibody-based) and cellular (CD8 T cell) responses by two to four orders of magnitude (see, e.g., Hawiger, et al., J. Exp. Med., 194(6):769-79 (2001); Bonifaz, et al., J. Exp. Med., 196(12):1627-38 (2002); Bonifaz, et al., J. Exp. Med., 199(6):815-24 (2004)).

[0320] Various other endocytic receptors, including mannose-specific lectin (mannose receptor) and IgGFc receptor, have also been targeted in this way, with similar enhancement of antigen presentation efficiency.Other suitable receptors that can be targeted include, but are not limited to, DC-SIGN, 33D1, SIGLEC-H, DCIR, CD11c, heat shock protein receptor, and scavenger receptor.

[0321] In some embodiments, the adjuvant is CpG. CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in vaccine settings. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cellular vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, it enhances the maturation and differentiation of dendritic cells, leading to enhanced Th1 cell activation and the generation of potent cytotoxic T lymphocytes (CTLs) even in the absence of CD4 T cells. The Th1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote a Th2 bias. CpG oligonucleotides further exhibit stronger adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, which are particularly necessary to induce a strong response when the antigen is relatively weak. They also enhance immune responses, and in some experiments, the antigen dose can be reduced by approximately two orders of magnitude with equivalent antibody responses to a total dose of vaccine without CpG (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, June 2006, 471-484). U.S. Patent No. 6,406,705 B1 describes the combination of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce antigen-specific immune responses. A commercially available CpG TLR9 antagonist is dSLIM (double stem-loop immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR binding molecules may also be used, such as RNA-binding TLR7, TLR8, and / or TLR9.

[0322] For example, xanthenone derivatives such as vadimezan or AsA404 (also known as 5,6-dimethylxanthenone-4-acetic acid (DMXAA)) may also be used as adjuvants in accordance with embodiments of the present invention. Alternatively, such derivatives may be administered in conjunction with the vaccines of the present invention, e.g., by systemic or intratumoral delivery, to stimulate immunity at the tumor site. Without being bound by theory, it is believed that such xanthene derivatives act by stimulating interferon (IFN) production via the stimulator of IFN genes (ISTING) receptor (see, e.g., Conlon et al. (2013) Mouse, but not Human STING, Binds and Signals in Response to the Vascular Disrupting Agent 5,6-Dimethylxanthenone-4-Acetic Acid, Journal of Immunology, 190:5216-25 and Kim et al. (2013) Anticancer Flavonoids are Mouse-Selective STING Agonists, 8: 1396-1401). Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), poly(I:C) (e.g., polyi:CI2U), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175. These may act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be easily determined by those skilled in the art without undue experimentation. Further adjuvants include colony stimulating factors such as granulocyte macrophage colony stimulating factor (GM-CSF, sarlamostim).

[0323] Poly-ICLC is a synthetically prepared double-stranded RNA consisting of poly-I and poly-C strands with an average length of approximately 5,000 nucleotides, stabilized against heat denaturation and hydrolysis by serum nucleases by the addition of polylysine and carboxymethylcellulose. This compound activates the RNA helicase domains of TLR3 and MDA5, both members of the PAMP family, leading to the activation of DCs and natural killer (NK) cells and the production of a "natural mixture" of type I interferons, cytokines, and chemokines. Furthermore, poly-ICLC exerts more direct and broader host-targeted anti-infective and, potentially, anti-tumor effects mediated by two IFN-inducible nuclear enzyme systems, 2'5'-OAS and Pl / eIF2a kinase (also known as PKR(4-6)), as well as RIG-I helicase and MDA5.

[0324] Such methods include, but are not limited to, producing sHDL nanoparticles in association with compositions comprising agents (e.g., DAMPs / PAMPs), antigens, and adjuvants (e.g., dendritic cell targeting molecules) that can stimulate an innate immune response in a subject upon administration to the subject. In some embodiments, the antigens and adjuvants are attached to the outer surface of the sHDL nanoparticles.

[0325] In some embodiments, sHDL nanoparticles are synthesized with thiol-reactive phospholipids, which allow reduction-sensitive conjugation of antigens and / or adjuvants. In some embodiments, loading of DCs into sHDL nanoparticles is facilitated by cholesterol modification of the DC molecules. In some embodiments, a lyophilization method is used to prepare homogeneous sHDL. In some embodiments, phospholipids and ApoA-mimetic peptides are dissolved in glacial acetic acid and lyophilized. In some embodiments, antigenic peptides are incubated with sHDL in a buffer (e.g., sodium phosphate buffer (pH 7.4)) (e.g., at room temperature for 3 hours) to allow conjugation of the antigenic peptide. In some embodiments, unbound antigenic peptides are removed using a desalting column (MWCO = 7000 Da). In some embodiments, incorporation of cholesterol-modified DCs (Cho-DCs) into sHDL involves incubation with sHDL at room temperature for approximately 30 minutes.

[0326] Such embodiments are not limited to a particular technique for characterizing sHDL bound to antigen and DCs. In some embodiments, the morphology of sHDL is observed by TEM. In some embodiments, the size distribution of sHDL is analyzed by dynamic light scattering (DLS) using a Malven Nanosizer instrument and a GPC assay.

[0327] sHDL nanoparticles (e.g., sHDL-αGalCer) configured to activate an immune response (e.g., Ag / DC-sHDL) are useful for activating T cells in subjects for prophylactic and therapeutic applications. Activation of T cells with the nanoparticle vaccine composition increases their proliferation, cytokine production, differentiation, effector function, and / or survival. Methods for measuring these are well known to those skilled in the art. T cells activated by the nanoparticle vaccine composition can be any cells that express T cell receptors, including α / β and γ / δ T cell receptors. T cells include all cells that express CD3, including T cell subsets that also express CD4 and CD8. T cells include both naive and memory cells, as well as effector cells such as CTLs. T cells also include regulatory cells such as Th1, Tc1, Th2, Tc2, Th3, Treg, and Tr1 cells. T cells also include NKT cells and similar distinct classes of T cell lineages. In some embodiments, the activated T cells are CD8+ T cells.

[0328] In general, compositions comprising sHDL nanoparticles (e.g., sHDL-STING agonist-αGalCer) configured to activate an immune response (e.g., Ag / DC-STING agonist-sHDL) are useful for treating subjects with or predisposed to any disease or disorder against which their own immune system mounts an immune response. The compositions are useful as prophylactic vaccines, which confer resistance to a subject against subsequent exposure to an infectious agent. The compositions are also useful as therapeutic vaccines that can be used to initiate or enhance a subject's immune response to pre-existing antigens, such as tumor antigens in subjects with cancer or viral antigens in subjects infected with a virus. The compositions are also useful as desensitization vaccines, which function to "tolerize" an individual to environmental antigens, such as allergens.

[0329] The ability of these compositions to target professional antigen-presenting cells, such as dendritic cells, and to induce T cell-mediated immune responses by causing cross-presentation of antigens makes them particularly useful for eliciting cell-mediated responses against disease-associated antigens to attack disease. Thus, in some embodiments, the type of disease to be treated or prevented is a malignancy or chronic infection caused by bacteria, viruses, protozoa, helminths, or other microbial pathogens that invade intracellularly, i.e., are attacked by cytotoxic T lymphocytes.

[0330] The desired outcome of a preventive, therapeutic, or desensitized immune response can vary depending on the disease, according to principles well known in the art. For example, an immune response against an infectious agent can completely prevent the colonization and replication of the infectious agent, resulting in "sterile immunity" and the absence of disease symptoms. However, a vaccine against an infectious agent can be considered effective if it reduces the number, severity, or duration of symptoms; reduces the number of individuals in a population with symptoms; or reduces the transmission of the infectious agent. Similarly, an immune response against cancer, allergens, or infectious agents can completely cure a disease, alleviate symptoms, or be an aspect of overall therapeutic intervention against a disease. For example, stimulating an immune response against cancer can be combined with surgical, chemotherapy, radiological, hormonal, and other immunological approaches to affect treatment.

[0331] Subjects with or exposed to infectious agents can be treated therapeutically or prophylactically with sHDL nanoparticles (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL) configured to activate an immune response as disclosed herein. Infectious agents include bacteria, viruses, and parasites. In some cases, subjects can be treated prophylactically, for example, if they may be at risk of developing a disease from an infectious agent. Individuals who travel to or live in areas with endemic infectious diseases may be considered at risk and may be candidates for vaccination against a particular infectious agent. Prophylactic treatment can be applied to any number of diseases where there is a known relationship between the particular disease and specific risk factors, such as geographic location or work environment.

[0332] Subjects with malignant tumors or at risk of developing malignant tumors can be treated therapeutically or prophylactically with sHDL nanoparticles (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL) configured to activate an immune response as disclosed herein. In adult animals, a balance between cell renewal and cell death is normally maintained in most organs and tissues. Various types of mature cells in the body have a given lifespan. When these cells die, new cells are generated by the proliferation and differentiation of various types of stem cells. Under normal circumstances, the production of new cells is regulated so that the number of cells of any particular type remains constant. However, occasionally, cells arise that no longer respond to normal growth control mechanisms. These cells expand to a considerable size and give rise to clones of cells capable of producing tumors or neoplasms. Tumors that are unable to grow indefinitely and do not extensively invade healthy surrounding tissue are benign. A tumor that continues to grow and progresses to become invasive is malignant. The term cancer specifically refers to a malignant tumor. In addition to uncontrolled growth, malignant tumors exhibit metastasis. In this process, small clusters of cancer cells migrate from the tumor, invade the blood or lymphatic vessels, and are transported to other tissues where they continue to grow. In this way, a primary tumor at one site can give rise to a secondary tumor at another site. sHDL nanoparticles configured to activate an immune response (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL) as disclosed herein are useful for treating subjects with malignant tumors.

[0333] Malignant tumors that can be treated are classified herein according to the embryonic origin of the tissue from which they originate. Carcinomas are tumors that arise from endodermal or ectodermal tissues, such as the skin or the epithelial lining of internal organs and glands. Melanoma is a type of carcinoma of the skin for which the present invention is particularly useful. Less frequent sarcomas originate from mesodermal connective tissues, such as bone, fat, and cartilage. Leukemia and lymphoma are malignant tumors of the hematopoietic cells of the bone marrow. Leukemias grow as single cells, while lymphomas tend to grow as tumor masses. Malignant tumors can appear and establish cancer in multiple organs or tissues of the body.

[0334] Cancer types that can be treated with sHDL nanoparticles (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL) configured and provided to activate an immune response include, but are not limited to, bladder, brain, breast, cervix, colorectal, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, etc. Administration is not limited to treating existing tumors or infections, but can also be used to prevent or reduce the risk of developing such diseases in individuals, i.e., for prophylactic use. Potential candidates for prophylactic vaccination include individuals at high risk of developing cancer, i.e., with a personal or family history of a particular type of cancer.

[0335] Subjects with allergens or at risk of exposure to allergens can be treated therapeutically or prophylactically with sHDL nanoparticles (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL) configured to activate an immune response as disclosed herein. Such sHDL nanoparticles can be administered to subjects to prevent and / or reduce allergic reactions, such as those leading to anaphylaxis. Allergic reactions can be characterized by a TH2 response to an antigen leading to the presence of IgE antibodies. Stimulation of a TH1 immune response and the production of IgG antibodies can alleviate allergic diseases. Thus, sHDL nanoparticles configured to activate an immune response (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL) as disclosed herein are useful for producing antibodies that prevent and / or reduce allergic reactions in subjects exposed to allergens.

[0336] Subjects with or at risk for immunosuppressive conditions can be treated therapeutically or prophylactically with sHDL nanoparticles (e.g., sHDL-STING agonist-αGalCer) configured to activate an immune response (e.g., Ag / DC-STING agonist-sHDL) as disclosed herein. The sHDL nanoparticle vaccines disclosed herein can be used for the treatment of conditions characterized by immunosuppression, including, but not limited to, AIDS or AIDS-related syndromes, idiopathic immunosuppression, drug-induced immunosuppression, other viral or environmentally induced conditions, and certain congenital immune deficiencies. Such sHDL nanoparticle vaccine compositions can also be used to increase immune function impaired by the use of immunosuppressants (e.g., certain chemotherapeutic agents) or radiation therapy. Thus, the sHDL nanoparticle vaccine compositions can be particularly useful when used in conjunction with such drugs or radiation therapy.

[0337] Generally, methods of administering vaccines as disclosed herein (e.g., sHDL nanoparticles (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL) configured to activate an immune response) are well known in the art. Any acceptable method known to one of skill in the art can be used to administer the formulation to a subject. Administration can be local (i.e., administration to a specific area, physiological system, tissue, organ, or cell type) or systemic. Vaccines can be administered by a number of routes, including, but not limited to, oral, inhalation (intranasal or pulmonary), intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means. Injections can be, for example, intravenous, intradermal, subcutaneous, intramuscular, or intraperitoneal. In some embodiments, injections can be administered at multiple locations.

[0338] The administration of the formulation can be achieved by any acceptable method that allows an effective amount of vaccine to reach the target. The specific mode selected will depend on factors such as the specific formulation, the severity of the condition of the subject being treated, and the dosage required to induce an effective immune response. As generally used herein, "effective amount" refers to the amount that can induce an immune response in the treated subject. The actual effective amount of the vaccine may vary depending on the specific antigen or combinations used, the specific composition formulated, the mode of administration, and the age, weight, condition, and route of administration and disease or disorder of the individual being vaccinated.

[0339] In certain embodiments, glycolipids encapsulated within sHDL nanoparticles are used as stimulators of natural killer T cell-mediated immune responses.

[0340] Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. Many of these cells recognize non-polymorphic CD1d molecules, antigen-presenting molecules that bind to self and foreign lipids and glycolipids. NKT cells comprise only approximately 0.1% of all peripheral blood T cells. NKT cells are a subset of T cells that co-express the αβ T cell receptor but also express various molecular markers typically associated with NK cells, such as NK1.1. The best-known NKT cells differ from conventional αβ T cells in that they have a much more limited diversity of T cell receptors ("invariant" or "type 1" NKT). They and other CD1d-restricted T cells ("type 2" NKT) recognize lipids and glycolipids presented by CD1d molecules, members of the CD1 family of antigen-presenting molecules, rather than peptide major histocompatibility complex (MHC). NKT cells express NK1.1 + and NK1.1 - Both CD4 + , CD4 - , CD8 + , and CD8 - Contains cells.

[0341] In certain embodiments, a composition comprising an agent (e.g., a DAMP / PAMP) capable of stimulating an innate immune response in a subject upon administration to the subject is further associated (e.g., by complexing, binding, encapsulating, absorbing, adsorbing, or mixing) with one or more therapeutic agents. Such embodiments are not limited to a particular type or variety of therapeutic agents.

[0342] In some embodiments, the therapeutic agent is configured to treat and / or prevent cancer. Examples of such therapeutic agents include, but are not limited to, chemotherapeutic agents, anti-tumor agents, anti-angiogenic agents, tumor suppressors, anti-microbial agents, etc.

[0343] In some embodiments, the therapeutic agent is configured to treat and / or prevent an autoimmune and / or inflammatory disorder. Examples of such therapeutic agents include, but are not limited to, disease-modifying antirheumatic drugs (e.g., leflunomide, methotrexate, sulfasalazine, hydroxychloroquine), biologics (e.g., rituximab, infliximab, etanercept, adalimumab, golimumab), nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, celecoxib, ketoprofen, naproxen, piroxicam, diclofenac), analgesics (e.g., acetaminophen, tramadol), immunomodulatory agents (e.g., anakinra, abatacept), glucocorticoids (e.g., prednisone, methylprednisone), TNF-α inhibitors (e.g., adalimumab, certolizumab pegol, etanercept, golimumab, infliximab), IL-1 inhibitors, and metalloprotease inhibitors. In some embodiments, therapeutic agents include but are not limited to infliximab, adalimumab, etanercept, parenteral gold, or oral gold.

[0344] In some embodiments, the therapeutic agent is configured for treating and / or preventing cardiovascular-related disorders (e.g., atherosclerosis, heart failure, arrhythmia, atrial fibrillation, hypertension, coronary artery disease, angina pectoris, etc.). Examples of therapeutic agents known to be useful for treating and / or preventing cardiovascular-related disorders include angiotensin-converting enzyme (ACE) inhibitors (e.g., benazepril, enalapril, lisinopril, perindopril, ramipril), adenosine, alpha blockers (alpha adrenergic antagonist drugs) (e.g., clonidine, guanabenz, labetalol, phenoxybenzamine, terazosin, doxazosin, guanfacine, methyldopa, prazosin), angiotensin II receptor inhibitors, and the like. Anticoagulants (ARBs) (e.g., candesartan, irbesartan, olmesartan medoxomil, telmisartan, eprosartan, losartan, tasosartan, valsartan), anticoagulants (e.g., heparin fondaparinux, warfarin, ardeparin, enoxaparin, reviparin, dalteparin, nadroparin, tinzaparin), antiplatelet agents (e.g., abciximab, clopidogrel, eptifibatide, ticlopidine, cilostazol, dipyridamole, sulfinpyrazone, tirofiba) beta-blockers (e.g., acebutolol, betaxolol, carteolol, metoprolol, penbutolol, propranolol, atenolol, bisoprolol, esmolol, nadolol, pindolol, timolol), calcium channel blockers (e.g., amlopidine, felodipine, isradipine, nifedipine, verapamil, diltiazem, nicardipine, nimodipine, nisoldipine), diuretics, aldosterone inhibitors, loop diuretics (e.g., bumetanide, nide, furosemide, ethacrynic acid, torasemide), potassium-sparing diuretics, thiazide diuretics (e.g., chlorothiazide, chlorthalidone, hydrochlorothiazide, hydroflumeazide, methyclothiazide, metolazone, polythiazide, quinatazone, trichlormethiazide), cardiac inotropes, bile acid sequestrants (e.g., cholestyramine, coletipol, colesevelam), fibrates (e.g., clofibrate, gemfibrozil, fenofibrate), statins (e.g.,Atorvastatin, lovastatin, simvastatin, fluvastatin, pravastatin), selective cholesterol absorption inhibitors (e.g., ezetimibe), potassium channel inhibitors (e.g., amidarone, ibutilide, dofetilide), sodium channel inhibitors (e.g., disopyramide, mexiletine, procainamide, quinidine, flecainide, moricizine, propafenone), thrombolytic agents (e.g., alteplase, reteplase, tenecteplase, anistreplase, streptokinase, urokinase), vasoconstrictors, vasodilators (e.g., hydralazine, minoxidil, mecamylamine, isorbidiol dintrate, isorbidiol mononitrate, nitroglycerin).

[0345] Generally, the nanoparticles so formed are spherical and have a diameter of about 5 nm to about 20 nm (e.g., 4-75 nm, 4-60 nm, 4-50 nm, 4-22 nm, 6-18 nm, 8-15 nm, 8-10 nm, etc.). In some embodiments, the sHDL nanoparticles are subjected to size exclusion chromatography to obtain a more homogeneous preparation.

[0346] In some embodiments, nanoparticles associated with a composition as described herein are further associated (e.g., by complexing, binding, encapsulation, absorption, adsorption, or mixing) with an agent useful for localizing the administered particle. Agents useful for this purpose include fluorescent tags, radionuclides, and imaging agents.

[0347] Suitable contrast agents include, but are not limited to, fluorescent molecules such as those described in Molecular Probes (Handbook of fluorescent probes and research products) (e.g., rhodamine, fluorescein, Texas Red, acridine orange, Alexa Fluor (various), allophycocyanin, 7-aminoactinomycin D, BOBO-1, BODIPY (various), Calcien, Calcium Crimson, Calcium green, Calcium Orange, 6-carboxyrhodamine 6G, Cascade blue, Cascade yellow, DAPI, DiA, DID, Di1, DiO, DiR, ELF 97, eosin, ER Tracker Blue-White,EthD-1, ethidium bromide, Fluo-3, Fluo-4, FM1-43, FM4-64, Fura-2, Fura Red, Hoechst 33258, Hoechst 33342, 7-hydroxy-4-methylcoumarin, Indo-1, JC-1, JC-9, JOE dye, Lissamine rhodamine B, Lucifer Yellow CH, LysoSensor Blue DND-167, LysoSensor Green, LysoSensor Yellow / Blu, Lysotracker Green FM, Magnesium Green, Marina Blue, Mitotracker Green FM, Mitotracker Orange CMTMRos, MitoTracker Red CMMXRos, Monobromobimane, NBD amines, NeruoTrace 500 / 525 green, Nile Red, Oregon Green, Pacific Blue, POP-1, propidium iodide, rhodamine 110, rhodamine red, R-phycoerythrin, Resorfin, RH414, Rhodamine-2, rhodamine green, rhodamine 123, ROX dye, Sodium Green, SYTO blue (various), SYTO green (various), SYTO orange (various), SYTOX blue, SYTOX green, SYTOX orange, tetramethylrhodamine B, TOT-1, TOT-3, X-rhod-1, YOYO-1, and YOYO-3. In some embodiments, ceramide is provided as an imaging agent. In some embodiments, an S1P agonist is provided as an imaging agent.

[0348] Additionally, radionuclides can be used as imaging agents. Suitable radionuclides include, but are not limited to, Fe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(II), indium, gallium, and technetium radioactive species. Other suitable imaging agents include metal ions commonly used for chelation in paramagnetic T1-type MIR contrast agents, including divalent and trivalent cations such as copper, chromium, iron, gadolinium, manganese, erbium, europium, dysprosium, and holmium. Metal ions that can be chelated and used for radionuclide imaging include, but are not limited to, metals such as gallium, germanium, cobalt, calcium, indium, iridium, rubidium, yttrium, ruthenium, yttrium, technetium, rhenium, platinum, thallium, and samarium. Additionally, metal ions known to be useful in neutron capture radiotherapy include boron and other metals with large nuclear cross sections. Additionally, metal ions useful in ultrasound and x-ray contrast compositions are also suitable.

[0349] Other examples of suitable contrast agents include radiopaque gases or gas-releasing compounds.

[0350] In some embodiments, nanoparticles associated with a composition as described herein are further associated with a targeting agent (e.g., by complexing, binding, encapsulation, absorption, adsorption, or mixing). In some embodiments, a targeting agent is used to aid in the delivery of nanoparticles associated with a composition as described herein to a desired body region (e.g., a body region suffering from a cardiovascular-related disorder). Examples of targeting agents include, but are not limited to, antibodies, receptor ligands, hormones, vitamins, and antigens, and the present invention is not limited by the nature of the targeting agent. In some embodiments, antibodies are specific for disease-specific antigens. In some embodiments, receptor ligands include, but are not limited to, ligands for CFTR, EGFR, estrogen receptor, FGR2, folate receptor, IL-2 receptor, glycoproteins, and VEGFR. In some embodiments, the receptor ligand is folate.

[0351] In some embodiments, nanoparticles associated with the compositions described herein can be delivered to a localized site in a patient by a medical device. Medical devices suitable for use in the present invention include known devices for localized delivery of therapeutic agents. Such devices include, but are not limited to, catheters such as injection catheters, balloon catheters, double-balloon catheters, microporous balloon catheters, channel balloon catheters, infusion catheters, and perfusion catheters (which may be coated with a therapeutic agent or through which a drug is administered); needle injection devices such as hypodermic needles and needle injection catheters; needleless injection devices such as jet injectors; coated stents, bifurcated stents, artificial blood vessels, stent grafts, and the like; and coated vascular occlusion devices such as wire coils.

[0352] Exemplary devices are described in U.S. Patent Nos. 5,935,114; 5,908,413; 5,792,105; 5,693,014; 5,674,192; 5,876,445; 5,913,894; 5,868,719; 5,851,228; 5,843,089; and 5,800,519. Nos. 5,800,508; 5,800,391; 5,354,308; 5,755,722; 5,733,303; 5,866,561; 5,857,998; 5,843,003; and 5,933,145, the entire contents of which are incorporated herein by reference. Exemplary stents that are commercially available and can be used in the present application include the RADIUS (SCIMED LIFE SYSTEMS, Inc.), the SYMPHONY (Boston Scientific Corporation), the Wallstent (Schneider Inc.), the PRECEDENT II (Boston Scientific Corporation), and NIR (Medinol Inc.). Such devices are delivered and / or implanted to target locations within the body using known techniques.

[0353] In some embodiments, the present invention also provides kits that include the compositions described herein. In some embodiments, the kits include one or more of the reagents and tools necessary to produce such compositions, as well as methods for using such compositions.

[0354] Nanoparticles associated with the compositions described herein can be characterized for size and uniformity by any suitable analytical technique, including, but not limited to, atomic force microscopy (AFM), electrospray ionization mass spectrometry, MALDI-TOF mass spectrometry, C-nuclear magnetic resonance spectroscopy, high-performance liquid chromatography (HPLC), size-exclusion chromatography (SEC) (equipped with multi-angle laser light scattering, dual UV, and refractive index detectors), capillary electrophoresis, and gel electrophoresis. These analytical methods ensure the uniformity of the sHDL nanoparticle population, which is important for manufacturing quality control for ultimate use in in vivo applications.

[0355] In some embodiments, gel permeation chromatography (GPC), which can separate sHDL nanoparticles from liposomes and free ApoA-I mimetic peptides, is used to analyze sHDL-TA nanoparticles. In some embodiments, size distribution and zeta potential are measured by dynamic light scattering (DLS), for example, using a Malven Nanosizer instrument.

[0356] When clinical use is contemplated, in some embodiments of the present invention, the sHDL nanoparticles are prepared as part of a pharmaceutical composition in a form suitable for the intended use. Generally, this requires the preparation of a composition that is essentially free of pyrogens and other impurities that may be harmful to humans or animals. However, in some embodiments of the present invention, the intact sHDL nanoparticle formulation can be administered using one or more of the routes described herein.

[0357] In a preferred embodiment, nanoparticles associated with the compositions described herein are used with appropriate salts and buffers to ensure stable delivery of the compositions, enabling uptake by target cells. A buffer is also used when sHDL nanoparticles are introduced into a patient. Aqueous compositions contain a cell-effective amount of sHDL nanoparticles dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions are also referred to as inocula. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

[0358] In some embodiments of the present invention, the active composition comprises a typical pharmaceutical formulation. These compositions according to the present invention can be administered via any common route, as long as the target tissue can be reached via that route. This includes oral, nasal, buccal, rectal, vaginal, or topical routes. Alternatively, administration can be via orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection.

[0359] The active nanoparticles associated with the composition as described herein can be administered parenterally or intraperitoneally or intratumorally.The solution of the active compound as a free base or pharmacologically acceptable salt is prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and their mixtures, and in oil.Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0360] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it may be preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0361] Sterile injection solution can be prepared by incorporating the required amount of active nanoparticles in association with the composition as described herein in suitable solvent with various other components as listed above as needed, and then sterilized by filtration.Generally, dispersion solution can be prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains basic dispersion medium and other components as listed above that are required.For the sterile powder that can be used to prepare sterile injection solution, the preferred method is vacuum drying and freeze-drying technology, which produces the powder of active ingredient and any additional desired components from the solution that has been previously sterilized and filtered.

[0362] Once formulated, the nanoparticles associated with the compositions described herein are administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug-release capsules, and the like. For parenteral administration in aqueous solution, for example, the solution is suitably buffered, and if necessary, the liquid diluent is first made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. For example, a single dosage can be dissolved in 1 ml of isotonic NaCl solution and then added to 1000 ml of subcutaneous infusion solution or injected at the proposed infusion site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). In some embodiments of the invention, the active particles or agent are formulated within the therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0, or about 10 milligrams per dose. Multiple doses may be administered.

[0363] Additional formulations suitable for other modes of administration include vaginal suppositories and pessaries. Rectal pessaries or suppositories may also be used. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina, or urethra. After insertion, the suppository softens, melts, or dissolves in the fluids of the body cavity. In general, for suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing 0.5% to 10%, preferably 1% to 2%, of the active ingredient. Vaginal suppositories or pessaries are usually spherical or oval, each weighing approximately 5 g. Vaginal medications are available in a variety of physical forms (e.g., creams, gels, or liquids) that depart from the typical concept of a suppository. sHDL nanoparticles may also be formulated as inhalants.

[0364] The present invention also encompasses methods comprising co-administering nanoparticles associated with a composition as described herein with one or more additional active agents. Indeed, it is a further aspect of the present invention to provide a method for enhancing prior art therapies and / or pharmaceutical compositions by co-administering the sHDL nanoparticles of the present invention. In co-administration procedures, the agents can be administered simultaneously or sequentially. In some embodiments, the sHDL nanoparticles associated with a composition as described herein are administered before the other active agent(s). The agent(s) to be co-administered will depend on the type of condition being treated.

[0365] The present disclosure further provides a composition comprising nanoparticles in association with the composition as described herein, or a kit comprising the components necessary to synthesize nanoparticles as described herein. In some embodiments, the kit contains all of the components necessary, sufficient, or useful for administering nanoparticles in association with the composition as described herein.

[0366] [Example] The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting its scope. As used herein, the terms "I," "We," "Our," and similar terms refer to the inventors.

[0367] Example 1 This example describes the synthesis and characterization of CDN / Zn, CDN / Zn@liposome NPs, and CDN@CaP / PEI-PEG.

[0368] As shown in Figure 1A, CDN-Zn NPs were prepared by simple coordination assembly. Zn, with its pyramidal coordination geometry, is assumed to be able to coordinate with both adenine and phosphate. To further enhance the stability of the resulting particles, CDN / Zn nanoparticles were modified with liposomes. There are several different methods for MOF surface modification, including coordination adjustment during MOF synthesis and post-synthesis modification by ligand exchange and silica or polymer shell coating. DOPA was added to the Zn during synthesis. 2+ Since it has been widely used to cap the base MOFs, here the coordination adjustment was applied for the synthesis of CDN / Zn@DOPA with lipid tails on the surface, which allowed for another lipid layer coating.

[0369] The morphology of the obtained CDN-Zn and CDN / Zn@liposome NPs is shown in TEM images (Figure 2). As shown in Figure 2A, the cdAMP-Zn NPs exhibited a spherical shape with higher TEM contrast at the surface. The fast nucleation of cdAMP-Zn in methanol resulted in the formation of Zn. 2+ Although this caused a lack of coordination, the particle surface was Zn 2+This is thought to have a saturated coordination of , increasing the surface contrast and resulting in a "core-shell"-like organization. Because nucleation occurs more slowly in water, we also found that synthesis in aqueous media resulted in uniform spherical structures (not shown). Consistent with TEM images, DLS and zeta potential data indicated that cdAMP-Zn was approximately 150 nm in size and had a neutral surface charge. As shown in Figure 2B, under the same synthesis conditions, cd-GMP NPs exhibited a uniform, irregular spherical structure approximately 100 nm in size and an electrically neutral surface charge. In contrast to cdAMP-Zn and cdGMP-Zn, the morphology and charge of cGAMP-Zn were different (Figure 2C). The spherical nanoparticles consisted of several accumulated small clusters, and the surface possessed a slight positive charge. To enhance the stability of CDN-Zn NPs, we modified CDN-Zn with liposomes. As shown in Figure 2D, cdAMP-Zn@liposomes were shown as a representative CDN-Zn@liposome structure. The results showed that CDN-Zn@liposomes exhibited more uniform and smaller sizes due to the DOPA capping effect, and their surfaces also showed a slight negative charge after liposome-PEG modification.

[0370] For the CaP / PEI-PEG formulation, we started with the clinically used adjuvant CaP hydrogel. 2+ and PO4 3- The CDN@CaP / PEI-PEG NPs were prepared by high-speed mixing with ethanol, resulting in the formation of needle-like nanostructures. To increase the loading of CDN into the CaP hydrogel, PEI-PEG was added to the CDN to increase the charge attraction to the CDN, thereby simultaneously increasing colloidal stability (Figure 1B). Unlike conventional CaP hydrogels, which tend to aggregate into gels, the CaP / PEI-PEG NPs dispersed well in water. As shown in Figure 2E, the CDN@CaP / PEI-PEG NPs exhibited a uniform needle-like cluster structure with a size of approximately 70 nm and a surface charge of approximately +15 mV. Based on their morphology, size, and surface properties, all of the formulations presented here have great potential for drug delivery applications.

[0371] Example 2 This example demonstrates the release profiles and in vitro STING activation of CDN-Zn and CDN@CaP / PEI-PEG.

[0372] As two important parameters of a drug delivery system, the drug loading and release characteristics of the CDN nanoformulations were further determined experimentally. The CDN loading efficiency in the nanoformulations exceeded 90% for the CDN-Zn formulation and 80% for the CDN / CaP-PEI-PEG formulation (Figure 3A). Regarding drug release, cdAMP / Zn and cdGMP / Zn showed similar release profiles (Figure 3B). For the first 18 h, release was near zero-order, followed by a slightly slower release phase. The zero-order drug release from cdAMP / Zn and cdGMP / Zn was likely the result of stable, constant dissociation of the framework. However, further studies under physiological conditions with different biomolecular interactions are required. For cGAMP / Zn NPs, a fast release phase was observed during the first 8 h of incubation, followed by a slower release phase (Figure 3B). The total release of cGAMP / Zn was faster than that of cdAMP / Zn and cdGMP / Zn, which may be related to its unique nanoparticle configuration. For CDN@CAP / PEI-PEG, there was a significantly rapid drug release phase, followed by another sustained release phase (Figure 3B). This profile is likely due to the CDN moiety attaching to the CAP / PEI-PEG surface through charge interactions and being readily released under high ionic strength and high pH conditions. The release profile of CDN-Zn@liposomes was not shown here because we have not developed a reliable method to quantify the drug loading after liposome coating on CDN-Zn. Liposomes on the CDN-Zn surface are expected to significantly increase particle stability and delay drug release. Sustained drug release helps increase in situ drug exposure and the degree of immune stimulation.

[0373] We performed an experiment to determine whether the CDN delivery system could effectively activate the STING pathway and induce immune responses in vitro. We transfected THP1-Blue cells with an IFN regulatory factor (IRF)-inducible SEAP reporter construct. TM Interferon-stimulated gene (ISG) cells were used in the experiments to monitor the activation of STING by the CDN formulation. As shown in Figure 3C, at 0.25–2 μg / ml of cdAMP, the degree of activation of the IFN signaling pathway by the cdAMP / Zn formulation was much higher than that of the soluble form of free cdAMP. A similar enhancement of stimulation was observed for the CDN@CaP / PEI-PEG formulation compared to the free form (Figure 3D). These in vitro evaluation results indicate that CDN-Zn and CDN@CaP / PEI-PEG have advantageous properties for in vivo therapeutic applications.

[0374] Example 3 This example describes the therapeutic effects of CDN-Zn and CDN@CaP / PEI-PEG.

[0375] Finally, we investigated the therapeutic effect of the CDN formulation on tumor-bearing mice. cdAMP(ps)2 was used as a representative CDN for demonstration. The tumor size was approximately 60 mm. 3When the tumor size reached 100 μg / dose, two doses of 25 μg / dose of cdAMP(ps)2 were administered intratumorally on days 10 and 15. To evaluate antigen-specific immune responses, PBMCs were collected for tetramer staining on day 17, and ELISPOT analysis was performed using the AH1 antigen peptide on day 22. As shown in Figure 4A, the mean tumor growth in mice treated with free CDN, CDN-Zn, and CDN@CaP / PEI-PEG was significantly delayed compared to the untreated group. Compared with CDN and CDN@CaP / PEI-PEG, CDN-Zn appeared to inhibit tumor growth better, but there was no statistical difference between them. Regarding mouse survival after treatment, the median survival times for the untreated, CDN, CDN-Zn, and CDN@CaP / PEI-PEG groups were 23 days, 42 days, 64 days, and not yet reached, respectively (Figure 4B). From the individual tumor growth curves (Figure 4C), complete tumor regression was observed in 0 of 5 mice in the untreated group; 2 of 5 mice in the free CDN and CDN-Zn groups; and 3 of 5 mice in the CDN@CaP / PEI-PEG group.

[0376] No significant differences were observed between the groups in the PBMC tetramer staining assay (Figure 4D). It is possible that PBMC tetramer staining was not sensitive enough to demonstrate antigen-specific T cell responses after nonspecific intratumoral CDN stimulation, or that the time point was not optimal. In contrast, ELISPOT evaluation on day 22 demonstrated significant antigen-specific immune responses (Figures 4E-4F). Seven days after the second CDN administration, significant AH1 antigen-specific T cell responses were observed in the free CDN, CDN-Zn, and CDN@CaP / PEI-PEG groups. The responses of CDN-Zn and CDN@CaP / PEI-PEG were also higher than those of free CDN, and statistical differences were observed between free CDN and CDN@CaP / PEI-PEG. Overall, these results demonstrated that the therapeutic activity of both CDN-Zn and CDN@CaP / PEI-PEG was comparable to or even better than that of free CDN. The therapeutic benefit of this formulation stems from the combined effects of slow release and increased cellular uptake. Based on this advantage, the present CDN-Zn@liposomes exhibit improved therapeutic efficacy due to more sustained release and improved in vivo stability.

[0377] Example 4 This example describes the materials and methods of Examples 1, 2, and 3.

[0378] Synthesis of CDN-Zn nanoparticles (NPs) cGAMP, cdAMP, and cdGMP were obtained from Invivogen, and cdAMP(ps)2 was obtained from MedchemExpress. CDN was dissolved in methanol before use. Meanwhile, ZnCl2 (Sigma-Aldrich) was dissolved in methanol to prepare a 100 mM stock solution. In a typical synthesis reaction, 10:1 (n / n) Zn 2+ The solution was added to a 1 mg / ml CDN working solution under vigorous stirring. The solution was stirred for an additional 24 hours at room temperature. The resulting CDN-Zn NPs were centrifuged at 20,000 × g for 15 minutes to separate the free CDN and Zn. 2+ was removed, followed by another wash with methanol.

[0379] Synthesis of CDN-Zn@liposomes CDN-Zn@liposomes were synthesized using two steps. First, CDN-Zn@DOPA NPs were synthesized by the coordination-modulation method. Briefly, a 10 molar ratio of Zn 2+ The solution was added to CDN / DOPA (Avanti Lipids) in chloroform under vigorous stirring. After 24 h of incubation, the CDN-Zn@DOPA NPs were isolated by centrifugation at 20,000 × g for 15 min. The CDN-Zn@DOPA NPs were then resuspended in a THF solution of DOPC, cholesterol, and DSPE-PEG2k (2:2:1, Avanti Lipids) and added to a solution of 30% (v / v) ethanol / HO at 60 °C. Finally, the THF was evaporated under reduced pressure, the final solution was cooled to room temperature, and the empty liposomes were removed by centrifugation at 20,000 × g for 20 min to obtain CDN-Zn@liposomes. The resulting CDN-Zn@liposomes were then resuspended in PBS for further use.

[0380] Synthesis of CDN@CaP / PEI-PEG NPs CDN@CaP / PEI-PEG NPs were prepared by a one-step precipitation method. Briefly, solutions of CaCl2 (Sigma-Aldrich) and Na2HPO4 (Sigma-Aldrich) were simultaneously added to the PEI-PEG and CDN mixture under continuous stirring. After overnight incubation, the CDN@CaP / PEI-PEG NPs were isolated by centrifugation at 18,000 × g for 15 min. The resulting NPs were washed twice with histidine buffer (pH 7.4).

[0381] In vitro release analysis The release profiles of CDN-Zn and CDN-Zn@liposomes were measured using a Slide-A-Lyzer TMThe devices were tested using a mini dialysis device with a 3.5K MWCO (Thermo Scientific). Briefly, 0.5 ml of CDN-Zn or CDN-Zn@liposomes was loaded into a regenerated cellulose membrane cup, and 14 ml of release buffer (PBS) was placed in a test tube. The dialysis cup was inserted into a conical tube and capped, after which the device was incubated at 37 °C with continuous shaking (200 rpm). When required, 300 μl of release medium was withdrawn and refilled with an equal volume of fresh PBS. The CDN concentration in the release medium was analyzed by HPLC (GPC). Finally, the release rate was calculated based on the CDN concentration in the release buffer, the buffer volume, and the total CDN loading.

[0382] Assessment of interferon-stimulated gene activation THP1-Blue purchased from Invivogen TM ISG (interferon stimulated gene) cells were handled and cultured according to the manufacturer's instructions. Briefly, cells were thawed immediately upon receipt and cultured in 25 cm of 5 ml of growth medium. 2 After one passage, the cells were maintained in growth medium and selective antibiotic was added every other passage, with 7 × 10 5 The cells were passaged every 3 days at a starting cell concentration of 100,000 cells / ml. To evaluate the biological activity of the CDN formulations, 20 μl of prewarmed formulation solution was added to a 96-well flat-bottom plate. 180 μl of cell suspension (∼100,000 cells per well) was then mixed with the CDN sample. After 18 hours of incubation at 37°C and 5% CO2, 20 μl of supernatant was collected and incubated with 180 μl of QUANTI-Blue solution (Invivogen) for colorimetry. THP1 activation was quantified by measuring absorbance at 620–655 nm.

[0383] Animal experiments All animals were cared for in accordance with federal, state, and local guidelines. All research conducted on animals complied with and was approved by the University Committee on Use and Care of Animals (UCUCA). Six- to eight-week-old female Balb / c mice (Jackson Laboratories) were inoculated with 1 × 10 5 CT26 colon cancer cells were inoculated. Tumor size was ~100 mm. 3 When the tumor volume reached 100 μg, two doses of 25 μg cdAMP(ps)2 in various formulations were administered by intratumoral route on days 10 and 15. Tumor size and survival were monitored every 2 or 3 days. Tumor size was calculated based on the formula: volume = length × width. 2 Animals were euthanized when tumors reached 1.5 cm in diameter or when they became moribund due to severe weight loss or ulceration. On day 17, the percentage of tumor antigen-specific CD8α T cells in PBMCs was analyzed using a peptide-MHC tetramer (H-2Kb restricted AH1) (NIH Tetramer Core Facility, Atlanta, GA) in a tetramer staining assay as described above. On day 22, ELISPOT assays were performed using PBMCs from mice treated as described above.

[0384] Example 5 This example describes the materials and methods of Examples 6-11.

[0385] Screening for metal ions that modulate innate immune stimulators in vitro Murine bone marrow-derived dendritic cells (BMDCs) were isolated and cultured. Briefly, bone marrow stem cells were harvested and plated in bacteriological Petri dishes with GM-CSF-containing medium. The cell culture medium was refreshed on days 3, 6, and 8. After 10 days of differentiation, immature DCs were harvested for use. To screen for metal ions that can modulate the cytokine profile of innate immune stimulators, we first seeded 100,000 BMDCs / 100 μl into each well of a 96-well plate. Various concentrations of various metal ions were then added together with various concentrations of various innate immune stimulators. At the same time, the same concentrations of free metal ions alone or free innate immune stimulators alone were used as controls. After 24 hours of incubation at 37°C and 5% CO2, the supernatants were collected for ELISA assays of various cytokines.

[0386] Combination formulation of cyclic innate immune stimulator and metal ion coordination polymer CDN-metal ion coordination polymers: cGAMP, cdAMP, and cdGMP were obtained from Invivogen, and cdAMP(ps)2 was obtained from MedchemExpress. CDNs were dissolved in methanol or endotoxin-free water before use. Meanwhile, metal ions were dissolved in methanol or water to prepare 100 mM stock solutions. In a typical synthesis reaction, a 10:1 (n / n) metal ion solution was added to a 1 mg / ml CDN working solution with vigorous stirring. The solution was stirred for an additional 24 h at room temperature. The resulting CDN-metal mixture was centrifuged at 20,000 × g for 15 min to remove free CDN and metal ions, followed by another wash with methanol.

[0387] CDN-metal ion@liposome: CDN-metal@liposome was synthesized using two steps. Here, we take CDN-Zn@liposome as an example. First, Zn-CDN / H11-DOPE NPs were synthesized by the coordination-modulation method. Briefly, a 10 molar ratio of Zn 2+The solution was added to CDN / H11-DOPE (Avanti Lipids) in chloroform under vigorous stirring. After 24 h of incubation, the Zn-CDN / H11-DOPE NPs were isolated by centrifugation at 20,000 × g for 15 min. The Zn-CDN / H11-DOPE NPs were then resuspended in a THF solution of DPPC, cholesterol, and DSPE-PEG5k (2:2:1, Avanti Lipids) and added to a solution of 50% (v / v) ethanol / HO. Finally, the THF was evaporated under reduced pressure, the final solution was cooled to room temperature, and the empty liposomes were removed by centrifugation at 20,000 × g for 20 min to obtain CDN-Zn@liposomes. The resulting CDN-Zn@liposomes were then resuspended in PBS for further use.

[0388] Metal ion-CDN / polyhistidine-PEG nanocoordination polymers (NCPs): Metal ion-CDN / polyhistidine-PEG NCPs were prepared by a one-step precipitation method. 2+ Take the case of CoCl2 (Sigma-Aldrich), CDN, polyhistidine-PEG, and HEPES buffer solution as an example. Briefly, a fixed ratio solution of CoCl2 (Sigma-Aldrich), CDN, polyhistidine-PEG, and HEPES buffer was added dropwise to the mixture solution under continuous stirring. After 24 hours of incubation, the Co 2+ -CDN / polyhistidine-PEG nanoparticles (NPs) were separated using a 10 kD centrifugal ultrafilter to remove free metal ions and CDN.

[0389] CDN@CaP / PEI-PEG NPs: CDN@CaP / PEI-PEG NPs were prepared by a one-step precipitation method. Briefly, solutions of CaCl2 (Sigma-Aldrich) and Na2HPO4 (Sigma-Aldrich) were simultaneously added to a mixture of PEI-PEG and CDN under continuous stirring. After overnight incubation, the CDN@CaP / PEI-PEG NPs were isolated by centrifugation at 18,000 × g for 15 min. The resulting NPs were washed twice with histidine buffer (pH 7.4).

[0390] Innate immune stimulator-metal mineral@anionic polypeptide-PEG: Innate immune stimulator-metal mineral@anionic polypeptide-PEG was prepared by a one-step precipitation method. For example, MnP@PGA-PEG NPs were prepared by adding MnCl2 (Sigma-Aldrich) and Na2HPO4 (Sigma-Aldrich) solutions simultaneously to a mixed solution of PGA-PEG and innate immune stimulator with continuous stirring. After overnight incubation, the innate immune stimulator-MnP@PGA-PEG NPs were isolated by centrifugation at 18,000 × g for 15 min. The resulting NPs were washed twice with histidine buffer (pH 7.4).

[0391] In vitro release analysis The release profile of the formulation was analyzed using a Slide-A-Lyzer TM The device was tested using a MINI dialysis device with a 3.5K MWCO (Thermo Scientific). Briefly, 0.5 ml of the formulation solution was filled into a regenerated cellulose membrane cup, and 14 ml of release buffer (PBS) was placed in a test tube. The dialysis cup was inserted into a conical tube and capped, after which the device was incubated at 37°C with continuous shaking (200 rpm). When required, 300 μl of the release medium was withdrawn and refilled with an equal volume of fresh PBS. The CDN concentration in the release medium was analyzed by HPLC (GPC). Finally, the release rate was calculated based on the CDN concentration in the release buffer, the volume of the buffer, and the total CDN loading.

[0392] Animal experiments All animals were cared for in accordance with federal, state, and local guidelines. All research conducted on animals complied with and was approved by the University Committee on Use and Care of Animals (UCUCA). Six- to eight-week-old female Balb / c mice (Jackson Laboratories) were inoculated with 1 × 10 5 CT26 colon cancer cells were inoculated. Tumor size was ~50 mm. 3When the tumor volume reached 100 mg / kg, the indicated drug or formulation was administered by the indicated route. Tumor size and survival were monitored every 2 or 3 days. Tumor size was calculated based on the formula: volume = length x width. 2 Animals were euthanized when tumors reached 1.5 cm in diameter or when they became moribund due to severe weight loss or non-healing ulcer formation. On day 17, the percentage of tumor antigen-specific CD8α T cells in PBMCs was analyzed using a peptide-MHC tetramer (H-2Kb restricted AH1) (NIH Tetramer Core Facility, Atlanta, GA) in a tetramer staining assay as previously described. On day 22, ELISPOT assays were performed using PBMCs from mice treated as previously described.

[0393] Example 6 This example describes the identification of metal ions that can enhance STING activation of STING agonists.

[0394] As shown in Figures 5A and 5B, mouse bone marrow-derived dendritic cells (BMDCs) were treated with different metal ions or co-treated with different metal ions and a STING agonist. We selected metal ions from essential and trace mineral elements in biological systems. Mn 2+ Mn alone was able to activate BMDCs at highly toxic doses. 2+ When combined with a STING agonist, Co significantly enhanced STING activation at much lower concentrations. 2+ However, 125 μM or 250 μM Co 2+When combined with 5 μM cGAMP, it significantly enhanced STING pathway activation. Both concentrations were well tolerated. To further confirm whether this phenomenon also works in human cells, we repeated the same experiment using the human monocytic cell line THP1 (Figure 5C). A similar trend was observed in human THP1 cells, demonstrating that this phenomenon is independent of the type of STING agonist.

[0395] Example 7 In this example, Co 2+ and Mn 2+ demonstrate enhanced STING activation and anti-cancer therapeutic efficacy.

[0396] We investigated whether the in vitro enhanced type I IFN response could benefit cancer treatment in vivo. We evaluated the combination of metal ions and STING agonists in a mouse tumor model. As shown in Figures 6a and 6c, Co 2+ -CDA and Mn 2+ -CDA delayed tumor growth. Notably, the metal-CDA group had significantly more tumor-free mice than the free-CDA group, as evidenced by an 80% survival rate in the metal-CDA group compared with a 20% survival rate in the free-CDA group (Fig. 6d). Furthermore, Co 2+ -CDA treatment resulted in significantly higher serum IFN-beta levels 8 hours after injection compared with free CDA treatment (Fig. 6b). 2+ The same phenomenon was not observed with the -CDA combination.

[0397] Example 8 This example demonstrates an in vivo immune response to a combination of a STING agonist and a metal.

[0398] To investigate the mechanism of action of the improved cancer therapeutic effect, we evaluated treated animals for antigen-specific T cell responses and performed tumor rechallenge 81 days after the initial treatment. 2+showed a better T cell specific response as shown in the ELISPOT results on day 22 of the experiment, whereas the T cell ELISPOT results were lower than those of CDA-Co 2+ The CDA-Co group and the free CDA group showed similar results (Fig. 7b). 2+ and CDA-Mn 2+ Survivors in the treatment group completely prevented the growth of a second CT26 tumor. 2+ The treatment group showed a significant increase in antigen-specific T cell responses.

[0399] Example 9 This example demonstrates the identification of metal ions that may regulate other innate immune stimulators.

[0400] Based on our results regarding the STING pathway, we also evaluated whether metal ions could regulate other innate immune stimulators. We treated mouse BMDCs with different metal ions or combinations of different metal ions and innate immune stimulators. We observed similar metal ion-innate immune stimulator synergy. However, different metal ions exhibited synergistic effects with different DAMPs or PAMPs, including TLR3 / 4 / 7 / 8 / 9 ligands, NOD1 / 2 ligands, TLR7 / 8 ligands, RIG-I & CDS agonists, and inflammasome inducers. For example, Co 3+ dramatically increased Poly IC-induced IFNb, TNFa, IL6, and IL2 production, whereas Mn 2+ Mn only increased IFNb production by Poly IC (Figures 8A-8D). 2+ increased IFNb and TNFa production in MPLA, but Ni 2+ Mn increased TNFα production in MPLA (Figures 8E-8F). 2+ increased IFNb and TNFa production in R848, but Ni 2+ increased TNFα production in R848 (Figures 7G-7H). 2+ and Mn 2+CpG increased the production of IFN-beta and TNF-alpha (Figures 8I-8J). The cytokine profile of NOD1 / 2 ligands, TLR 7 / 8 ligands, RIG-I & CDS agonists, and pro-inflammatory substances was significantly increased by Mn. 2+ , Co 2+ , Al 3+ , Cu 2+ , Fe 3+ , Ni 2+ These results demonstrate that our metal ion-based approach is a simple yet effective method for modulating the cytokine profile of a wide range of immune stimulators. Based on this discovery, we anticipate that pharmaceutically acceptable formulations may be developed to create better, more potent vaccine adjuvants or cancer immunotherapeutics. For example, specific metal salts of DAMPs / PAMPs may perform better than their parent forms. Coordination polymers composed of selected metal ions and DAMPs / PAMPs with or without pharmaceutically acceptable coordinating molecules may result in optimized metal ion-DAMP / PAMP combinations. Other pharmaceutically acceptable formulations, including but not limited to metal hydroxide / carbonate / phosphate minerals, liposomes, lipid nanoparticles, PLGA particles, hydrogels, emulsions, etc., for the co-delivery of metal ions and DAMPs / PAMPs are also possible.

[0401] Example 10 This example describes a representative formulation of a metal ion-innate immune stimulator.

[0402] To codeliate metal ions and innate immune stimulators with ideal release profiles to target tissues, appropriate formulations based on the physical and chemical properties of specific metal salts of DAMPs / PAMPs, coordination complexes, and other pharmaceutically acceptable formulations (hydroxide / carbonate / phosphate minerals, liposomes, lipid nanoparticles, PLGA, hydrogels, emulsions, etc.) can be designed. Here, we present several representative examples of coordination complexes, including manganese-CDA-H11-DOPE@liposome nanoparticles (Mn-CDA / H11@liposome, Figure 13), Co-CDA / H33-PEG coordination nanoparticles (Co-CDA / H33-PEG, Figure 14), and CDA@Co. 2+ The CDA@4aH11-Co hydrogel (Figure 15) provides a 4-arm-PEG-His11 hydrogel. CDA itself is a 4-arm-PEG-His11 hydrogel. 2+ and Mn 2+ Nanoparticle structures (Figures 13-14) or hydrogels (Figure 15) can be generated by various building module designs and can be further stabilized by polyhistidine. 2+ / Mn 2+ The loading effect could be adjusted by optimizing the ratio and concentration of CDA:polyhistidine-PEG, reaction time, and pH. 2+ / Mn 2+The reduction was approximately 30% for CDA and over 70% for CDA. We further tested these coordination formulations in a murine CT26 colon cancer model. As shown in Figures 13-15, these nanoparticle or hydrogel formulations were able to significantly enhance STING activation in vivo compared with free CDA or free CDA plus metal ions. In particular, the liposome-coated nanoparticle, CDA-Mn-His11-DOPE@liposome (Mn-CDA / H11@lip), could be used for systemic delivery of STING agonists and eradicated 60% of established CT26 colon cancer cells (Figure 13); Co-CDA / His33-PEG significantly prolonged IFNb production, which was detectable even 4 days after injection (Figure 14); and the injectable CDA@4aH11-Co hydrogel induced a very strong local ablative immune response and significant ulcers formed after the first injection (Figure 15F). These improved therapeutic effects were also characterized by increased antigen-specific T cell responses, type I IFN responses, and proinflammatory cytokine release.

[0403] In addition to the formulations mentioned above, there are many other formulations that can be synthesized to deliver metal-innate immune stimulators. Here, we provide several examples with morphologies shown in TEM images (...

Claims

1. 1. A nanoparticle composition comprising: one or more DAMPs or PAMPs, a lipid-poly-histidine, Zn ２＋ , and Mn ２＋ one or more cations selected from the group consisting of one or more lipid molecules selected from 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0 PA), and 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (18:1 PA); Including, The nanoparticles comprise a particle size in the range of 20 to 500 nm. Nanoparticle compositions.

2. the one or more DAMPs or PAMPs are selected from STING agonists, purine-containing or purine-derivative factors, Toll-like receptor (TLR) agonists, NOD-like receptor (NLR) agonists, RIG-I-like receptor (RLR) agonists, cytoplasmic DNA sensor (CDS) agonists, C-type lectin receptor (CLR) agonists, and inflammasome inducers; The nanoparticle composition of claim 1.

3. The one or more STING agonists include cGAMP, cdiAMP, cdiGMP, cAIMP, 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP difluoro, cAIM(PS)2, difluoro(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluorinated, c-di-AMP fluorinated, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluorinated, 2'3'-c-di-GMP, c-di-IMP, 【Chemical 1】 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluoro, cAIM (PS)2, difluoro (Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM (PS)2 (Rp / Sp), 3'3'-cGAMP fluoride, c-di-AMP fluoride, 2'3'-c-di-AMP, 2'3'-c-di-AM (PS)2 (Rp, Rp), c-di-GMP fluoride, 2'3'-c-di-GMP, c-di-IMP, cGAMP, 2' 3'-cGAMP, 2'2'-cGAMP, 3'3'-cGAMP, cGAM(PS)2, 2'3'-cGAM(PS)2(Rp / Sp), 2'2'-cGAM(PS)2, 2'3'-cGAM(PS)2, cGAMP fluoride, 3'3'-cGAMP fluoride, 2'3'-cGAMP fluoride, 2'2'-cGAMP fluoride, c-di-AMP, 2'3'-cdAMP, 2'2'-cdAMP, 3'3'-cdAMP, c-di-AM(PS)2, 2'3'-c-di-AM(PS)2(Rp,Rp), 2'2'-c-di-AM(PS)2, 3'3'-c-di-AM(PS)2, c-di-AMP fluoride, 2'3'-cdAMP fluoride, 2'2'-cdAMP fluoride, 3'3'-cdAMP fluoride, cdGMP, 2'3'-cdGMP, 2'2'-cdGMP, 3'3'-cdGMP, c-di-GM(PS)2, 2'3'-c-di- GM(PS)2, 2'2'-c-di-GM(PS)2, 3'3'-c-di-GM(PS)2, cdGMP fluoride, 2'3'-cdGMP fluoride, 2'2'-cdGMP fluoride fluoride, 3'3'-cdGMP fluoride, cAIMP, 2'3'-cAIMP, 2'2'-cAIMP, 3'3'-cAIMP, cAIMP difluoro (3'3'-cAIMP fluoride) fluoride, 2'3'-cAIMP fluoride, 2'2'-cAIMP fluoride, cAIM(PS)2 difluoro, 3'3'-cAIM(PS)2 difluoro (Rp / Sp), 2'3' -cAIM(PS)2 difluoro, 2'2'-cAIM(PS)2 difluoro, c-di-IMP, 2'3'-cdIMP, 2'2'-cdIMP, 3'3'-cdIMP, c-di- IM(PS)2, 2'3'-c-di-IM(PS)2, 2'2'-c-di-IM(PS)2, 3'3'-c-di-IM(PS)2, c-di-IMP fluoride, 2'3'-cdIMP fluoride, 2'2'-cdIMP fluoride, 3'3'-cdIMP fluoride, and amide benzimidazole (ABZI)-based compounds, The nanoparticle composition of claim 2.

4. The TLR agonist is selected from a TLR-3 agonist, a TLR-4 agonist, a TLR-5 agonist, a TLR-7 agonist, a TLR-8 agonist, and a TLR-9 agonist. The nanoparticle composition of claim 2.

5. The NLR agonist is an NLRP3 agonist; The nanoparticle composition of claim 2.

6. The purine-containing factor or purine-derivative factor may be 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluoro, cAIM(PS)2, difluoro(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluoride, c-di-AMP fluoride, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluoride, 2'3'-c-di-GMP, c-di-I MP, cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, 3'3'-cGAMP, cGAM(PS)2, 2'3'-cGAM(PS)2(Rp / Sp), 2'2'-cGAM(PS)2, 2'3'-cGAM(PS)2, cGAMP fluoride, 3'3'-cGAMP fluoride, 2'3'-cGAMP fluoride, 2'2'-cGAMP fluoride, c-di-AMP, 2'3'-cdAMP, 2'2'-cdAMP, 3'3'-cdAMP, c-di-AM(PS)2, 2'3'-c-di-AM(PS)2(Rp,Rp), 2'2'-c-di-AM(PS)2, 3'3'-c-di-AM(PS)2, c-di-AMP fluoride, 2'3'-cdAMP fluoride, 2'2'-cdAMP fluoride, 3'3'-cdAMP fluoride, cdGMP, 2'3'-cdGMP, 2'2'-cdGMP, 3'3'-cdGMP, c-di-GM(PS)2, 2'3'-c-di- GM(PS)2, 2'2'-c-di-GM(PS)2, 3'3'-c-di-GM(PS)2, cdGMP fluoride, 2'3'-cdGMP fluoride, 2'2'-cdGMP fluoride fluoride, 3'3'-cdGMP fluoride, cAIMP, 2'3'-cAIMP, 2'2'-cAIMP, 3'3'-cAIMP, cAIMP difluoro (3'3'-cAIMP fluoride) Fluorinated compound, 2'3'-cAIMP fluorinated compound, 2'2'-cAIMP fluorinated compound, cAIM(PS)2 difluoro, 3'3'-cAIM(PS)2 difluoro (Rp / Sp), 2'3 '-cAIM(PS)2 difluoro, 2'2'-cAIM(PS)2 difluoro, c-di-IMP, 2'3'-cdIMP, 2'2'-cdIMP, 3'3'-cdIMP, c-d i-IM(PS)2, 2'3'-c-di-IM(PS)2, 2'2'-c-di-IM(PS)2, 3'3'-c-di-IM(PS)2, c-di-IMP fluoride, 2'3'-cdIMP fluoride, 2'2'-cdIMP fluoride, 3'3'-cdIMP fluoride, imiquimod, resiquimod, 6-(4-amino-imidazoquinolyl)-norleucine, 【Chemistry 2】 selected from RNA, siRNA, microRNA, interfering RNA, mRNA, replicon mRNA, RNA analogs, DNA, and purine-based PI3K inhibitors; The nanoparticle composition of claim 2.

7. The average particle size of the nanoparticles is between 30 and 500 nm. The nanoparticle composition according to any one of claims 1 to 6.

8. The average particle size of the nanoparticles is between 50 and 500 nm. The nanoparticle composition of claim 7.

9. The average particle size of the nanoparticles is between 75 and 250 nm. The nanoparticle composition of claim 8.

10. The average particle size of the nanoparticles is between 40 and 120 nm. The nanoparticle composition of claim 7.

11. The lipid molecule is 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA). The nanoparticle composition according to any one of claims 1 to 6.

12. The lipid molecule is 1,2-distearoyl-sn-glycero-3-phosphate (18:0 PA). The nanoparticle composition according to any one of claims 1 to 6.

13. The lipid molecule is 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (18:1 PA). The nanoparticle composition according to any one of claims 1 to 6.

14. The lipid-poly-histidine is DOPE-H11. The nanoparticle composition according to any one of claims 1 to 6.

15. The cation is Mn ２＋ That is, The nanoparticle composition according to any one of claims 1 to 6.

16. The cation is Zn ２＋ That is, The nanoparticle composition according to any one of claims 1 to 6.

17. A nanoparticle composition described in any one of claims 1 to 6 for use in stimulating an innate immune response in a subject.

18. Stimulating an innate immune response includes stimulating an innate cytokine response in the subject that is mediated via cytokines.

18. A nanoparticle composition for use according to claim 17.

19. The innate cytokine response is mediated through type 1 interferon.

19. A nanoparticle composition for use according to claim 18.

20. the subject has or is at risk of having cancer; 20. A nanoparticle composition for use according to claim 19.

21. 10. The nanoparticle composition of claim 1 for use in treating cancer in a subject, wherein the treatment comprises administering to the subject the composition and one or more of a chemotherapeutic agent, an anti-immunosuppressant, and an immunostimulatory agent.

22. the immunostimulatory agent is selected from an inhibitor of anti-CTLA-4 antibody, anti-PD-1, anti-PD-L1, anti-TIM-3, anti-BTLA, anti-VISTA, anti-LAG3, anti-CD25, anti-CD27, anti-CD28, anti-CD137, anti-OX40, anti-GITR, anti-ICOS, anti-TIGIT, and IDO; 22. A nanoparticle composition for use according to claim 21.

23. The chemotherapeutic agent includes aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alfa, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, and idarubicin. , ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (TAXOL), pilocarpine, prochlorperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate; 22. A nanoparticle composition for use according to claim 21.

24. the cancer is one or more selected from bladder cancer, brain cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, abdominal cancer, head and neck cancer, testicular cancer, melanoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, B-cell lymphoma, and uterine cancer; 22. The use according to claim 21.

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