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

Abstract

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

Description

[0001] This application claims priority to U.S. Provisional Application No. 62 / 697,092, filed on July 12, 2018, the entire contents of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under Grant CA210273 from the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0004] The present disclosure provides compositions and methods for stimulating an innate immune response in a subject with an agent capable of stimulating an innate immune response in the subject following administration to the subject (e.g., damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs)). In particular, the present invention relates to compositions of DAMPs / PAMPs and metal ions, and systems and methods utilizing such nanoparticles (e.g., in diagnostic and / or therapeutic settings). Background Art

[0005] The innate immune system is the first line of defense in humans, and its activation can induce the secretion of proinflammatory cytokines and coordinate the adaptive immune system. DAMPs and PAMPs represent two major stimulants of innate immunity. DAMPs are endogenous host biomolecules released after tissue damage and include heat shock proteins and HMGB1 (high-mobility group protein 1), ATP, uric acid, hyaluronic acid fragments, heparan 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), stimulator of interferon genes (STING) agonists, purine-containing or purine-derived agents, and ligands of C-type lectin receptors (CLRs).

[0006] DAMPs and PAMPs can induce proinflammatory cytokine production and proinflammatory phenotypic changes in immune cells, which are crucial for both cancer and autoimmune diseases. On the one hand, proinflammatory phenotypic changes may disrupt the immunosuppressive tumor microenvironment, thereby transforming "cold tumors" into "hot tumors." Consequently, TLR-3, TLR4, TLR7, TLR9, NLRP3, and STING agonists are currently undergoing clinical trials for cancer immunotherapy. In particular, the recent discovery that the tumor-derived DNA-cGAS-STING pathway is crucial for tumor immune surveillance has shown significant cancer immunotherapy efficacy in preclinical studies, leading to numerous Phase I clinical trials of STING agonists. On the other hand, DAMPs and PAMPs are widely involved in the development and progression of autoimmune diseases. Inhibiting aberrant innate immune activation is emerging as an effective treatment for many incurable autoimmune diseases. Modulating DAMP- and PAMP-mediated immune responses will provide new therapeutic approaches for a variety of human diseases, including cancer and autoimmune diseases.

[0007] The present invention addresses this need. Summary of the Invention

[0008] 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 around 30%. This may be attributed to some tumors, characterized as "cold tumors," being less visible to the immune system. Characteristics of such tumors include a low inflammatory response by T cells and other proinflammatory immune cells, a low mutational burden, and insufficient tumor infiltration. In contrast, "hot tumors," with more pronounced inflammatory features recognizable by the immune system, have better responses to cancer immunotherapy. Therefore, understanding how to transform "cold tumors" into "hot" ones is crucial.

[0009] Accumulated evidence indicates that immune surveillance of tumors mediated by the innate immune system recognizes the presence of tumors by detecting tumor cell-derived DNA through the STING pathway. Activation of the STING pathway can trigger innate immune cascades, such as type I interferon responses and other pro-inflammatory phenotypic changes, which further trigger adaptive anti-tumor responses. Therefore, STING is regarded as a "trigger" for reversing from a "cold tumor" to a "hot tumor." For example, intratumoral administration of STING agonists can induce anti-tumor immune responses against both local and metastatic tumors. In clinical settings, type 1 interferon responses were found to be a clear feature of better cancer treatment prognosis, similar to antigen-specific T cell infiltration. Therefore, the development of STING agonists with high in vivo stability, favorable pharmacokinetic properties and an acceptable safety profile is of great significance and high translational value.

[0010] However, most human STING agonists currently under evaluation are based on cyclic dinucleotides and their derivatives, whose small molecular weight, poor pharmacokinetic parameters, and severe side effects greatly limit their systemic application.

[0011] Experiments conducted during the course of developing embodiments of the present invention have shown that in the presence of Zn 2+ In the presence of Zn 2+ Such CDNs assembled into uniform nanoparticles in the presence of calcium phosphate and a copolymer of cationic poly(ethyleneimine) (PEI) and polyethylene glycol (PEG) were further stabilized with lipid vesicles. Additional experiments showed that CDNs could be formulated into nanoparticles in the presence of calcium phosphate and a copolymer of cationic poly(ethyleneimine) (PEI) and polyethylene glycol (PEG). It was further shown that such CDN-nanoparticle assemblies (e.g., CDNs formulated into nanoparticles in the presence of calcium phosphate and a copolymer of PEI-PEG) (e.g., in the presence of Zn 2+ CDNs formulated into nanoparticles in the presence of liposomes provide increased cancer cell uptake and more precise targeting to the tumor microenvironment (e.g., TME), enabling increased delivery efficacy and reduced toxicity of STING agonists.

[0012] For the CDN-Zn embodiment, such results indicate the following unique features compared to previous drug delivery systems: 1) reversible assembly for sustained drug release without loss of bioactivity, 2) high loading efficacy and loading capacity, 3) increased cellular uptake, 4) pH-sensitive release at low pH, 5) good biocompatibility, 6) flexible surface chemistry for surface modification and functionalization, and 7) low cost and ease of scale-up.

[0013] For the CDN@CaP / PEI-PEG embodiment, such results indicate the following unique features compared to previous drug delivery systems: 1) increased cellular uptake, 2) high loading efficacy, 3) pH-sensitive release at low pH, 4) biocompatibility, and 5) low cost and ease of scale-up.

[0014] Such results have important clinical implications, as these nanoparticles conjugated to CDNs can induce tumor-specific immune responses through systemic administration, thus avoiding the need for direct local injection into tumors.

[0015] Additional experiments conducted during the course of developing embodiments of the present invention determined that specific metal ions can significantly enhance STING activation and type I IFN responses by STING agonists. For example, it was shown that under optimal conditions, Mn 2+ or Co 2+cGAMP enhanced STING activation by more than 60-fold. 2+ or Co 2+ Administration of the combination to murine tumors significantly improved the therapeutic effect, characterized by elevated serum type I IFN levels, higher tumor eradication efficacy, and longer animal survival. After treatment, 80% of tumor-bearing mice eradicated established tumors, and they were resistant to a second tumor attack 80 days later, indicating long-term immunity against tumor recurrence. In addition, it was found that this phenomenon can be generalized to various other innate immune pathways, including but not limited to TLR 3 / 4 / 7 / 8 / 9 ligands, NOD1 / 2 ligands, TLR 7 / 8 ligands, RIG-I & CDS agonists, and inflammasome-inducers. For example, Co 3+ Significantly increased poly-IC-mediated IFNb, TNFa, IL6, and IL2 production by dendritic cells, while Mn 2+ Increased poly IC-mediated IFNb production. Mn 2+ MPLA-mediated IFNb and TNFa production was increased, while Ni 2+ Increased MPLA-mediated TNFa production. Mn 2+ R848-mediated IFNb and TNFa production was increased, while Ni 2+ Increased R848-mediated TNFa production. 2+ and Mn 2+ Increased CpG-mediated IFNb and TNFa production.

[0016] Based on such results, several pharmaceutically acceptable formulations have been developed to precisely deliver metal-innate immune stimulator combinations to the desired targets and promote immune activation. For example, liposome-coated nanoparticles CDA-Mn-His11-DOPE@liposomes (Mn-CDA / H11@lip) can be used for systemic delivery of STING agonists and eradicate 60% of established CT26 colon tumors. Co-CDA / His33-PEG can greatly prolong the production of IFNb, which can be detected even 4 days after injection. In addition, experiments were conducted to test whether chelating intracellular metal ions can inhibit innate immune responses. Through unbiased screening, several chelators were identified that can effectively inhibit DNA-induced cGAS-STING-type I IFN / NFkB responses and poly IC-induced TLR3-cGAS-STING-type I IFN, which may be useful for the treatment of autoimmune diseases. Overall, the results represent a simple but effective approach to addressing several unmet medical challenges, such as improving the efficacy of vaccine adjuvants, developing cancer immunotherapies, and controlling autoimmune diseases.

[0017] Thus, such results and embodiments demonstrate a new class of drug delivery systems for local and systemic delivery of agents capable of stimulating an innate immune response in a subject upon administration to the subject.

[0018] 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 relates to such compositions comprising an agent capable of stimulating an innate immune response in the subject upon administration to the subject, methods for synthesizing such compositions, and systems and methods utilizing such compositions (e.g., in diagnostic and / or therapeutic settings).

[0019] Thus, in certain embodiments, the present invention provides compositions comprising one or more DAMPs or PAMPs, and

[0020] a) calcium phosphate and copolymers of cationic poly(ethyleneimine) (PEI) and 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); or

[0021] b) one or more cations selected from the group consisting of: 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+ 、Pd2+ , Pt 4+ 、Na + , K + , and related phosphates or carbonates.

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

[0023] Therefore, in certain embodiments, the present invention provides a method for treating cancer in a subject, comprising administering to the subject a pharmaceutically effective amount of a composition comprising an agent (e.g., a DAMP / PAMP) that can stimulate an innate immune response in the subject upon administration to the subject. In some embodiments, the innate immune response is an innate cytokine response in the subject mediated by a cytokine. In some embodiments, the innate cytokine response is mediated by type 1 interferon in the subject.

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

[0025] In some embodiments, the composition is co-administered with a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is one or more of: aldesleukin, altretinoin, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), actinomycin D, docetaxel, doxorubicin, dronabinol, epoetin alpha, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfidine, dapoxetine ... amide, interferon alfa, irinotecan, lansoprazole, levamisole, leucovorin, megestrol acetate, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (TAXOL), pilocarpine, prochloroperazine, 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, the DAMP and PAMP agonists are selected from STING agonists, purine-containing or purine-derived agents, 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, the 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-derived agents. In some embodiments, the purine-containing or purine-derived agent is selected from 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), fluorinated 3'3'-cGAMP, fluorinated c-di-AMP, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), fluorinated c-di-GMP, 2'3'-c-di-GMP, c-di-IMP, cGAMP AMP, 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, fluorinated cGAMP, fluorinated 3'3'-cGAMP, fluorinated 2'3'-cGAMP, fluorinated 2'2'-cGAMP, 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, fluorinated c-di-AMP, fluorinated 2'3'-cdAMP, fluorinated 2'2'-cdAMP, fluorinated 3'3'-cdAMP, 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, fluorinated cdGMP, fluorinated 2'3'-cdGMP, fluorinated 2'2'-cdGMP, fluorinated 3'3'-cdGMP, cAIMP, 2'3'-cAIMP, 2'2'-cAIMP, 3'3'-cAIMP, cAIMP difluoro(fluorinated 3' 3'-cAIMP, fluorinated 2'3'-cAIMP, fluorinated 2'2'-cAIMP, 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, fluorinated c-di-IMP, fluorinated 2'3'-cdIMP, fluorinated 2'2'-cdIMP, fluorinated 3'3'-cdIMP, imiquimod, resiquimod, 6-(4-amino-imidazoquinolinyl)-norleucine, RNA, siRNA, microRNA, interfering RNA, mRNA, replicon mRNA, RNA analogs, DNA, and purine-based PI3K inhibitors.

[0028] Such compositions are not limited to a specific type or class of STING agonists. 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 includes cGAMP, cdiAMP, cdiGMP, and cAIMP. Additional examples of cyclic purine dinucleotides are described in detail in, for example, U.S. Patent Nos. 7,709,458 and 7,592,326; WO 2007 / 054279; and Yan et al., Bioorg. Med. Chem Lett. 18:5631 (2008), each of which is incorporated herein by reference. In some embodiments, the additional STING agonist is selected from the group consisting of 5,6-dimethylxanthone-4-acetic acid (DMXAA), methoxyvone, 6,4′-dimethoxyflavone, 4′-methoxyflavone, 3′,6′-dihydroxyflavone, 7,2′-dihydroxyflavone, daidzein, formononetin, and retusin 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), fluorinated 3'3'-cGAMP, fluorinated c-di-AMP, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), fluorinated c-di-GMP, 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 selected from:

[0030]

[0031] SB11285 (Spring Bank Pharmaceuticals), gemcitabine STING-agonist-C11 STING agonist-1 STING agonist G10 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), fluorinated 3'3'-cGAMP, fluorinated c-di-AMP, 2'3'-c-di-AMP, 2'3'-c-di-AM (PS)2(Rp,Rp), fluorinated c-di-GMP, 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, fluorinated cGAMP , fluorinated 3'3'-cGAMP, fluorinated 2'3'-cGAMP, fluorinated 2'2'-cGAMP, 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, Fluorinated c-di-AMP, fluorinated 2'3'-cdAMP, fluorinated 2'2'-cdAMP, fluorinated 3'3'-cdAMP, 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,

[0032] Fluorinated cdGMP, fluorinated 2'3'-cdGMP, fluorinated 2'2'-cdGMP, fluorinated 3'3'-cdGMP, cAIMP, 2'3'-cAIMP, 2'2'-cAIMP, 3'3'-cAIMP, cAIMP difluoro (fluorinated 3'3'-cAIMP, fluorinated 2'3'-cAIMP, fluorinated 2'2'-cAIMP, 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, fluorinated c-di-IMP, fluorinated 2'3'-cdIMP, fluorinated 2'2'-cdIMP and fluorinated 3'3'-cdIMP as well as amidobenzimidazole (ABZI)-based compounds.

[0033] As mentioned above, CDNs have two key limitations for use as cancer drugs: 1) poor pharmacokinetics and severe off-target side effects. Regarding poor pharmacokinetics, if administered via intratumoral injection, CDNs will readily diffuse due to their small molecular weight and high hydrophilicity; if administered via intravenous injection, CDNs will exhibit low bioavailability to tumor tissue due to in vivo instability, low lipophilicity, and rapid excretion. Regarding severe off-target side effects, STING, as an immunological sensor of viral infection, is widely distributed throughout the body. Thus, high doses of STING agonists or systemically administered STING agonists will nonspecifically activate the innate immune system and induce 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).

[0034] In fact, in some embodiments, the small molecule agonist of a DAMP and / or PAMP is a prodrug of a small molecule agonist of a DAMP and / or PAMP. For example, in some embodiments, the prodrug of a 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 a small molecule agonist of a DAMP and / or PAMP is attached to a hydrophobic moiety that facilitates loading into the nanoparticle and / or facilitates tissue retention.

[0035] In some embodiments, CDNs are modified with cleavable lipid moieties to create CDN prodrugs. For example, as shown in the following schemes, three synthetic routes for lipid-CDN prodrugs are contemplated. Each is activated by a different mechanism: esterase-based activation for Route 1, phosphoamidase-based activation for Route 2, and activation that reduces environmental sensitivity for Route 3.

[0036] Scheme 1. Synthetic route of lipid-CDN prodrugs.

[0037]

[0038] Scheme 2. Synthetic route of lipid-CDN prodrugs.

[0039]

[0040] Scheme 3. Synthetic route of lipid-CDN prodrugs.

[0041]

[0042] After modification, lipid-CDN prodrugs are expected to be administered in free form or in liposomal formulations. Such an embodiment would significantly improve the pharmacokinetics of CDNs and reduce side effects. For example, it is expected that injected lipid-CDN prodrugs will be retained at the injection site and slowly release the CDN in the tumor, thereby conferring high bioavailability and reducing side effects on normal tissues. For example, lipid-CDN prodrugs formulated into liposomes can be administered intravenously or topically. Such liposomally formulated lipid-CDNs can significantly prolong drug circulation in the blood and increase tumor accumulation and lymph node drainage. More importantly, CDNs are inactive after lipid modification and can only be reactivated when cleaved by esterases. Furthermore, previous studies have shown that metastatic nodes can be distinguished from tumor-free lymph nodes by high esterase levels, which would enable selective activation of lipid-CDN prodrugs at the tumor site.

[0043] In some embodiments, STING activating compounds are provided (see, e.g., WO 2017011920, WO 2017027646, WO 2017011622, U.S. Patent Application Publication No. 20160287623, WO 2016100261, U.S. Patent Application Publication No. 20160074507, and WO 2015161762).

[0044] In some embodiments, cGAS modulating compounds are provided (see, e.g., WO 2014179335).

[0045] In some embodiments, STING inhibitory compounds are provided (see, e.g., U.S. Patent Application Publication No. 20170037400).

[0046] In some embodiments, compounds are provided that are capable of killing STING-deficient and / or cGAS-deficient cancer cells (see, e.g., WO 2016201450).

[0047] In some embodiments, a STING pathway agonist is provided in combination with a pharmaceutically active ingredient (see, e.g., STING activation / chemotherapy (WO 2016096577), STING activation / selected vaccine formulations that stimulate immune responses (U.S. Patent Application Publication Nos. 20150056224 and 20140205653), and STING activation / cytokine production (WO2013185052)).

[0048] In some embodiments, such compositions comprising agents (e.g., DAMPs / PAMPs) capable of stimulating an innate immune response in a subject upon administration to the subject are associated with (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, incorporated into) nanoparticles.

[0049] In some embodiments, such compositions combined with nanoparticles are further combined (e.g., complexed, put together, encapsulated, absorbed, adsorbed, blended) with calcium phosphate and a copolymer of PEI / PEG, PH-PEG, PK-PEG or PGA-PEG. In fact, in some embodiments, the combination of an agent capable of stimulating an innate immune response in a subject with the nanoparticles is carried out in the presence of a copolymer of calcium phosphate and PEI / PEG, PH-PEG, PK-PEG or PGA-PEG.

[0050] In some embodiments, such compositions associated with nanoparticles are further associated (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, incorporated) with one or more cations selected from the group consisting of: 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+ 、Co3+ 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 + Indeed, in some embodiments, the binding of an agent capable of stimulating an innate immune response in a subject to the nanoparticle is in the presence of such a cation (e.g., Zn 2+ 、Co 2+ or Mn 2+ ) is carried out in the presence of .

[0051] In some embodiments, nanoparticles and one or more cations (e.g., Zn 2+ 、Co 2+ or Mn 2+ ) or calcium phosphate-bound compositions are further bound (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, incorporated) to a hydrophobic molecule.

[0052] 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.

[0053] Examples of lipid molecules suitable for use in embodiments of the present invention include, but are not limited to, phospholipids such as phosphatidylcholine, phosphatidylethanolamine, lysolecithin, lysolecithin, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylglycerol (DPPG), palmitoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylcholine (DP ... Oleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably derived from C 10 -C 24 Acyl groups of carbon-chain fatty acids, for example, lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl.

[0054] Other non-limiting examples of lipid molecules include sterols, such as cholesterol and its derivatives, such as cholestanol, cholestanone, cholesterenone, coprosterol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

[0055] Other examples of lipid molecules suitable for use in the present invention include lipids that do not contain phosphorus, such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glyceryl ricinoleate, cetyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkyl aryl sulfate polyethoxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, sphingomyelin, and the like.

[0056] Other examples of lipid molecules suitable for use in the present invention include fatty acids and their derivatives or analogs. These include oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, glyceryl monooleate (1-monooleoyl-rac-glyceride), dilaurin, caprylic acid, arachidonic acid, 1-monocaprin, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, and C-1,4-diol. 1-10 Alkyl esters (e.g., methyl, isopropyl, and tert-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).

[0057] Other examples applicable to lipid molecules of the present invention include lipid molecules (PEG-lipid) modified with PEG.The example of PEG-lipid includes but is not limited to PEG (PEG-DAA) as described in for example PCT Publication No. WO05 / 026372 being coupled to dialkyloxypropyl class, PEG (PEG-DAG) as described in for example U.S. Patent Publication No. 20030077829 and 2005008689 being coupled to diacylglycerol, PEG such as phosphatidylethanolamine (PEG-PE) as described in for example U.S. Patent number 5,885,613 being coupled to PEG on ceramide, PEG being coupled to cholesterol or its derivatives, and its mixture.For all purposes, the disclosure of these patent documents is incorporated herein in full by quoting with it.Other PEG-lipid includes but is not limited to PEG-C-DOMG, 2KPEG-DMG and mixture thereof.

[0058] PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEG is classified by its molecular weight; for example, PEG 2000 has an average molecular weight of approximately 2,000 daltons, while PEG 5000 has an average molecular weight of approximately 5,000 daltons. PEG is commercially available from Sigma Chemical and other companies and includes, for example, the following: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH2), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). Other PEGs, such as those described in U.S. Patent Nos. 6,774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are also used to prepare the PEG-lipid conjugates of the present invention. The disclosures of these patents are incorporated herein by reference in their entirety for all purposes. In addition, monomethoxypolyethylene glycol acetic acid (MePEG-CH2COOH) is particularly useful for preparing PEG-lipid conjugates, including, for example, PEG-DAA conjugates.

[0059] The PEG portion of the PEG-lipid conjugates described herein can include an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In some cases, the PEG portion 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 a preferred embodiment, the PEG portion has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0060] In some cases, PEG can be optionally substituted by an alkyl, alkoxy, acyl or aryl group. PEG can be directly conjugated to the lipid or can be connected to the lipid through a linker moiety. Any linker moiety suitable for connecting PEG to the lipid can be used, including, for example, a linker moiety that does not contain an ester and a linker moiety that contains an ester. In a preferred embodiment, the linker moiety is a linker moiety that does not contain an ester. As used herein, the term "linker moiety that does not contain an ester" refers to a linker moiety that does not contain a carboxylate 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 (—S—S—), ether (—O—), succinyl (—(O)CCH2CH2C(O)—), succinimidyl (—NHC(O)CH2CH2C(O)NH—), ether, disulfide, and combinations thereof (e.g., linkers comprising both a carbamate linker moiety and an amide linker moiety). In a preferred embodiment, the PEG is attached to the lipid using a carbamate linker.

[0061] In other embodiments, PEG is attached to the lipid using an ester-containing linker moiety. Suitable ester-containing linker moieties include, for example, carbonate (—OC(O)O—), succinyl, phosphate (—

[0062] O—(O)POH—O—), sulfonic acid esters and combinations thereof.

[0063] Phosphatidylethanolamines with various acyl chain groups of varying chain lengths and degrees of saturation can be conjugated 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. Preferred are those with C 10 to C 20 Phosphatidylethanolamines comprising saturated or unsaturated fatty acids having carbon chain lengths within a range of 1 to 400 nm can also be used. Phosphatidylethanolamines with mono- or di-unsaturated fatty acids and 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), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0064] In some embodiments, the nanoparticles associated with such compositions comprising agents capable of stimulating an innate immune response in a subject (e.g., DAMPs / PAMPs) upon administration to the subject are further associated (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, incorporated) with one or more agents configured to target cancer cells.

[0065] In some embodiments, the agent configured to target cancer cells is a tumor antigen 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, 2 and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, Mage-A1,2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100 (Pmel 17), 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 (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, CA 19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), human EGFR protein or its fragment,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 mutant, p53 mutant, proteinase 3 (PR1), survivin, hTERT, sarcoma translocation breakpoint, 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, fucose 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, AKAP-4, XAGE 1, B7H3, leguminin, Tie 2, 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, aldehyde dehydrogenase and any derivatives thereof.

[0066] In some embodiments, one or more agents configured to target cancer cells are conjugated to the outer surface of the nanoparticle. In some embodiments, one or more agents configured to target cancer cells are encapsulated within the nanoparticle.

[0067] In some embodiments, the nanoparticles associated with such compositions comprising an agent (e.g., DAMP / PAMP) capable of stimulating an innate immune response in a subject upon administration to the subject are further associated (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, incorporated) with an adjuvant.

[0068] In some embodiments, the adjuvant is selected from the group consisting of: CPG, poly IC, poly-ICLC, 1018ISS, aluminum salts (e.g., aluminum hydroxide, aluminum phosphate), Amplivax, BCG, CP-870,893, CpG7909, CyaA, dSLIM, cytokines (such as GM-CSF, IL-2, IFN-a, Flt-3L), IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, delivery system, PLGA microparticles, imiquimod, resiquimod, gardiquimod, 3M-052, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, β-glucan, Pam3Cys, Aquila's QS21 stimulator, vadimezan, AsA404 (DMXAA), 3M MEDI9197, glucopyranosyl lipid adjuvant (GLA), GLA-SE, CD1d ligands (such as C20:2, OCH, AH04-2, α-galactosylceramide, α-C-galactosylceramide, α-mannosylceramide, α-fructoseceramide, β-galactosylceramide, β-mannosylceramide), STING agonists (e.g., cyclic dinucleotides, including 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 di-AMP, cyclic di-GMP), CL401, CL413, CL429, Flagellin, RC529, E6020, imidazoquinoline-based small molecule TLR-7 / 8a (including lipidated analogs thereof), virosomes, AS01, AS02, AS03, AS04, AS15, IC31, CAF01, ISCOM, cytokines (such as GM-CSF, IL-2, IFN-a, Flt-3L) and bacterial toxins (such as CT and LT). 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.

[0069] Such compositions comprising an agent (eg, DAMP / PAMP) that is capable of stimulating an innate immune response in a subject upon administration to the subject, in conjunction with the nanoparticles, are not limited to a particular type of nanoparticle.

[0070] In some embodiments, the nanoparticles are sHDL nanoparticles. In some embodiments, the nanoparticles are selected from the group consisting of sHDL nanoparticles, fullerenes, endohedral metallofullerene buckyballs, trimetallic 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, and methods of synthesizing a molecule using a layer-by-layer process, a self-assembly process, or a polymerase chain reaction. Any nanostructure, microstructure or derivative thereof formed by electrolytes, 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 and sHDL-polyhistidine and metal-organic framework (MOF) coordination polymers (CP).

[0071] In some embodiments, the average size of the nanoparticles is between 6 and 500 nm.

[0072] 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 (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 phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, -sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-bis-(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.

[0073] In some embodiments, the ApoA-I mimetic is described by any of SEQ ID NO:1-336, and 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), PLGEEMRDRARAHVDALRTHLA (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:36), ALDKLKEFGNTLEDKARELIS (SEQ IDNO:361), VVALLALLASARASEAEDASLL (SEQ ID NO:362), HLRKLRKRLLRDADDLQKRLAVYOA (SEQID NO:363), AQAWGERLRARMEEMGSRTRDR (SEQ ID NO:364), LDEVKEQVAEVRAKLEEQAQ (SEQID 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).

[0074] In some embodiments, the average particle size of the sHDL nanoparticles is between 6-70 nm.

[0075] In some embodiments, the nanoparticles combined with such a composition comprising an agent (e.g., DAMP / PAMP) capable of stimulating an innate immune response in a subject upon administration to the subject are further combined (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, incorporated) with one or more neoantigenic peptides. Each of the one or more neoantigenic peptides is specific for a neoantigenic mutation identified in a neoplastic biological sample obtained from the subject. In some embodiments, the subject is human.

[0076] In some embodiments, the one or more neoantigenic peptides range from about 5 to about 50 amino acids in length. In some embodiments, the one or more neoantigenic peptides range from about 15 to about 35 amino acids in length. In some embodiments, the one or more neoantigenic peptides range from about 18 to about 30 amino acids in length. In some embodiments, the one or more neoantigenic peptides range from about 6 to about 15 amino acids in length.

[0077] In some embodiments, the nanoparticles associated with such compositions comprising an agent capable of stimulating an innate immune response in a subject (e.g., a DAMP / PAMP) upon administration to the subject are further associated (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, incorporated) with one or more biomacromolecular agents.

[0078] Such compositions are not limited to specific biomacromolecule agents.

[0079] In some embodiments, the biomacromolecule agent is a nucleic acid. Such embodiments include any type of nucleic acid molecule, including but not limited to RNA, siRNA, microRNA, interfering RNA, mRNA, replicon mRNA, RNA analogs and DNA.

[0080] In some embodiments, the biomacromolecule agent is a peptide.

[0081] In some embodiments, the peptide is adrenocorticotropic hormone (ACTH), growth hormone peptide, melanocyte stimulating hormone (MSH), oxytocin, vasopressin, corticotropin-releasing factor (CRF), CRF-related peptide, gonadotropin-releasing hormone-related peptide (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 pro-peptide, calcitonin gene-related peptide (CGRP), parathyroid hormone (PTH), thyroid 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-inhibiting 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 peptides, opioid peptides, thymosin, adrenomedullin peptide, allostatin peptide, amyloid beta protein fragment, antimicrobial peptide, antioxidant peptide, apoptosis-related peptide, bag cell peptide (BCP), bombesin, osteocalcin (Gla) protein peptide, cocaine and amphetamine-related transcript (CART) peptide, cell adhesion peptide, chemotactic peptide, complement inhibitory factor, cortistatin peptide, fibronectin fragment, fibrin-related peptide, FMRF, FMRFamide-related peptide (FaRP), galanin, galanin-related peptide, growth factor, growth factor-related peptide, G therapeutic peptide binding protein fragment, Gualylin, uroguanylin (Ur oguanylin), inhibin peptide, interleukin (IL), interleukin receptor protein, laminin fragment, leptin fragment peptide, cockroach kinin, pituitary adenylate cyclase activating polypeptide (PAPCAP), pancreatic inhibin, polypeptide repeat chain, signal transducer, thrombin inhibitor, toxin, trypsin inhibitor, virus-related peptide, adjuvant peptide analog, α mating factor, antiarrhythmic peptide, anorexigenic peptide, α-1 antitrypsin, bovine pinecone anti-reproductive peptide, bursin, C3 peptide P16, cadherin peptide, chromogranin A fragment, contraceptive tetrapeptide, conantokin G,Cone snail sleep peptide T, Crustacean cardioactive peptide, C-terminal peptide, cytochrome b588 peptide, antithrombotic peptide (Decorsin), delicious peptide, delta sleep-inducing peptide, diazempam binding inhibitor fragment, nitric oxide synthase blocking peptide, OVA peptide, platelet calpain inhibitor (P1), plasminogen activator inhibitor 1, Rigin, schizophrenia-related peptide, sodium potassium atherapeutic peptidase inhibitor 1, sperm activation peptide (Speract), sperm activation peptide, systemin, thrombin receptor agonist, tuftsin, lipokine, uremic pentapeptide, antifreeze peptide, tumor necrosis factor (TNF), leech [Des Asp10] antithrombotic peptide (Leech [Des Asp10] Decorsin), L-ornithine taurine hydrochloride, P-aminophenylacetyl tuftsin, 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, D DDDD, 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 Kallikrei N-1, placental ribonuclease inhibitor, Sarcolecin binding protein, surfactant protein D, Wilms' tumor suppressor factor, GABAB 1b receptor peptide, prion-related peptide (iPRP13), choline binding protein fragment, telomerase inhibitor, cardiostatin peptide, endostatin-derived peptide, prion inhibitory peptide, N-methyl D-aspartate receptor antagonist and C-peptide analog.

[0082] In some embodiments, the peptide is selected from 177Lu-DOTA0-Tyr3-Octreotate, Abarelix acetate, ADH-1, Afamelanotidec, melanotan-1, CUV1647, Albiglutide, Aprotinin, Arginine, Atosiban acetate, Bacitracin, Bentyramide, BH3 domain, Bivalirudin, Bivalirudin trifluoroacetate hydrate, Blisibimod, Bortezomib, Buserelin, Buserelin acetate, Calcitonin, Carbetocin, Carbetocin acetate, Cetrorelix, Cetrorelix acetate, Cyclosporin, Cilengitidec, EMD121974, Corticorelin acetate injection, hCRF, Corticorelin trifluoroate ovine triflutate), cortirellin trifluoroacetate, adrenocorticotropin, corticotropin, ACTH 1-24, teicosin hexaacetate, dalbavancin, daptomycin, degarelix acetate, depoxetine trifluoroacetate (with sodium pertechnetate), desmopressin acetate, desmopressin DDAVP, dulaglutide, ecallantide, eduterotide (with yttrium-90), elcatonin acetate, enalapril maleate (or 2-succinate), enfuvirtide, eptifibatide, exenatide, ganirelix acetate, glatiramer acetate 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, leuprolide, liraglutide, lisinopril, lixisenatide, lysine vasopressin, magainin 2, MALP-2Sc, synthetic macrophage Cytokine-activated lipopeptide-2, nafarelin acetate, nesiritide, NGR-hTNF, octreotide acetate, octreotide, oxytocin, pasireotide, penisatide, pentagastrin, pentreotide (with indium-111), phenypressin, pleurocidin, pramlintide, prorelin, thyrotropin-releasing hormone, TRH, TRF, salmon calcitonin, salami acetate, secretin (human), secretin (porcine), semaglutide, selacotide acetate, ACTH, adrenocorticotropic hormone, sermorelin acetate, GRF1-29, sinapeptide, KL4 in lucinactant, sincalide, somatostatin acetate, GHRH, GHRF, GRF, somatostatin acetate, spaglumat magnesium (or sodium) salt, substance P, taltirelin hydrate, terduglutide, teicoplanin, telavancin, teriparatide, terlipressin acetate, teicosin, thymosin alpha-1, thymopentin, trebananib, triptorelin, triptorelin pamoate, tyroserleutide, ularitide, vancomycin, vapreotide acetate, vasoactive intestinal peptide acetate, Vx-001c, TERT572Y, ziconotide acetate, α5-α6 Bax peptide, and β-defensin peptide.

[0083] In some embodiments, the peptide is any peptide that will aid in achieving the desired purpose with the composition. For example, in some embodiments, the peptide is any peptide that will facilitate the treatment of any type of disease and / or disorder.

[0084] In some embodiments, the peptide is an antigen.

[0085] In some embodiments, the antigen is selected from the group consisting of: peptide-based antigens, protein-based antigens, polysaccharide-based antigens, carbohydrate-based antigens, lipid-based antigens, glycolipid-based antigens, nucleic acid-based antigens, inactivated organism-based antigens, attenuated organism-based antigens, viral antigens, bacterial antigens, parasite antigens, allergen-derived antigens, and tumor antigens.

[0086] In some embodiments, the antigen is a tumor antigen as described herein.

[0087] In some embodiments, the antigen is any type of viral, bacterial, or self-antigen, including but not limited to FimH for urinary tract infection; soluble F protein from respiratory syncytial virus (RSV); NEF, GAG, and ENV proteins from HIV; Streptococcus pneumoniae proteins; HMGB1 proteins; hemagglutinin and neuroamidase proteins against influenza; viral antigens from HPV types 16 and 18; gL2, ICP4, gD2ΔTMR, gD2ΔTMR, or ICP4.2 from HSV-2; antigens from Streptococcus pneumoniae, such as pneumolysoid, choline binding protein A (CbpA), or pneumococcal surface protein A (PspA), SP1912, SP1912, SP1912L, SP0148 (with or without signal sequence), SP2108 (with or without signal sequence); antigens from Chlamydia trachomatis. trachomatis), such as CT209 polypeptide antigen, CT253 polypeptide antigen, CT425 polypeptide antigen, CT497 polypeptide antigen and CT843 polypeptide antigen; amyloid beta peptide.

[0088] In some embodiments, the antigen is conjugated to the outer surface of the nanoparticle. In some embodiments, the antigen is encapsulated within the nanoparticle.

[0089] 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 to make intracellular metal ions unavailable for cGAS-STING-type I IFN activation.

[0090] In certain embodiments, the present invention provides compositions capable of modulating innate immune activation that include one or more cell-permeable chelators (eg, metal ion chelators) to render intracellular metal ions unavailable to innate immune pathways.

[0091] In some embodiments, such cell permeable chelators (e.g., metal ion chelators) include, but are not limited to, polyphenol-based chelators (-)-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, Crofelemer, and the like.

[0092] In some embodiments, such compositions capable of inhibiting cGAS-STING activation and type I IFN responses are used to treat a subject having or at risk for an autoimmune disorder.

[0093] Thus, the present invention provides a method for treating an autoimmune disorder by administering to a subject (e.g., a human subject) a composition capable of modulating innate immune activation, the composition comprising one or more cell permeable chelators (e.g., metal ion chelators) to make 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, polyphenol-based chelators (-)-epigallocatechin gallate (EGCG), punicalagin, (-)-catechin gallate, (-)-catechin, tannic acid, tannin, punica granatum, ellagitannin, procyanidin C1, geraniol, theaflavin 3,3′-digallate, lipid-modified NTA, porphyrin, EDTA, NOTA, DOTA, TPEN, Crofelemer, and the like.

[0094] Examples of autoimmune disorders include, but are not limited to, systemic lupus erythematosus, Aicardi–Goutières syndrome, acute pancreatitis, age-dependent 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.

[0095] In some embodiments, additional therapeutic agents are co-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, pegseroma, etanercept, golimumab, infliximab), IL-1 inhibitors, and metalloproteinase inhibitors. In some embodiments, the therapeutic agent includes but is not limited to infliximab, adalimumab, etanercept, parenteral gold, or oral gold.

[0096] In certain embodiments, the present invention provides a method for treating cancer in a subject, comprising administering to the subject a composition as 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 immunostimulant, and an antigen (as described herein). In some embodiments, the subject is a human subject.

[0097] In some embodiments, the immunostimulatory agent is selected from anti-CTLA-4 antibodies, 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 inhibitors.

[0098] In some embodiments, the chemotherapeutic agent is selected from the group consisting of aldesleukin, altretinoin, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), actinomycin D, docetaxel, doxorubicin, dronabinol, epoetin alfa, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxy Urea, idarubicin, ifosfamide, interferon alfa, irinotecan, lansoprazole, levamisole, leucovorin, megestrol acetate, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (TAXOL), pilocarpine, prochlorperazine, rituximab, tamoxifen, Taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate.

[0099] In some embodiments, the cancer is one or more selected from the group consisting of 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, gastric cancer, head and neck cancer, testicular cancer, melanoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia and B-cell lymphoma, and uterine cancer.

[0100] Additional embodiments will be apparent to persons skilled in the relevant art(s) based on the teachings contained herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 : Schematic diagram of the synthesis of CDN-Zn, CDN-Zn@liposome and CDNs@CaP / PEI-PEG. (A) Zn 2+Coordinate crosslinking with CDNs enables the assembly of CNDs-Zn NPs, which are then further modified with liposomes. (B) CDNs can be loaded into CaP / PEI-PEG NPs during synthesis via charge interactions between CDNs and the PEI-PEG backbone.

[0102] Figure 2 Characterization of CDN-Zn, CDN-Zn@liposomes, and CDN@CaP / PEI-PEG. TEM images (top panel), size (middle panel), and zeta potential (bottom panel) of cdAMP-Zn (a), cdGMP-Zn (b), cGAMP-Zn (c), CDN-Zn@liposomes (d), and CDN@CaP / PEI-PEG (e).

[0103] Figure 3 : Release profiles and in vitro STING activation of different CDN formulations. (A) CDN loading efficacy of relevant formulations. The red line represents CDN absorbance before loading, while the blue line represents the absorbance of unloaded free CDN in the supernatant after loading. (B) CDN release kinetics from nanoformulations. (C) Representative THP1 activation evaluation by different concentrations of free CDN and CDN-Zn. The CDN used here is cdAMP. (D) Representative THP1 activation by different concentrations of free CDN and CDN@CaP / PEI-PEG. The CDN used here is cdAMP(ps)2.

[0104] Figure 4 : The therapeutic effect of CDN formulation in CT26 tumor model. (AC) 6-7 week old Balb / c mice were inoculated with 1.5×10 5 CT26 tumor cells. On days 10 and 15, tumor-bearing mice were treated intratumorally with the indicated formulations containing 25 μg / dose of adAMP(ps)2. Shown are (A) average tumor growth curves of tumor-bearing mice; (B) survival rates of mice after different treatments; and (C) tumor growth curves of individual mice in different groups. (D-E) Seven days after the second dose of CDN treatment, PBMCs were harvested for (D) tetramer staining and (E) ELISPOT analysis with AH1 peptide. (F) Seven days after the first dose of CDN treatment, PBMCs were harvested for ELISPOT analysis with AH1 peptide.

[0105] Figure 5Figure 2: Metal ions enhance cGAS-STING-type I interferon activation in vitro. Figures ac) Bone marrow-derived dendritic cells (BMDCs) (ab) and the human monocytic cell line THP1 (c) were incubated with varying concentrations of metal ions with or without STING agonists. STING activation was quantified by interferon-β (IFN-β) release in the cell culture medium.

[0106] Figure 6 :In vivo through Co 2+ and Mn 2+ Enhanced STING activation and cancer therapeutic efficacy. a) Individual tumor growth curves after three intratumoral injections of the indicated formulations on days 9, 12, and 15 after tumor inoculation. b) Serum IFN-β concentrations 8 hours after the first dose of the indicated formulations. cd) Individual tumor growth (c) and survival (d) of tumor-bearing mice treated with the indicated formulations.

[0107] Figure 7 :After passing through Co 2+ and Mn 2+ Enhanced STING activation leads to improved antigen-specific immune responses. a) Percentage of AH1-specific CD8+ T cells in PBMCs on day 16. b) IFN-γ-secreting cell counts per 5E4 PBMCs after stimulation with AH1 peptide on day 22. ce) Timeline (c), tumor growth curve (d), and percentage of AH1-specific CD8+ T cells in splenic CD8+ T cells (e) from day 81 of the tumor rechallenge study.

[0108] Figure 8: Cytokine profiles of representative PAMPs modulated by metal ions in vitro. (a) Bone marrow-derived dendritic cells (BMDCs) were incubated with various concentrations of metal ions (with or without the TLR3 agonist poly-IC). (e) BMDCs were incubated with various concentrations of metal ions (with or without the TLR4 agonist MPLA). (gh) BMDCs were incubated with various concentrations of metal ions (with or without the TLR7 / 8 agonist R848). (ij) 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.

[0109] Figure 9Figure 2: Modulation of immune responses to representative NOD-like receptor (NLR) ligands by metal ions in vitro. (a-f) Bone marrow-derived dendritic cells (BMDCs) were incubated with varying concentrations of metal ions with or without the NOD1 agonist C12-iE-DAP. (g-1) BMDCs were incubated with varying concentrations of metal ions with or without the NOD2 agonist C18-MDP. Cytokine levels in the cell culture medium were quantified by ELISA. Control: relative PAMPs in saline.

[0110] Figure 10 : Modulation of immune responses to representative RIG-I-like receptor (RLR) ligands by metal ions in vitro. af) Bone marrow-derived dendritic cells (BMDCs) were incubated with different concentrations of metal ions (with or without the RLR ligand poly(dA:dT) / LyoVec TM The cells were incubated with 1% paraformaldehyde (PAMP) and 1% paraformaldehyde (PAMP) (Invivogen). Cytokine levels in the cell culture medium were quantified by ELISA. Control: relative PAMP in saline.

[0111] Figure 11 : In vitro modulation of immune responses to representative inflammasome inducers by metal ions. af) Bone marrow-derived dendritic cells (BMDCs) were pretreated with 300 ng / ml phorbol 12-myristate 13-acetate (PMA) for 3 h, then washed twice and treated with 10-200 mg / ml alum crystals. NLRP3 inflammasome formation can be characterized by IL-1b secretion. (gk) BMDCs were incubated with Escherichia coli (E. coli) outer membrane vesicles, an atypical inflammasome inducer, and various metal ions at varying concentrations. Cytokine levels in the cell culture medium were quantified by ELISA. Control: relative PAMPs in saline.

[0112] Figure 12 Figure 3: In vitro immunological effects of individual metal ions. a) Bone marrow-derived dendritic cells (BMDCs) were exposed to various concentrations of metal ions. Cytokine levels in the cell culture medium were quantified by ELISA. Control: saline.

[0113] Figure 13Representative formulations consisting of innate immune stimulants and metal ions 1. a) Scheme of the metal ion-polyHis-DOPE@liposome nanoparticle composition. b) TEM image of manganese-CDA-H11-DOPE@liposome nanoparticles (Mn-CDA / H11@liposome). ce) Tumor growth curves and the number of cured tumor-free mice out of five mice treated with the indicated formulations: intratumoral (IT) injection of c) three doses of 5 μg free CDA / Mn2+ or Mn-CDA / H11@liposomes containing 5 μg CDA and d) three doses of 1 μg free CDA / Mn2+ or Mn-CDA / H11@liposomes containing 1 μg CDA on days 9, 12, and 15 after tumor inoculation; and intravenous (IV) injection of e) three doses of 20 μg free CDA / Mn2+ or Mn-CDA / H11@liposomes containing 20 μg CDA on days 9, 12, and 15 after tumor inoculation. f) AH-1 antigen-specific T cell ratio in PBMCs 7 days after the first dose. g) ELISPOT counts per 100,000 PBMCs 14 days after the first dose. hj) Serum IFN-β, IP10, and TNF-α levels four hours after injection of the indicated formulations.

[0114] Figure 14 : Representative formulations consisting of innate immune stimulators and metal ions. 2. a) Scheme of metal ion-poly-His-PEG nanoparticle compositions. b) TEM images of Co-CDA / H33-PEG nanoparticles. c) In vitro STING activation of BMDCs treated with the indicated formulations. d) Serum IFN-β after a single intratumoral injection of the indicated formulations in a B16F10 melanoma model. ef) Tumor growth (e) and individual tumor growth (f) in mice treated with the indicated formulations. Three doses of 5 μg free CDA / Mn2+ or Mn-CDA-H33-PEG containing 5 μg CDA were injected (IT) into CT16 tumors on days 9, 12, and 15 after tumor inoculation. gh) AH-1 antigen-specific T cell ratio in PBMCs 7 days after the first dose (g) and ELISPOT counts per 100,000 PBMCs 14 days after the first dose.

[0115] Figure 15: Representative formulations composed of innate immune stimulants and metal ions 3. a) Schematic composition of metal ion-4-arm-PEG-polyHis coordination hydrogel. Shown is CDA@Co2+-4-arm-PEG-His11 hydrogel (CDA@4aH11-Co hydrogel). b) Injectable trypan blue@4aH11-Co hydrogel retained at the injection site 6 hours after injection. ce) Individual tumor growth in mice treated with the indicated formulations. On days 9, 12, and 15 after tumor inoculation, three doses of 20 μg free CDA / Mn2+ or hydrogel containing 20 μg CDA were injected intratumorally (IT). f) Photographs of representative tumors after treatment with CDA@4a H11-Co hydrogel.

[0116] Figure 16 Other representative formulations for delivering metal ions and PAMPs include: a) Metal ion and CDN self-assemblies; b) Liposome-coated CDN-metal ion coordination nanoparticles; c) Polyhistidine-coated nanoparticles; and e) Polymer-stabilized metal-CDN coordination nanoparticles or metal mineral nanoparticles. Examples include poly(histidine)-polyethylene glycol copolymers (PH-PEG or pHis-PEG), poly(ethyleneimine)-polyethylene glycol (PEI-PEG), poly(lysine)-polyethylene glycol (PEG) (PK-PEG), and anionic poly(glutamic acid)-polyethylene glycol (PGA-PEG).

[0117] Figure 17: From Figure 12 The therapeutic effects of selected formulations in the CT26 colon tumor model. a) Representative THP1 activation evaluation by free CDN and CDN-Zn at different concentrations. The CDN used here is cdAMP. b) Representative THP1 activation by free CDN and CDN@CaP / PEI-PEG at different concentrations. The CDN used here is cdAMP(ps)2. b) 6-7 week old Balb / c mice were inoculated with 1.5×10 5 CT26 tumor cells. On days 10 and 15, tumor-bearing mice were treated intratumorally with the indicated formulations containing 25 μg / dose of adAMP(ps)2. Shown are: c) average tumor growth curves of tumor-bearing mice; d) survival rates of mice after different treatments; and e) tumor growth curves of individual mice in different groups. (fg) Tetramer staining seven days after the first dose (f) and ELISPOT analysis seven days after the second dose (g).

[0118] Figure 18: Chelating metal ions to inhibit the cGAS-STING-type I IFN pathway. a) Molecular structures of representative chelators that can inhibit the cGAS-STING-type I IFN pathway. b) THP 1 bi-KI-hSTING treated with DNA / lipofectamine 2000 (ThermoFisher, 11668027). WT(R232) Dose-inhibition curves of the indicated compounds on IFN-I responses (b) and NF-κB inflammatory responses (c) in reporter cells (Invivogen, THP-D-R232). d) Cell viability of bc. e) THP-I-ISGhSTING treated with DNA / Lipofectamine 2000 (ThermoFisher, 11668027). HAQ Dose-inhibition curves of the indicated compounds on IFN-I responses in reporter cells (Invivogen, thp-isg). (f) Double-KI-hSTING in cGAMP-treated THP 1 cells. WT(R232) Dose-inhibition curves of the indicated compounds on IFN-I responses in reporter cells (Invivogen, thpd-r232).

[0119] Figure 19 : Chelating metal ions to inhibit the TLR3-type I IFN pathway. Dose-inhibition curves of the indicated compounds on IFN-I responses in THP1 dual-STING KO reporter cells (Invivogen) treated with polyIC / Lipofectamine 2000 (ThermoFisher).

[0120] Figure 20 : Molecular structures of other representative potent polyphenol chelating agents.

[0121] definition

[0122] To facilitate understanding of the present invention, a number of terms and phrases are defined below:

[0123] As used herein, the term "lipid" or "lipid molecule" refers to a water-insoluble fatty substance, and includes fats, oils, waxes and related compounds. They can be made in the blood (endogenous) or taken in (exogenous) in the diet. Lipid is essential for normal body function, and no matter whether it is produced from an exogenous or endogenous source, they must be transported and then released for use by cells. The production, transport and release of lipids for cell use are referred to as lipid metabolism. Although there are several classes of lipids, the main two classes are cholesterol and triglycerides. Cholesterol can be taken in the diet and is manufactured by the cells of most organs and tissues in the body (mainly in the liver). Cholesterol can exist in its free form, or more commonly in combination with fatty acids, referred to as cholesterol esters. As used herein, "lipid" or "lipid molecule" refers to any lipophilic compound. Non-limiting examples of lipid compounds include fatty acids, cholesterol, phospholipids, complex lipids and derivatives or analogs thereof. They are generally divided into at least three categories: (1) "simple lipids," which include fats and oils as well as waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derivatized lipids," such as steroids. Lipids or lipid molecules suitable for use in the present invention include both membrane-forming and non-membrane-forming lipids.

[0124] As used herein, the term "lipoprotein" refers to a spherical compound whose structure is such that water-insoluble lipids are contained in a partially water-soluble shell. Depending on the type of lipoprotein, the content includes varying amounts of free and esterified cholesterol, triglycerides, and apoproteins or apolipoproteins. There are five major types of lipoproteins, which differ in function and in their lipid and apoprotein content and are classified according to their increased 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 bound to lipoproteins.

[0125] As used herein, the term "HDL" or "high-density lipoprotein" refers to high-density lipoprotein. HDL comprises a complex of approximately equal amounts of lipids and proteins that acts as a transporter of cholesterol in the blood. HDL is primarily synthesized and secreted in the liver and intestinal epithelial cells. Immediately after secretion, HDL takes the form of disc-shaped particles containing apolipoprotein AI (also known as apoA-I) and phospholipids as its main components, and is also referred to as nascent HDL. This nascent HDL receives free cholesterol from the cell membranes of surrounding cells or from the free cholesterol produced during the hydrolysis of other lipoproteins in the blood and forms mature spherical HDL, while retaining cholesterol esters converted from the cholesterol by the action of LCAT (phosphatidylcholine cholesterol acyltransferase) at its hydrophobic center. HDL plays an extremely important role in the lipid metabolism process known as "reverse cholesterol transport," which absorbs cholesterol from peripheral tissues in the blood and transports it to the liver. High levels of HDL are associated with a reduced risk of atherosclerosis and coronary heart disease (CHD), as reverse cholesterol transport is considered one of the major mechanisms of HDL's protective effect against atherosclerosis.

[0126] As used herein, the terms "synthetic HDL," "sHDL," "reconstituted HDL," or "rHDL" refer to particles that are structurally similar to native HDL and are composed of one or more lipids bound to at least one of the proteins of HDL (preferably Apo AI) or its mimetics. Typically, the components of sHDL can be derived from blood or produced by recombinant technology.

[0127] As used herein, the term "complexation" as used herein refers to the non-covalent interaction of a biomacromolecular agent (eg, antigen, adjuvant, etc.) with a nanoparticle and / or microparticle.

[0128] As used herein, the term "conjugation" as used herein refers to the covalent bonding between a biomacromolecular agent (eg, antigen, adjuvant, etc.) and a nanoparticle and / or microparticle.

[0129] As used herein, the term "encapsulated" refers to the location of a biomacromolecular agent (eg, antigen, adjuvant, etc.) surrounded or completely contained within the interior of a nanoparticle and / or microparticle.

[0130] As used herein, the term "absorption" refers to the biomacromolecular agent (eg, antigen, adjuvant, etc.) being brought into and stably retained in the interior of the nanoparticle and / or microparticle, ie, inside the outer surface.

[0131] As used herein, the term "adsorption" refers to the attachment of a biomacromolecule 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 bonding between two or more oppositely charged or ionic chemical groups. Typically, adsorption is reversible.

[0132] As used herein, the term "incorporated" refers to dissolving, dispersing or suspending a biomacromolecule agent (e.g., antigen, adjuvant, etc.) in a nanoparticle and / or microparticle. In some cases, the biomacromolecule agent can be uniformly incorporated in the nanoparticle and / or microparticle.

[0133] As used herein, the term "biological biomacromolecule" or "biomacromolecule" or "biomacromolecule agent" as used herein refers to a molecule with a molecular weight greater than 1 kDa that can be isolated from an organism or cell culture, such as a eukaryotic (e.g., mammalian) cell culture or a prokaryotic (e.g., bacterial) cell culture. In some embodiments, the use of the term refers to polymers, such as biopolymers, such as 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 "biomacromolecule" refers to a protein. In some embodiments, the term "biomacromolecule" refers to a recombinant protein or a fusion protein. In some embodiments, the protein is soluble. In some embodiments, the biomacromolecule is an antibody, such as a monoclonal antibody. In some embodiments, the biomacromolecule is an adjuvant, an antigen, a therapeutic agent, an imaging agent, etc.

[0134] As used herein, the term "antigen" is defined herein as a molecule comprising one or more epitopes that stimulate the host immune system to produce 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 cells 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. The antigen can be a natural or synthetic antigen, such as a hapten, a polytope, a flanking epitope, and other antigens of recombinant or synthetic origin (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)).

[0135] As used herein, the term "neoantigen" or "neoantigenic" refers to a class of tumor antigens that are derived from one or more tumor-specific mutations that alter the amino acid sequence of the genome-encoded protein.

[0136] As used herein, the term "tumor-specific antigen" is defined herein as an antigen that is unique to tumor cells and is not found in or on other cells in the body.

[0137] As used herein, the term "tumor-associated antigen" is defined herein as an antigen that is not specific to tumor cells and is also expressed in or on normal cells under conditions that fail to induce an immune response to the antigen.

[0138] As used herein, the term "adjuvant" is defined herein as a substance that increases the immune response to other antigens when administered together with the other antigens. Adjuvants are also referred to herein as "immunoenhancers" and "immunomodulators."

[0139] As used herein, the term "antigen presenting cell" is defined herein as a highly specialized cell that processes antigens and displays their peptide fragments on the cell surface along with molecules required for lymphocyte activation. The major antigen presenting cells for T cells are dendritic cells, macrophages, and B cells. The major antigen presenting cells for B cells are follicular dendritic cells.

[0140] As used herein, the term "cross-presentation" is defined herein as the ability of antigen-presenting cells to take up, process, and present extracellular antigens with MHC class I molecules to CD8 T cells (cytotoxic T cells). This process induces cellular immunity against most tumors and against viruses that do not infect antigen-presenting cells. Cross-presentation is also required to induce cytotoxic immunity by vaccination with protein antigens (e.g., in tumor vaccination).

[0141] As used herein, the term "immune," "immunological," or "immune" response is the development of a humoral and / or cellular response to an antigen.

[0142] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of sHDL nanoparticles as described herein (e.g., a composition comprising sHDL nanoparticles encapsulating siRNA) (e.g., a composition comprising sHDL nanoparticles configured to activate an immune response), such a delivery system includes a system and / or supporting materials (e.g., written instructions for using the materials, etc.) that allow storage, transport from one location to another, or delivery of such a composition. For example, a kit includes one or more enclosures (e.g., boxes) containing neccary agents and / or supporting materials. As used herein, the term "fragmented kit" refers to a delivery system comprising two or more separate containers, each of which contains a sub-portion of all the 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 the ingredients necessary to synthesize such sHDL nanoparticles, while a second container contains a second reagent (e.g., siRNA, antigen, adjuvant) (e.g., antibiotic or spray applicator). In fact, the term "fragmented kit" includes any delivery system that includes two or more separate containers, each of which contains a sub-portion of all the kit components. In contrast, a "combination kit" refers to a delivery system that contains all the components necessary to synthesize and utilize any of the sHDL nanoparticles as described (e.g., in a single box containing each required component). The term "kit" includes both fragmented kits and combination kits.

[0143] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that is to be the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein with reference to a human subject.

[0144] As used herein, the term "sample" is used in its broadest sense. In a sense, this is meant to include samples or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and include fluids, solids, tissues and gases. Biological samples include blood products, such as plasma, serum, etc. Environmental samples include environmental materials, such as surface materials, soil, water, crystals and industrial samples. However, such examples should not be interpreted as limiting the sample types applicable to the present invention.

[0145] As used herein, the term "in vitro" refers to an artificial environment and processes or reactions that occur in 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 cell) and processes or reactions that occur in a natural environment.

[0146] As used herein, the term "drug" or "therapeutic agent" is intended to include any molecule, molecular complex, or substance that is administered to an organism for diagnostic or therapeutic purposes, including medical imaging, monitoring, contraception, cosmetic, nutraceutical, pharmaceutical, and prophylactic applications. The term "drug" is further intended to include any such molecule, molecular complex, or substance that is chemically modified and / or operatively attached to a biological or biocompatible structure.

[0147] As used herein, the term "solvent" refers to the medium in which a reaction is carried out. The solvent can be, but is not limited to, a liquid. Solvent classes include, but are not limited to, non-polar, polar, protic, and aprotic. DETAILED DESCRIPTION

[0148] CDN 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 patterns) that bind to a PRR (pathogen recognition receptor) called STING. STING is an adaptor protein in the cytoplasm of host mammalian cells that activates the TANK binding kinase (TBK1)-IRF3 signaling axis, leading to the induction of IFN-β and other IRF-3 dependent gene products, which strongly activate innate immunity. It is now recognized that STING is a component of the host cytoplasmic surveillance pathway that senses infection by intracellular pathogens and induces IFN-β production 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.

[0149] Immunotherapy is advancing cancer treatment in many ways. Recently, it has been discovered that activating the innate immune system through cyclic GAM-AMP (cGAMP), a stimulator of the IFN gene (STING) pathway, can initiate a strong anti-tumor immune response. In addition to cGAMP, various other cyclic dinucleotides (CDNs), such as cdiAMP, cdiGMP, and cAIMP, can also activate the STING pathway, which is considered to be an essential immune defense mechanism against tumors and exogenous pathogens. However, due to the small molecular weight, poor pharmacokinetic properties, and severe off-target cytotoxicity, STING agonists need to be injected directly into the tumor locally. Experiments conducted during the process of developing embodiments of the present invention found that in the presence of (1) Zn 2+Or (2) in the presence of calcium phosphate and PEI-PEG, CDN can be assembled into uniform nanoparticles. Based on these results, two types of drug delivery systems for delivering CDN were developed. In the subcutaneous CT26 tumor model, the formulations showed significant inhibition of tumor growth and achieved complete regression rates of 40% and 60%. Therefore, these formulations represent a new class of drug delivery systems for both local and systemic delivery of STING agonists.

[0150] Such results have important clinical implications, as these nanoparticles conjugated to CDNs can induce tumor-specific immune responses through systemic administration, thus avoiding the need for direct local injection into tumors.

[0151] Additional experiments conducted during the course of developing embodiments of the present invention determined that certain metal ions such as Mn 2+ and Co 2+ STING agonists can enhance STING activation and type I IFN responses. In the mouse CT26 colon tumor model, Mn 2+ / Co 2+ The combination of -STING agonists exhibited elevated serum type I IFN levels, produced higher tumor eradication efficacy, and promoted longer survival of tumor-bearing mice, of which 80% were cured and resistant to a second tumor challenge after 80 days. In addition, this phenomenon was found to be prevalent in various other innate immune pathways, including but not limited to Toll-like receptor (TLR) 3 / 4 / 7 / 8 / 9 ligands, NOD1 / 2 ligands, TLR 7 / 8 ligands, RIG-I & CDS agonists, and inflammasome inducers. Based on this discovery, some pharmaceutically acceptable formulations, such as metal salts of DAMP / PAMP, coordination formulations and other metal-loaded formulations (hydroxide / carbonate / phosphate minerals, liposomes, self-assembled nanoparticles, PLGA, hydrogels, emulsions, etc.), can be developed to precisely deliver and release metal-innate immune stimulator combinations to the desired target in an ideal manner. Finally, we found that some chelators could effectively inhibit DNA-induced cGAS-STING-type I IFN / NFkB responses and poly-IC-induced TLR3-cGAS-STING-type I IFN.

[0152] Thus, such results and embodiments demonstrate a new class of drug delivery systems for local and systemic delivery of agents capable of stimulating an innate immune response in a subject upon administration to the subject.

[0153] 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 relates to such compositions comprising an agent capable of stimulating an innate immune response in the subject upon administration to the subject, methods for synthesizing such compositions, and systems and methods utilizing such compositions (e.g., in diagnostic and / or therapeutic settings).

[0154] Thus, in certain embodiments, the present invention provides compositions comprising one or more DAMPs and / or PAMPs, and

[0155] a) calcium phosphate and copolymers of cationic poly(ethyleneimine) (PEI) and 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); or

[0156] b) one or more cations selected from the group consisting of: 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 related phosphates or carbonates.

[0157] Such compositions are not limited to specific DAMP or PAMP agonists.

[0158] In some embodiments, the DAMP and PAMP agonists are selected from STING agonists, purine-containing or purine-derived agents, 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.

[0159] In some embodiments, the DAMP and PAMP agonist is 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 a NLRP3 agonist.

[0160] Such compositions are not limited to specific purine-containing or purine-derived agents. In some embodiments, the purine-containing or purine-derived agent is selected from 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), fluorinated 3'3'-cGAMP, fluorinated c-di-AMP, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), fluorinated c-di-GMP, 2'3'-c-di-GMP, c-di-IMP, cGAMP AMP, 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, fluorinated cGAMP, fluorinated 3'3'-cGAMP, fluorinated 2'3'-cGAMP, fluorinated 2'2'-cGAMP, 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, fluorinated c-di-AMP, fluorinated 2'3'-cdAMP, fluorinated 2'2'-cdAMP, fluorinated 3'3'-cdAMP, 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, fluorinated cdGMP, fluorinated 2'3'-cdGMP, fluorinated 2'2'-cdGMP, fluorinated 3'3'-cdGMP, cAIMP, 2'3'-cAIMP, 2'2'-cAIMP, 3'3'-cAIMP, cAIMP difluoro(fluorinated 3' 3'-cAIMP, fluorinated 2'3'-cAIMP, fluorinated 2'2'-cAIMP, 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, fluorinated c-di-IMP, fluorinated 2'3'-cdIMP, fluorinated 2'2'-cdIMP, fluorinated 3'3'-cdIMP, imiquimod, resiquimod, 6-(4-amino-imidazoquinolinyl)-norleucine, and purine-based PI3K inhibitors.

[0161] Such compositions are not limited to a specific STING agonist. In some embodiments, the STING agonist is a cyclic dinucleotide. For example, in some embodiments, the cyclic dinucleotide is cdi-AMP, cGAMP, or cGMP, 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), fluorinated 3'3'-cGAMP, fluorinated c-di-AMP, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), fluorinated c-di-GMP, 2'3'-c-di-GMP, c-di-IMP, SB11285, STING-agonist-C11, STING agonist-1, STING agonist G10, gemcitabine, and additional STING agonists as described herein.

[0162] 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. Additional examples of cyclic purine dinucleotides are described in detail in, for example, U.S. Patent Nos. 7,709,458 and 7,592,326; WO 2007 / 054279; and Yan et al., Bioorg. Med. Chem Lett. 18:5631 (2008), each of which is incorporated herein by reference.

[0163] Additional suitable STING agonists for use in the disclosed methods include, but are not limited to, flavonoids. In some embodiments, the STING agonist can include a flavonoid. In other embodiments, the STING agonist can consist of a flavonoid. Suitable flavonoids include, but are not limited to, 10-(carboxymethyl)-9(10H)acridone (CMA), 5,6-dimethylxanthone-4-acetic acid (DMXAA), isooxyflavones, 6,4′-dimethoxyflavones, 4′-methoxyflavones, 3′,6′-dihydroxyflavones, 7,2′-dihydroxyflavones, daidzein, formononetin, crotalurine 7-methyl ether, xanthone, or any combination thereof. In some aspects, the STING agonist can be 10-(carboxymethyl)-9(10H)acridone (CMA). In some aspects, the STING agonist can be 5,6-dimethylxanthone-4-acetic acid (DMXAA). In some aspects, the STING agonist can be an isoxiflavone. In some aspects, the STING agonist can be 6,4′-dimethoxyflavone. In some aspects, the STING agonist can be 4′-methoxyflavone. In some aspects, the STING agonist can be 3′,6′-dihydroxyflavone. In some aspects, the STING agonist can be 7,2′-dihydroxyflavone. In some aspects, the STING agonist can be daidzein. In some aspects, the STING agonist can be formononetin. In some aspects, the STING agonist can be crotalurine 7-methyl ether. In some aspects, the STING agonist can be a xanthone. In some aspects, the STING agonist can be any combination of the above-mentioned flavonoids. Thus, for example, in some embodiments, the flavonoid comprises DMXAA.

[0164] 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), fluorinated 3'3'-cGAMP, fluorinated c-di-AMP, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), fluorinated c-di-GMP, 2'3'-c-di-GMP, c-di-IMP, SB11285, STING-agonist-C11, STING agonist-1, STING agonist G10, and gemcitabine.

[0165] 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 to make intracellular metal ions unavailable for cGAS-STING-type I IFN activation.

[0166] In certain embodiments, the present invention provides compositions capable of modulating innate immune activation that include one or more cell-permeable chelators (eg, metal ion chelators) to render intracellular metal ions unavailable to innate immune pathways.

[0167] In some embodiments, such cell permeable chelators (e.g., metal ion chelators) include, but are not limited to, polyphenol-based chelators (-)-epigallocatechin gallate (EGCG), punicalagin, (-)-catechin gallate, (-)-catechin, tannic acid, tannin, punicalagin, quercetin, procyanidin C1, geraniol, theaflavin 3,3′-digallate, lipid-modified NTA, porphyrin, EDTA, NOTA, DOTA, TPEN, Crofelemer, and the like.

[0168] In some embodiments, such compositions capable of inhibiting cGAS-STING activation and type I IFN responses are used to treat a subject having or at risk for an autoimmune disorder.

[0169] Thus, the present invention provides a method for treating an autoimmune disorder by administering to a subject (e.g., a human subject) a composition capable of modulating innate immune activation, the composition comprising one or more cell permeable chelators (e.g., metal ion chelators) to make 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, polyphenol-based chelators (-)-epigallocatechin gallate (EGCG), punicalagin, (-)-catechin gallate, (-)-catechin, tannic acid, tannin, punica granatum, ellagitannin, procyanidin C1, geraniol, theaflavin 3,3′-digallate, lipid-modified NTA, porphyrin, EDTA, NOTA, DOTA, TPEN, Crofelemer, and the like.

[0170] Examples of autoimmune disorders include, but are not limited to, systemic lupus erythematosus, Aicardi–Goutières syndrome, acute pancreatitis, age-dependent 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.

[0171] In some embodiments, additional therapeutic agents are co-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, pegseroma, etanercept, golimumab, infliximab), IL-1 inhibitors, and metalloproteinase inhibitors. In some embodiments, the therapeutic agent includes but is not limited to infliximab, adalimumab, etanercept, parenteral gold, or oral gold.

[0172] In certain embodiments, a composition comprising an agent (e.g., DAMP / PAMP) capable of stimulating an innate immune response in a subject upon administration to the subject is associated with (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, incorporated into) a nanoparticle.

[0173] In some embodiments, such compositions combined with nanoparticles are further combined (e.g., complexed, put together, encapsulated, absorbed, adsorbed, blended) with calcium phosphate and a copolymer of PEI / PEG, PH-PEG, PK-PEG or PGA-PEG. In fact, in some embodiments, the combination of an agent capable of stimulating an innate immune response in a subject with the nanoparticles is carried out in the presence of a copolymer of calcium phosphate and PEI / PEG, PH-PEG, PK-PEG or PGA-PEG.

[0174] In some embodiments, such compositions associated with nanoparticles are further associated (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, incorporated) with one or more cations selected from the group consisting of: 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 + Indeed, in some embodiments, the binding of an agent capable of stimulating an innate immune response in a subject to the nanoparticle is in the presence of such a cation (e.g., Zn 2+ 、Co 2+ or Mn 2+ ) is carried out in the presence of .

[0175] Those skilled in the art know that STING (stimulator of interferon genes) is an adaptor of multiple cytoplasmic DNA receptors and is a pattern recognition receptor (PRR) that recognizes the bacterial second messengers cyclic diadenosine monophosphate (c-di-AMP) and cyclic diguanosine 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 and activates the adaptor protein STING and induces IFN. STING includes five putative transmembrane regions, is primarily located in the endoplasmic reticulum, and is able to activate both the NF-κB and IRF3 transcriptional pathways to induce the expression of type I interferons (IFN-α and IFN-β) and exert an effective antiviral state after expression.

[0176] Thus, DAMPs and PAMPs (eg, STING agonists) can stimulate intrinsic cytokine responses in cancer cells. Thus, in some embodiments, DAMPs and PAMPs (eg, STING agonists) can stimulate intrinsic cytokine responses in cancer cells.

[0177] The innate cytokine response stimulated by a DAMP or PAMP is mediated by a cytokine. In some embodiments, for example, the innate cytokine response can be mediated by type 1 interferon.

[0178] As described, the present disclosure provides compositions and methods for stimulating an innate immune response in cancerous cells with an agent (e.g., DAMP / PAMP) capable of stimulating an innate immune response in a subject to suppress and / or inhibit the growth of such cancer cells (e.g., tumor cells) following administration to a subject. In particular, the present invention relates to compositions comprising nanoparticles associated with (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, incorporated into) an agent (e.g., DAMP / PAMP) capable of stimulating an innate immune response in the subject following administration to a subject, methods for synthesizing such nanoparticles, and systems and methods utilizing such nanoparticles (e.g., in diagnostic and / or therapeutic settings).

[0179] Indeed, experiments conducted during the course of developing embodiments of the present invention have shown that in the presence of Zn 2+ In the presence of Zn 2+ Such CDNs assembled into uniform nanoparticles in the presence of calcium phosphate and a copolymer of cationic poly(ethyleneimine) (PEI) and polyethylene glycol (PEG) are further stabilized with lipid vesicles. Additional experiments have shown that CDNs can be formulated into nanoparticles in the presence of calcium phosphate and a copolymer of cationic poly(ethyleneimine) (PEI) and polyethylene glycol (PEG). It is further shown that such CDN-nanoparticle assemblies (e.g., CDNs formulated into nanoparticles in the presence of calcium phosphate and a copolymer of PEI-PEG) (e.g., in the presence of Zn 2+ 、Co 2+ or Mn 2+ CDNs formulated into nanoparticles in the presence of liposomes provide increased cancer cell uptake and more precise targeting to the tumor microenvironment (e.g., TME), enabling increased delivery efficacy and reduced toxicity of STING agonists.

[0180] The present invention is not limited to a particular type or class of nanoparticles that are associated (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, incorporated) with such compositions comprising agents (e.g., DAMPs / PAMPs) that are capable of stimulating an innate immune response in a subject upon administration to the subject.

[0181] 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, fullerene (also known as C 60 、C 70 、C 76 、C 80 、C 84), endohedral metallofullerenes (EMI) buckyballs (which contain additional atoms, ions, or clusters within their fullerene cages), trimetallic nitride templated endohedral metallofullerenes (TNT EMEs, high symmetry tetraatomic molecular cluster intercalates formed in trimetallic nitride templates within carbon cages), 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 metallofullerenes and / or other internal chemical structures), carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, quantum dots, superparamagnetic nanoparticles, nanorods, and cellulose nanoparticles. Particle embodiments may also include microparticles with the ability to enhance efficacy or selectivity. Other non-limiting exemplary nanoparticles include glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold, silver, carbon, and iron nanoparticles.

[0182] In some embodiments, the nanoparticles are modified micelles. In these embodiments, the modified micelles include a polyol polymer modified to include a hydrophobic polymer block. The term "hydrophobic polymer block" as used in this disclosure refers to a segment of a polymer that will itself be hydrophobic. As used herein, the term "micelle" refers to an aggregate of molecules dispersed in a liquid. Typical micelles in aqueous solutions form aggregates with a hydrophilic "head" region in contact with the surrounding solvent, thereby isolating a hydrophobic single tail region in the center of the micelle. In some embodiments, the head region can be, for example, a surface region of a polyol polymer, and the tail region can be, for example, a hydrophobic polymer block region of the polyol polymer.

[0183] The present invention further includes the use of micron-sized particles in addition to nanometer-sized particles. In the case of using microparticles, it is preferred that they are relatively small, on the order of 1-50 microns. For ease of discussion, the use of "nanoparticles" herein encompasses true nanoparticles (having a size of about 1 nm to about 1000 nm), microparticles (e.g., about 1 micron to about 50 microns), or both.

[0184] Examples of nanoparticles include, by way of example and without limitation, paramagnetic nanoparticles, superparamagnetic nanoparticles, metallic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers, dendrimers with covalently attached metal chelates, nanofibers, nanohorns, nano-onions, nanorods, nanoropes, and quantum dots. In some embodiments, the nanoparticles are metallic nanoparticles (e.g., nanoparticles of gold, palladium, platinum, silver, copper, nickel, cobalt, iridium, or alloys of two or more thereof). As in core-shell nanoparticles, the nanoparticles may include a core or a core and a shell.

[0185] In some embodiments, the nanoparticles are sHDL nanoparticles. Typically, sHDL nanoparticles are composed of a mixture of HDL apolipoprotein and amphiphilic lipids.

[0186] The present invention is not limited to the use of a specific type or species of HDL apolipoproteins. HDL apolipoproteins include, 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 the group consisting of preproapolipoprotein, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, preApoA-II, ApoA-II, preproApoA-IV, preApoA-IV, ApoA-V, preproApoE, preApoE, ApoE, preproApoA-1Milano, preApoA-1Milano, ApoA-1Milano, preproApoA-IParis, preApoA-IParis, and ApoA-IParis, and peptide mimetics of mixtures thereof. Preferably, the carrier particle is composed of Apo AI or Apo A-II, however, other lipoproteins including apolipoprotein A4, apolipoprotein Cs, or apolipoprotein E can be used alone or in combination to formulate a carrier particle mixture for delivery of the therapeutic agent. In some embodiments, mimetics of such HDL apolipoproteins are used.

[0187] ApoA-I is synthesized by the liver and small intestine as a preproapolipoprotein, which is secreted as a proprotein that is rapidly cleaved to generate a mature polypeptide with 243 amino acid residues. ApoA-I is mainly composed of 6 to 8 different 22 amino acid repeats, which are separated by a linker portion that is usually proline, and in some cases, consists of a sequence segment (stretch) consisting of several residues. ApoA-I forms three types of stable complexes with lipids: a small lipid-poor complex, called pre-β-1HDL (pre-β-1HDL); a flat disc-shaped particle containing polar lipids (phospholipids and cholesterol), called pre-β-2HDL; and a spherical particle containing both polar and non-polar lipids, called spherical or mature HDL (HDL3 and HDL2). Most HDL in the circulating population contains both ApoA-I and ApoA-II (the second major HDL protein).

[0188] In some embodiments, an ApoA-I agonist or mimetic is provided. In some embodiments, such an ApoA-I mimetic is capable of forming an amphipathic α-helix that mimics ApoA-I activity and has a specific activity that approaches or exceeds that of the native molecule. In some, the ApoA-I mimetic is a peptide or peptide analog that forms an amphipathic helix (in the presence of lipids), binds lipids, forms pre-β-like or HDL-like complexes, activates phosphatidylcholine: cholesterol acyltransferase (LCAT), increases serum levels of the HDL fraction, and promotes cholesterol efflux.

[0189] The present invention is not limited to the use of a specific ApoA-I mimetic. In some embodiments, any ApoA-I mimetic described in Srinivasa, et al., 2014 Curr. Opinion Lipidology Vol. 25(4): 304-308 is used. In some embodiments, any ApoA-I mimetic described in U.S. Patent Application Publication Nos. 20110046056 and 20130231459 is used.

[0190] In some embodiments, the "22A" ApoA-I mimetic (PVLDLFRELLNELLEALKQKLK) (SEQ ID NO: 4) is used (see, e.g., U.S. Patent No. 7,566,695). In some embodiments, any of the following ApoA-I mimetics as shown in Table 1 described in U.S. Patent No. 7,566,695 are used:

[0191] Table 1. ApoA-I mimetics

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199] * indicates an N-terminally acetylated and C-terminally amidated peptide; * indicates an N-terminally dansylated peptide; sp indicates a peptide that exhibited solubility issues under experimental conditions; X is Aib; Z is Nal; O is Orn; He (%) indicates percent helicity; mics indicates micelle; and ~ indicates a missing amino acid.

[0200] In some embodiments, an ApoA-I mimetic having the following sequence: Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu AlaPhe (SEQ ID NO: 255) is utilized as described in U.S. Patent No. 6,743,778.

[0201] In some embodiments, any of the following ApoA-I mimetics as shown in Table 2 described in U.S. Patent Application Publication No. 2003 / 0171277 are used:

[0202] Table 2.

[0203]

[0204]

[0205]

[0206]

[0207] In some embodiments, an ApoA-I mimetic having the following sequence: FAEKFKEAVKDYFAKFWD (SEQ ID NO: 333) is utilized as described in US Patent Application Publication No. 2006 / 0069030.

[0208] In some embodiments, an ApoA-I mimetic having the following sequences as described in US Patent Application Publication No. 2009 / 0081293 is utilized: DWFKAFYDKVAEKFKEAF (SEQ ID NO: 334); DWLKAFYDKVAEKLKEAF (SEQ ID NO: 335); PALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ (SEQ ID NO: 336).

[0209] 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), PLGEEMRDRARAHVDALRTHLA (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 IDNO:361), VVALLALLASARASEAEDASLL (SEQ ID NO:362), HLRKLRKRLLRDADDLQKRLAVYOA (SEQID NO:363), AQAWGERLRARMEEMGSRTRDR (SEQ ID NO:364), LDEVKEQVAEVRAKLEEQAQ (SEQIDNO: 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).

[0210] Amphiphilic lipids include, for example, any lipid molecule having both a hydrophobic portion and a hydrophilic portion. Examples include phospholipids or glycolipids. Examples of phospholipids that can be used in sHDL-TA nanoparticles include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-bis-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] ... Hexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-diphexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-bis-(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 complexed with an imaging 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-diphexadecanoyl-sn-glycero-3-phosphothioethanol, or N-4-(p-maleimidophenyl)butyryl)dipalmitoylphosphatidylethanolamine (MPB-DPPE)).

[0211] In some embodiments, exemplary phospholipids include, but are not limited to, small alkyl chain phospholipids, egg yolk phosphatidylcholine, soybean phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, dilauroylphosphatidylglycerolphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol such as dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, Dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, egg sphingomyelin, milk sphingomyelin, palmitoyl sphingomyelin, phytosphingomyelin, dipalmitoyl sphingomyelin, distearoyl sphingomyelin, dipalmitoylphosphatidylglycerol salt, phosphatidic acid, galactocerebroside, ganglioside, cerebroside, dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3) diacylglycerol, aminophenyl glycoside, 3-cholesteryl-6′-(glycosylthio)hexyl ether glycolipid, and cholesterol and its derivatives. The phospholipid moiety comprising SM and palmitoylsphingomyelin may optionally include minor amounts of any type of lipid, including but not limited to lysophospholipids, sphingomyelins other than palmitoylsphingomyelin, galactocerebrosides, gangliosides, cerebrosides, glycerides, triglycerides, and cholesterol and its derivatives.

[0212] In some embodiments, the sHDL nanoparticles have a phospholipid / HDL apolipoprotein molar ratio of 2 to 250 (e.g., 10 to 200, 20 to 100, 20 to 50, 30 to 40).

[0213] Typically, 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 produce a more uniform preparation.

[0214] Compared with other strategies, including conventional nanoparticle carriers, sHDL nanoparticles have impressive biocompatibility and cargo loading capacity. For example, their ultrasmall but tunable size (e.g., 10-20 nm) enables sHDL nanoparticles to be efficiently excreted into lymph nodes and deliver cargo peptide antigens and nucleic acid-based adjuvants to lymph node-resident dendritic cells, positioning them as a highly effective platform for co-delivery of STING agonists and adjuvants for tumor immunotherapy.

[0215] In certain embodiments, the present invention provides compositions comprising nanoparticles conjugated to such compositions comprising one or more agents (e.g., DAMPs / PAMPs) capable of stimulating an innate immune response in a subject upon administration to a subject, wherein any type of biomacromolecular agent (e.g., nucleic acids, peptides, glycolipids, etc.) is conjugated to the nanoparticles.

[0216] In some embodiments, the biomacromolecule agent is a peptide.

[0217] For example, in some embodiments, the peptide is an antigen.

[0218] In some embodiments, the antigen is a tumor antigen. The antigen can be a tumor antigen, including tumor-associated or tumor-specific antigens, such as, but not limited to: 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, 2 and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, Mage-A1,2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100 (Pmel 17), 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, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), human EGFR protein or its fragment,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 mutant, p53 mutant, proteinase 3 (PR1), survivin, hTERT, sarcoma translocation breakpoint, 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, fucose 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, AKAP-4, XAGE 1, B7H3, leguminin, Tie 2, 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, and aldehyde dehydrogenase.

[0219] In some embodiments where the biomacromolecule is an antigen, the composition further comprises an adjuvant (as described herein).

[0220] In some embodiments, the peptide is adrenocorticotropic hormone (ACTH), growth hormone peptide, melanocyte stimulating hormone (MSH), oxytocin, vasopressin, corticotropin-releasing factor (CRF), CRF-related peptide, gonadotropin-releasing hormone-related peptide (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 pro-peptide, calcitonin gene-related peptide (CGRP), parathyroid hormone (PTH), thyroid 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-inhibiting 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 peptides, opioid peptides, thymosin, adrenomedullin peptide, allostatin peptide, amyloid beta protein fragment, antimicrobial peptide, antioxidant peptide, apoptosis-related peptide, bag cell peptide (BCP), bombesin, osteocalcin (Gla) protein peptide, cocaine and amphetamine-related transcript (CART) peptide, cell adhesion peptide, chemotactic peptide, complement inhibitory factor, cortistatin peptide, fibronectin fragment, fibrin-related peptide, FMRF, FMRFamide-related peptide (FaRP), galanin, galanin-related peptide, growth factor, growth factor-related peptide, G therapeutic peptide binding protein fragment, Gualylin, uroguanylin (Ur oguanylin), inhibin peptide, interleukin (IL), interleukin receptor protein, laminin fragment, leptin fragment peptide, cockroach kinin, pituitary adenylate cyclase activating polypeptide (PAPCAP), pancreatic inhibin, polypeptide repeat chain, signal transducer, thrombin inhibitor, toxin, trypsin inhibitor, virus-related peptide, adjuvant peptide analog, α mating factor, antiarrhythmic peptide, anorexigenic peptide, α-1 antitrypsin, bovine pinecone anti-reproductive peptide, bursin, C3 peptide P16, cadherin peptide, chromogranin A fragment, contraceptive tetrapeptide, conantokin G,Cone snail sleep peptide T, Crustacean cardioactive peptide, C-terminal peptide, cytochrome b588 peptide, antithrombotic peptide (Decorsin), delicious peptide, delta sleep-inducing peptide, diazempam binding inhibitor fragment, nitric oxide synthase blocking peptide, OVA peptide, platelet calpain inhibitor (P1), plasminogen activator inhibitor 1, Rigin, schizophrenia-related peptide, sodium potassium atherapeutic peptidase inhibitor 1, sperm activation peptide (Speract), sperm activation peptide, systemin, thrombin receptor agonist, tuftsin, lipokine, uremic pentapeptide, antifreeze peptide, tumor necrosis factor (TNF), leech [Des Asp10] antithrombotic peptide (Leech [Des Asp10] Decorsin), L-ornithine taurine hydrochloride, P-aminophenylacetyl tuftsin, 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, D DDDD, 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 Kallikrei N-1, placental ribonuclease inhibitor, Sarcolecin binding protein, surfactant protein D, Wilms' tumor suppressor factor, GABAB 1b receptor peptide, prion-related peptide (iPRP13), choline binding protein fragment, telomerase inhibitor, cardiostatin peptide, endostatin-derived peptide, prion inhibitory peptide, N-methyl D-aspartate receptor antagonist and C-peptide analog.

[0221] In some embodiments, the peptide is selected from 177Lu-DOTA0-Tyr3-Octreotate, Abarelix acetate, ADH-1, Afamelanotidec, melanotan-1, CUV1647, Albiglutide, Aprotinin, Arginine, Atosiban acetate, Bacitracin, Bentyramide, BH3 domain, Bivalirudin, Bivalirudin trifluoroacetate hydrate, Blisibimod, Bortezomib, Buserelin, Buserelin acetate, Calcitonin, Carbetocin, Carbetocin acetate, Cetrorelix, Cetrorelix acetate, Cyclosporin, Cilengitidec, EMD121974, Corticorelin acetate injection, hCRF, Corticorelin trifluoroate ovine triflutate), cortirellin trifluoroacetate, adrenocorticotropin, corticotropin, ACTH 1-24, teicosin hexaacetate, dalbavancin, daptomycin, degarelix acetate, depoxetine trifluoroacetate (with sodium pertechnetate), desmopressin acetate, desmopressin DDAVP, dulaglutide, ecallantide, eduterotide (with yttrium-90), elcatonin acetate, enalapril maleate (or 2-succinate), enfuvirtide, eptifibatide, exenatide, ganirelix acetate, glatiramer acetate 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, leuprolide, liraglutide, lisinopril, lixisenatide, lysine vasopressin, magainin 2, MALP-2Sc, synthetic macrophage Cytokine-activated lipopeptide-2, nafarelin acetate, nesiritide, NGR-hTNF, octreotide acetate, octreotide, oxytocin, pasireotide, penisatide, pentagastrin, pentreotide (with indium-111), phenypressin, pleurocidin, pramlintide, prorelin, thyrotropin-releasing hormone, TRH, TRF, salmon calcitonin, salami acetate, secretin (human), secretin (porcine), semaglutide, selacotide acetate, ACTH, adrenocorticotropic hormone, sermorelin acetate, GRF1-29, sinapeptide, KL4 in lucinactant, sincalide, somatostatin acetate, GHRH, GHRF, GRF, somatostatin acetate, spaglumat magnesium (or sodium) salt, substance P, taltirelin hydrate, terduglutide, teicoplanin, telavancin, teriparatide, terlipressin acetate, teicosin, thymosin alpha-1, thymopentin, trebananib, triptorelin, triptorelin pamoate, tyroserleutide, ularitide, vancomycin, vapreotide acetate, vasoactive intestinal peptide acetate, Vx-001c, TERT572Y, ziconotide acetate, α5-α6 Bax peptide, and β-defensin peptide.

[0222] In some embodiments, the peptide is any peptide that will aid in achieving the desired purpose with the composition. For example, in some embodiments, the peptide is any peptide that will facilitate the treatment of any type of disease and / or disorder.

[0223] In some embodiments, the biomacromolecule agent is a nucleic acid. Such embodiments include any type of nucleic acid molecule, including but not limited to RNA, siRNA, microRNA, interfering RNA, mRNA, replicon mRNA, RNA analogs and DNA.

[0224] In certain embodiments, such compositions and antigen-bound nanoparticles that are included in a reagent (e.g., DAMP / PAMP) that can stimulate the innate immune response in a subject after being administered to the subject are used to cause an immune response. In some embodiments, such nanoparticles are further combined (e.g., complexing, conjugating, encapsulating, absorbing, adsorbing, blending) with an adjuvant (e.g., the dendritic cells (DC) of the targeting molecule). In some embodiments, nanoparticles are co-administered together with an adjuvant. In some embodiments, antigen is combined (e.g., complexing, conjugating, encapsulating, absorbing, adsorbing, blending) with an adjuvant. In some embodiments, antigen is not combined (e.g., complexing, conjugating, encapsulating, absorbing, adsorbing, blending) with an adjuvant. In some embodiments, antigen is combined with a hydrophobic molecule. In some embodiments, adjuvant is combined with a hydrophobic molecule. In some embodiments, the average size of the nanoparticles is between 6 and 500 nm.

[0225] 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.

[0226] Examples of lipid molecules suitable for use in embodiments of the present invention include, but are not limited to, phospholipids such as phosphatidylcholine, phosphatidylethanolamine, lysolecithin, lysolecithin, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylglycerol (DPPG), palmitoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylcholine (DP ... Oleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably derived from C 10 -C 24 Acyl groups of carbon-chain fatty acids, for example, lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl.

[0227] Other non-limiting examples of lipid molecules include sterols, such as cholesterol and its derivatives, such as cholestanol, cholestanone, cholesterenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

[0228] Other examples of lipid molecules suitable for use in the present invention include lipids that do not contain phosphorus, such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glyceryl ricinoleate, cetyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkyl aryl sulfate polyethoxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, sphingomyelin, and the like.

[0229] Other examples of lipid molecules suitable for use in the present invention include fatty acids and their derivatives or analogs. These include oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, glyceryl monooleate (1-monooleoyl-rac-glyceride), dilaurin, caprylic acid, arachidonic acid, 1-monocaprin, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, and C-1,4-diol. 1-10 Alkyl esters (e.g., methyl, isopropyl, and tert-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).

[0230] Other examples applicable to lipid molecules of the present invention include lipid molecules (PEG-lipid) modified with PEG.The example of PEG-lipid includes but is not limited to PEG (PEG-DAA) as described in for example PCT Publication No. WO05 / 026372 being coupled to dialkyloxypropyl class, PEG (PEG-DAG) as described in for example U.S. Patent Publication No. 20030077829 and 2005008689 being coupled to diacylglycerol, PEG such as phosphatidylethanolamine (PEG-PE) as described in for example U.S. Patent number 5,885,613 being coupled to PEG on ceramide, PEG being coupled to cholesterol or its derivatives, and its mixture.For all purposes, the disclosure of these patent documents is incorporated herein in full by quoting with it.Other PEG-lipid includes but is not limited to PEG-C-DOMG, 2KPEG-DMG and mixture thereof.

[0231] PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEG is classified by its molecular weight; for example, PEG 2000 has an average molecular weight of approximately 2,000 daltons, while PEG 5000 has an average molecular weight of approximately 5,000 daltons. PEG is commercially available from Sigma Chemical and other companies and includes, for example, the following: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH2), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). Other PEGs, such as those described in U.S. Patent Nos. 6,774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are also used to prepare the PEG-lipid conjugates of the present invention. The disclosures of these patents are incorporated herein by reference in their entirety for all purposes. In addition, monomethoxypolyethylene glycol acetic acid (MePEG-CH2COOH) is particularly useful for preparing PEG-lipid conjugates, including, for example, PEG-DAA conjugates.

[0232] The PEG portion of the PEG-lipid conjugates described herein can include an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In some cases, the PEG portion 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 a preferred embodiment, the PEG portion has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0233] In some cases, PEG can be optionally substituted by an alkyl, alkoxy, acyl or aryl group. PEG can be directly conjugated to the lipid or can be connected to the lipid through a linker moiety. Any linker moiety suitable for connecting PEG to the lipid can be used, including, for example, a linker moiety that does not contain an ester and a linker moiety that contains an ester. In a preferred embodiment, the linker moiety is a linker moiety that does not contain an ester. As used herein, the term "linker moiety that does not contain an ester" refers to a linker moiety that does not contain a carboxylate 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 (—S—S—), ether (—O—), succinyl (—(O)CCH2CH2C(O)—), succinimidyl (—NHC(O)CH2CH2C(O)NH—), ether, disulfide, and combinations thereof (e.g., linkers comprising both a carbamate linker moiety and an amide linker moiety). In a preferred embodiment, the PEG is attached to the lipid using a carbamate linker.

[0234] In other embodiments, PEG is attached to the lipid using an ester-containing linker moiety. Suitable ester-containing linker moieties include, for example, carbonate (—OC(O)O—), succinyl, phosphate (—O—(O)POH—O—), sulfonate, and combinations thereof.

[0235] Phosphatidylethanolamines of various acyl chain groups with different chain lengths and degrees of saturation can be conjugated 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.

[0236] It is preferred to have C 10 to C 20 Phosphatidylethanolamines comprising saturated or unsaturated fatty acids having carbon chain lengths within a range of 1 to 400 nm can also be used. Phosphatidylethanolamines with mono- or di-unsaturated fatty acids and 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), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0237] Such embodiments are not limited to specific antigens. In practice, 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 viruses, bacteria, parasites, plants, protozoa, fungi, tissues, or transformed cells, such as cancer cells or leukemia cells, and can be whole cells or immunogenic components thereof, such as cell wall components or molecular components thereof.

[0238] In some embodiments, antigen is known in the art and available from commercial government and scientific sources. In some embodiments, antigen is a complete inactivated or attenuated organism. These organisms can be infectious organisms, such as viruses, parasites and bacteria. These organisms can also be tumor cells. Antigen can be a purified or partially purified polypeptide derived from a tumor or a viral or bacterial source. The standard for identifying and selecting effective antigenic peptides (for example, the minimum peptide sequence that can elicit an immune response) can be found in the art. Antigen can be a recombinant polypeptide produced by expressing a DNA encoding a polypeptide antigen in a heterologous expression system. Antigen can be a DNA encoding all or part of an antigen protein. DNA can be in the form of a vector DNA, such as a plasmid DNA.

[0239] Antigens can be provided as a single antigen or can be provided in combination. Antigens can also be provided as a complex mixture of polypeptides or nucleic acids.

[0240] In some embodiments, the antigen is a self-antigen. As used herein, the term "self-antigen" refers to an immunogenic antigen or epitope that is natural to a mammal and that may be involved in the pathogenesis of an autoimmune disease.

[0241] In some embodiments, the antigen is a viral antigen. The viral antigen can be isolated from any virus, including but not limited to viruses from any of the following virus 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 herpesviruses 1, 3, 4, 5, and 6, and cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microphages Microviridae, Orthomyxoviridae (e.g., influenza viruses A, B, and C), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and foot-and-mouth disease virus), Poxviridae (e.g., vaccinia and variola viruses), 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.

[0242] Viral antigens can be derived from specific strains such as papillomaviruses, herpes viruses, i.e., herpes simplex 1 and 2; hepatitis viruses, e.g., hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV), tick-borne encephalitis viruses; parainfluenza, varicella-zoster, cytomeglavirus, Epstein-Barr, rotavirus, rhinovirus, adenovirus, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choriomeningitis.

[0243] In some embodiments, the antigen is a bacterial antigen. Bacterial antigens can be derived from any bacteria, including but not limited to: Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Helicobacter pylori, Haemophilus, Hemophilus influenzae type B. type B) (HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, B, and C, Methanobacterium, Micrococcus, Mycobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus teus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, Treponema, Vibrio, and Yersinia.

[0244] In some embodiments, the antigen is a parasite antigen. Parasite antigens can be obtained from parasites such as, but not limited to, antigens derived from Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni. mansoni). These include sporozoan antigens, Plasmodium antigens, such as all or part of the circumsporozoite protein, sporozoite surface proteins, liver stage antigens, apical membrane-associated proteins or merozoite surface proteins.

[0245] In some embodiments, the antigens are allergens and environmental antigens, such as, but not limited to, antigens derived from naturally occurring allergens, such as pollen allergens (tree, herb, weed and grass pollen allergens), insect allergens (inhalant, saliva and venom allergens), animal hair and dandruff allergens and food allergens. Important pollen allergens from trees, grasses and herbs are from the taxonomic orders Fagales, Oleales, Pinales and Platanaceae, including, i.e., birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedars (Cryptomeria and Juniperus), plane trees (Platanaceae). anus), the Poaceae order includes grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, and the Asterales and Urticales orders include herbaceous plants of the genera Ambrosia, Artemisia, and Parietaria. Other allergen antigens that may be used include: allergens from house dust mites of the genera Dermatophagoides and Euroglyphus, storage mites such as Lepidoglyphys, Glycyphagus and Tyrophagus, allergens from cockroaches, Chironomus and fleas such as Blatella, Periplaneta, Chironomus and Ctenocepphalides, allergens from mammals such as cats, dogs and horses, birds, venom allergens including such allergens derived from stinging or biting insects such as allergens from the taxonomic order Hymenoptera which includes bees (superfamily Apidae), wasps (superfamily Vespidae) and mites. Vespidea) and ants (superfamily Formicoidae).Still other allergen antigens that may be used include inhalation allergens from fungi, such as allergens from Alternaria and Cladosporium.

[0246] In some embodiments, the antigen is a tumor antigen (described herein).

[0247] One of the key obstacles in developing curative and tumor-specific immunotherapies is the identification and selection of highly specific and restrictive tumor antigens to avoid autoimmunity. Tumor neoantigens that arise as a result of genetic changes within malignant cells (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.) represent the most tumor-specific class of antigens.

[0248] In some embodiments, the antigen is a neoantigen. The term neoantigen is used herein to define any newly expressed antigenic determinant. When the protein conformation changes, the neoantigen can be produced as a newly expressed determinant (especially on the surface of a transformed or infected cell), as a result of the formation of a complex of one or more molecules or as a result of molecular cleavage, to obtain a display of the neoantigenic determinant. Therefore, as used herein, the term neoantigen encompasses antigens expressed during infection (e.g., viral infection, protozoan infection, or bacterial infection) in prion-mediated diseases, during cell transformation (cancer), in which case the neoantigen can be referred to as a tumor-associated antigen.

[0249] The present invention is not limited to a particular manner of identifying neoantigens. In some embodiments, identification of neoantigens involves identifying all or nearly all mutations in a neoplasia / tumor at the DNA level using whole genome sequencing, whole exome (e.g., captured exons only) sequencing, or RNA sequencing of the tumor compared to a matched germline sample from each patient. In some embodiments, identification of neoantigens involves analyzing these identified mutations with one or more peptide-MHC binding prediction algorithms to generate a plurality of candidate neoantigen T cell epitopes that are expressed within the neoplasia / tumor and that can bind to patient HLA alleles. In some embodiments, identification of neoantigens involves synthesizing a plurality of candidate neoantigen peptides selected from a collection of all novel open reading frame peptides and predicted binding peptides for use in cancer vaccines.

[0250] Thus, the present invention is based, at least in part, on the ability to identify all or nearly all mutations (e.g., translocations, inversions, large and small deletions and insertions, missense mutations, splice site mutations, etc.) within a neoplasia / tumor. Specifically, these mutations are present in the genome of a subject's neoplasia / tumor cells but not in normal tissue from the subject. Such mutations are particularly interesting if they result in changes that produce a protein with an altered amino acid sequence that is unique to the patient's neoplasia / tumor (e.g., a neoantigen). For example, useful mutations may include: (1) non-synonymous mutations that produce a different amino acid in the protein; (2) read-through mutations where the stop codon is modified or deleted, resulting in translation of a longer protein with a novel tumor-specific sequence at the C-terminus; (3) splice site mutations that result in the inclusion of introns in the mature mRNA and thus a unique tumor-specific protein sequence; (4) chromosomal rearrangements that result in a chimeric protein with a tumor-specific sequence at the junction of two proteins (i.e., a gene fusion); (5) frameshift mutations or deletions that result in a new open reading frame with a novel tumor-specific protein sequence; and the like. Peptides or mutant polypeptides with mutations resulting from, for example, splice site, frameshift, read-through, or gene fusion mutations in tumor cells can be identified by sequencing DNA, RNA, or protein in tumor and normal cells.

[0251] Also within the scope of the present invention are personalized neoantigen peptides derived from common tumor driver genes and may further include previously identified tumor-specific mutations.

[0252] Preferably, any suitable sequencing while synthesis platform can be used to identify mutations. Four main sequencing while synthesis platforms are currently available: the genome sequencer from Roche / 454Life Sciences, the HiSeq analyzer from Illumina / Solexa, the SOLiD system from Applied BioSystems and the Heliscope system from HelicosBiosciences. Pacific Biosciences and VisiGen Biotechnologies have also described sequencing while synthesis platforms. Each of these platforms can be used in the method of the present invention. In some embodiments, a plurality of nucleic acid molecules to be sequenced are combined with a support (for example, a solid support). In order to fix nucleic acid on a support, capture sequence / universal priming site can be added at the 3' end and / or 5' end of the template. Nucleic acid can be combined with a support by hybridizing the capture sequence with a complementary sequence covalently attached to the support. Capture sequence (also referred to as universal capture sequence) is a nucleic acid sequence complementary to the sequence attached to the support, and it can serve as a universal primer doubly.

[0253] As an alternative to the capture sequence, a member of a coupling pair (such as, for example, antibody / antigen, receptor / ligand or avidin-biotin pair as described in, for example, U.S. Patent Application No. 2006 / 0252077) can be connected to each fragment to be captured on the surface, and the surface is coated with the corresponding second member of the coupling pair. After capture, the sequence can be analyzed, for example, by single molecule detection / sequencing, such as described in the Examples and U.S. Patent No. 7,283,337, including template-dependent sequencing while synthesis. In sequencing while synthesis, the surface-bound molecule is exposed to a plurality of labeled triphosphate nucleotides in the presence of a polymerase. The sequence of the template is determined by the order of the labeled nucleotides incorporated into the 3' end of the growing chain. This can be completed in real time or can be completed in a step-by-step repeat mode. For real-time analysis, different optical labels for each nucleotide can be incorporated, and a plurality of lasers can be used to stimulate the incorporated nucleotides.

[0254] Any cell type or tissue can be used to obtain nucleic acid samples used in the sequencing methods described herein. In some embodiments, DNA or RNA samples are obtained from neoplasia / tumors or body fluids (e.g., 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).

[0255] A variety of methods can be used to detect the presence of a specific mutation or the presence of an allele in the DNA or RNA of an individual. Progress in this field has provided accurate, easy and inexpensive large-scale SNP genotyping. Recently, for example, several new technologies have been described, including dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrophosphate sequencing, oligonucleotide specific ligation, TaqMan system and multiple DNA " chip " technologies such as Affymetrix SNP chip. These methods require typically amplifying the target genetic region by PCR. Based on other newly developed methods generated by small signal molecules of mass spectrometry or fixed padlock probes and rolling circle amplification by invasive cutting, the needs to PCR may be finally eliminated. Some methods known in the art for detecting specific single nucleotide polymorphisms are summarized below. Method of the present invention should be understood to include all available methods.

[0256] PCR-based detection methods can include simultaneous multiplex amplification of multiple labels. For example, it is well known in the art to select PCR primers to generate PCR products that do not overlap in size and can be analyzed simultaneously.

[0257] Alternatively, different markers can be amplified with primers, which are differentially labeled and thus can be detected differentially. Certainly, detection means based on hybridization allow a plurality of PCR products in the difference detection sample. Other technologies are known in the art, to allow the multiplex analysis of a plurality of markers.

[0258] Several methods have been developed to facilitate the analysis of single nucleotide polymorphisms in genomic DNA or cellular RNA. In one embodiment, single base polymorphisms can be detected by using special exonuclease-resistant nucleotides, as disclosed in, for example, U.S. Patent number 4,656,127. According to the method, primers complementary to the allele sequence next to the polymorphic site 3' allow hybridization with the target molecule obtained from a specific animal or human. If the polymorphic site on the target molecule comprises nucleotides complementary to the specific exonuclease-resistant nucleotide derivative present, that derivative will be incorporated into the end of the hybridization primer. Such incorporation causes the primer to be resistant to exonucleases and thereby allows its detection. Because the identity of the exonuclease-resistant derivative of the sample is known, the discovery that the primer has become resistant to exonucleases reveals that the nucleotides present in the polymorphic site of the target molecule are complementary to the nucleotide derivatives used in the reaction. The method has the advantage that it does not need to measure a large amount of external sequence data.

[0259] In another embodiment of the present invention, a solution-based method is used to determine the identity of the nucleotide at the polymorphic site (see, for example, French Patent No. 2,650,840; PCT Application No. WO1991 / 02087). As in the method of U.S. Patent No. 4,656,127, a primer complementary to the allele sequence immediately 3' to the polymorphic site can be used. This method uses labeled dideoxynucleotide derivatives to determine the identity of the nucleotide at the site. These derivatives, if complementary to the nucleotide at the polymorphic site, will be incorporated into the end of the primer.

[0260] An alternative method, called Genetic Bit Analysis or Described in PCT Application No. WO 1992 / 15712. A mixture of a labeled terminator and a primer complementary to the sequence 3' of the polymorphic site is used. The incorporated labeled terminator is thus determined by the nucleotide present in the polymorphic site of the target molecule being evaluated and the terminator is complementary to that nucleotide. In contrast to the method of Cohen et al. (French Patent 2,650,840; PCT Application No. WO 1991 / 02087), this The method is preferably a heterogeneous assay, wherein the primers or the target molecule are immobilized on a solid phase. Recently, several primer-directed nucleotide incorporation procedures have been described for determining polymorphic sites in DNA (see, e.g., Komher, J. S. 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. N. 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., Genomics 9:114-117 (1992)). al., Anal. Biochem. 208: 171-175 (1993). These methods are similar to The difference between these two methods is that they all rely on the incorporation of labeled deoxynucleotides to distinguish bases at polymorphic sites. In such a format, since the signal is proportional to the number of deoxynucleotides incorporated, polymorphisms occurring in a run of the same nucleotide can produce a signal proportional to the run length (see, e.g., Syvanen, A.-C, et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

[0261] An alternative approach 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 proteomics methods allow for rapid, 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 neoantigens (see, e.g., Guthals et al. (2012) Shotgun Protein Sequencing with Meta-contig Assembly, Molecular and Cellular Proteomics 11(10): 1084-96).

[0262] Tumor-specific neoantigens can 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 can be performed using MHC tetramer-based screening technologies (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 contemplated within the scope of the present invention that such tetramer-based screening techniques can be used to initially identify tumor-specific neoantigens, or alternatively as a secondary screening protocol to assess which neoantigens a patient may have been exposed to, thereby aiding in the selection of candidate neoantigens for the vaccines of the present invention.

[0263] The present invention further includes isolated peptides (e.g., neoantigenic peptides comprising tumor-specific mutations identified by the methods, peptides comprising known tumor-specific mutations, and mutant polypeptides identified by the methods or fragments thereof). These peptides and polypeptides are referred to herein as "neoantigenic peptides" or "neoantigenic polypeptides." These polypeptides or peptides can be of various lengths and will at a minimum include a small region ("epitope") that is predicted to bind to the patient's HLA molecule and additional adjacent amino acids extending in both the N-terminal and C-terminal directions. These polypeptides or peptides can be in their native (uncharged) form or in the form of their salts and contain no modifications such as glycosylation, side chain oxidation, or phosphorylation, or contain such modifications, subject to the proviso that the modification does not destroy the biological activity of the polypeptide as described herein.

[0264] In certain embodiments, the size of at least one neoantigenic peptide molecule can 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 a specific embodiment, the neoantigenic peptide molecule is equal to or less than 50 amino acids. In a preferred embodiment, the neoantigenic peptide molecule is equal to about 20 to about 30 amino acids.

[0265] Thus, the present invention provides nanoparticles that are bound to such compositions comprising an agent (e.g., DAMP / PAMP) that can stimulate an innate immune response in a subject upon administration to a subject, and one or more neoantigenic peptides. In some embodiments, the nanoparticles are bound to two neoantigenic peptides. In some embodiments, the nanoparticles are bound to at least 5 or more neoantigenic peptides. In some embodiments, the nanoparticles are bound to 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 bound to at least 20 different peptides.

[0266] These neoantigenic peptides, polypeptides and analogs can be further modified to include additional chemical moieties that are not normally part of the protein. Those derivatized moieties can improve the solubility, biological half-life, absorption or binding affinity of the protein. These moieties can also reduce or eliminate any desired side effects of the protein, etc. A review of those moieties can be found in Remington's Pharmaceutical Sciences, 20 th ed., Mack Publishing Co., Easton, PA (2000). For example, if necessary, neoantigenic peptides and polypeptides with desired activity can be modified to provide certain desired properties, such as improved pharmacological characteristics, while increasing or at least substantially retaining all biological activities of the unmodified peptide to bind to the desired MHC molecule and activate appropriate T cells. For example, these neoantigenic peptides and polypeptides can be subjected to various changes, such as conservative or non-conservative substitutions, where such changes may provide certain advantages in their use, such as improved MHC binding. Such conservative substitutions can encompass the replacement of one amino acid residue with another that is biologically and / or chemically similar, for example, replacing a hydrophobic residue with another hydrophobic residue, or replacing a polar residue with another polar residue. D-amino acids can also be used to explore the effects of single amino acid substitutions. Such modifications can be made using well-known peptide synthesis procedures, as described in, for example, 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).

[0267] In some embodiments, neoantigenic peptides and polypeptides can be modified with linkers to facilitate binding to nanoparticles (e.g., sHDL nanoparticles). The present invention is not limited to a particular type or class of linkers. In some embodiments, the linker is a cysteine-serine-serine (CSS) molecule.

[0268] In some embodiments where the nanoparticle is 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), wherein upon mixing, the DOPE-PDP and CSS associate, resulting in complexing (linking) of the CSS-Ag to the sHDL.

[0269] These neoantigenic peptides and polypeptides can also be modified by extending or reducing the amino acid sequence of the compound (for example, by adding or deleting amino acids). These neoantigenic peptides, polypeptides or analogs can also be modified by changing the order or composition of certain residues. It will be appreciated by those skilled in the art that certain amino acid residues necessary for biological activity, for example, those at key contact sites or conserved residues, can generally not be changed without side effects on biological activity. Non-critical amino acids are not necessarily limited to those naturally occurring in proteins, such as La-amino acids or their D-isomers, but may also include non-natural amino acids such as β-γ-δ-amino acids and many derivatives of La-amino acids.

[0270] Typically, a series of peptides with single amino acid substitutions can be used to optimize neoantigen polypeptides or peptides to determine the effects of electrostatic charge, hydrophobicity, etc. on MHC binding. For example, a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions can be made along the length of the peptide to reveal different sensitivity patterns to various MHC molecules and T cell receptors. In addition, multiple substitutions using small, relatively neutral moieties (such as Ala, Gly, Pro, or similar residues) can be used. These substitutions can be homo-oligomers or hetero-oligomers. The number and type of residues substituted or added depends on the required spacing between the necessary contact points and certain functional properties sought (e.g., hydrophobicity versus hydrophilicity). Such substitutions can also achieve increased binding affinity for MHC molecules or T cell receptors compared to the affinity of the parent peptide. In any case, such substitutions should utilize amino acid residues or other molecular fragments selected to avoid, for example, steric and charge interference that may disrupt binding. Amino acid substitutions are typically single residues. Substitutions, deletions, insertions, or any combination thereof can be combined to obtain the final peptide.

[0271] Those of ordinary skill in the art will recognize that there are a variety of ways to generate such tumor-specific neoantigens. Generally, such tumor-specific neoantigens can be generated in vitro or in vivo. Tumor-specific neoantigens can be generated in vitro as peptides or polypeptides, which can then be formulated into personalized neoplasia vaccines and administered to a subject. Such in vitro generation can occur by a variety of methods known to those of ordinary skill 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.

[0272] Alternatively, tumor-specific neoantigens can be produced in vivo by introducing molecules encoding the tumor-specific neoantigens (e.g., DNA, RNA, viral expression systems, etc.) into a subject, followed by expression of the encoded tumor-specific neoantigens.

[0273] Proteins or peptides can be prepared by any technique known to those skilled in the art, including expression of proteins, polypeptides or peptides by standard molecular biological techniques, isolation of proteins or peptides from natural sources, or chemical synthesis of proteins or peptides. Nucleotide and protein, polypeptide and peptide sequences corresponding to various genes have been previously disclosed and can be found in computerized databases known to those of ordinary skill in the art. One such database is the Genbank and GenPept databases at the National Center for Biotechnology Information, located on the website of the National Institutes of Health. The coding regions of known genes can be amplified and / or expressed using techniques disclosed herein or that should be known to those of ordinary skill in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of ordinary skill in the art.

[0274] Peptides can be readily synthesized chemically using reagents that are free of contaminating bacterial or animal matter (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of atetrapeptide. J. Am. Chem. Soc. 85: 2149-54, 1963).

[0275] Another aspect of the present invention provides a nucleic acid (e.g., a polynucleotide) encoding a neoantigenic peptide of the present invention, which can be used to produce neoantigenic peptides in vitro. The polynucleotide can be, for example, a natural or stable form of single-stranded and / or double-stranded DNA, cDNA, PNA, CNA, RNA or polynucleotide, such as, for example, a polynucleotide with a thiophosphate backbone or a combination thereof, and it may or may not contain introns, as long as it encodes the peptide. Still another aspect of the present invention provides an expression vector capable of expressing a polypeptide according to the present invention. Expression vectors for different cell types are well known in the art and can be selected without excessive experimentation. Typically, DNA is inserted into an expression vector (such as a plasmid) in the proper direction and in the correct reading frame for expression. If necessary, the DNA can be linked to an appropriate transcriptional and translational regulatory control nucleotide sequence recognized by the desired host (e.g., bacteria), although such control is typically available in the expression vector. The vector is then introduced into host bacteria 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).

[0276] The present invention further includes variants and equivalents that are substantially homologous to the tumor-specific neoantigens identified herein. These may include, for example, conservative substitution mutations, i.e., replacement of one or more amino acids with similar amino acids. For example, a conservative substitution refers to replacing one amino acid with another within the same general class, such as, for example, replacing an acidic amino acid with another acidic amino acid, replacing a basic amino acid with another basic amino acid, or replacing a neutral amino acid with another neutral amino acid. Conservative amino acid substitutions are well known in the art.

[0277] The present invention also includes expression vectors comprising isolated polynucleotides and host cells comprising expression vectors. It is also contemplated within the scope of the present invention that these neoantigenic peptides can be provided in the form of RNA or cDNA molecules encoding the desired neoantigenic peptides. The present invention also provides that one or more neoantigenic peptides of the present invention can be encoded by a single expression vector. The present invention also provides that one or more neoantigenic peptides of the present invention can be encoded and expressed in vivo using a virus-based system (e.g., an adenovirus system).

[0278] The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that include only coding sequences for the polypeptide as well as polynucleotides that include additional coding and / or non-coding sequences. The polynucleotides of the present invention may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding (antisense) strand.

[0279] In embodiments, these polynucleotides may include a coding sequence for a tumor-specific neoantigenic peptide fused in the same reading frame to a polynucleotide that, for example, facilitates expression and / or secretion of a polypeptide by a host cell (e.g., serves as a leader sequence for a secretory sequence that controls transport of the polypeptide from the cell). Polypeptides with leader sequences are preproteins and the leader sequence can be cleaved by the host cell to form the mature form of the polypeptide.

[0280] In some embodiments, these polynucleotides may include a coding sequence for a tumor-specific neoantigenic peptide fused in the same reading frame to a marker sequence that, for example, allows purification of the encoded polypeptide, which can then be incorporated into a personalized neoplasia vaccine. For example, in the case of a bacterial host, the marker sequence may be a hexahistidine tag provided by the pQE-9 vector to provide for purification of the mature polypeptide fused to the marker, or when a mammalian host (e.g., COS-7 cells) is used, the marker sequence may be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein. Additional tags include, but are not limited to, calmodulin tags, FLAG tags, Myc tags, S tags, SBP tags, Softag 1, Softag 3, V5 tags, Xpress tags, Isopeptag, SpyTag biotin carboxyl carrier protein (BCCP) tags, GST tags, fluorescent protein tags (e.g., green fluorescent protein tags), maltose binding protein tags, Nus tags, Strep-tags, thioredoxin tags, TC tags, Ty tags, and the like. In embodiments, these polynucleotides may include coding sequences for one or more of the tumor-specific neoantigenic peptides fused in the same reading frame to generate a single concatamerized neoantigenic peptide construct capable of producing multiple neoantigenic peptides.

[0281] 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 neoantigenic peptide of the present invention.

[0282] 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 may include up to five point mutations per 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 may be deleted or substituted with another nucleotide, or a plurality of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the amino-terminal position or the carboxyl-terminal position of the reference nucleotide sequence, or anywhere between those terminal positions, either individually interspersed among nucleotides in the reference sequence or in one or more contiguous groups.

[0283] As a practical matter, known computer programs can be used to routinely determine whether any particular nucleic acid molecule is at least 80% consistent, at least 85% consistent, at least 90% consistent with the reference sequence, and in some embodiments, at least 95%, 96%, 97%, 98% or 99% consistent, these computer programs are such as Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses local homology algorithm (Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981)) to find the best homology segment between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is for example 95% consistent with the reference sequence according to the present invention, these parameters are set so that on the full length of the reference nucleotide sequence, percent identity is calculated and a homology gap of 5% of the total nucleotides in the reference sequence is allowed.

[0284] The isolated tumor-specific neoantigen peptides described herein can be produced in vitro (e.g., in a laboratory) by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing DNA sequences encoding isolated polypeptide sequences and expressing those sequences in suitable transformed hosts. In some embodiments, recombinant technology is used to construct DNA sequences by isolating or synthesizing DNA sequences encoding wild-type proteins of interest. Optionally, the sequence can be mutagenized by site-specific mutagenesis to provide functional analogs thereof. See, for example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81: 5662-5066 (1984) and U.S. Patent No. 4,588,585.

[0285] In an embodiment, an oligonucleotide synthesizer is used to construct a DNA sequence encoding a polypeptide of interest by chemical synthesis. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and by selecting those codons that are preferred in the host cell that will produce the recombinant polypeptide of interest. Standard methods can be used to synthesize the isolated polynucleotide sequence of the isolated polypeptide of interest for encoding. For example, complete amino acid sequence can be used to build a back-translated gene. In addition, a DNA oligomer can be synthesized that comprises the nucleotide sequence of the polypeptide of the specific separation of encoding. For example, the little oligonucleotides of the part of the polypeptide of the desired several encodings can be synthesized and then connected. Independent oligonucleotides typically comprise 5' or 3' overhangs for complementary components.

[0286] Once assembled (e.g., by synthesis, site-directed mutagenesis or other methods), the polynucleotide sequence encoding the specific isolated polypeptide of interest will be inserted into an expression vector and optionally operably linked to expression control sequences suitable for expressing the protein in the desired host. Appropriate assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of the biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of the transfected gene in the host, the gene can be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host. Recombinant expression vectors can be used to amplify and express DNA encoding tumor-specific neoantigen peptides. Recombinant expression vectors are replicable DNA constructs that have synthetic or cDNA-derived DNA fragments encoding tumor-specific neoantigen peptides or biologically equivalent analogs that are operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit typically includes a collection of the following: (1) one or more genetic elements that have a regulatory role in gene expression, such as a transcriptional promoter or enhancer; (2) a structure or coding sequence that is transcribed into mRNA and translated into protein; and (3) appropriate transcription and translation start and stop sequences, as described in detail below. Such regulatory elements may include an operator sequence for controlling transcription. An origin of replication, which generally confers the ability to replicate in a host, and a selection gene that aids in identifying 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 that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of that sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to allow translation. Generally, operably linked means continuous, and in the case of a secretory leader, continuous and in reading frame. Structural elements intended for use in yeast expression systems include a leader sequence that allows the host cell to secrete the translated protein extracellularly. Alternatively, in the case where a recombinant protein is expressed without a leader or transit sequence, it may include an N-terminal methionine residue. This residue can then be optionally cleaved from the expressed recombinant protein to provide the final product.

[0287] The selection of expression control sequences and expression vectors will depend on the selection of the host. A variety of expression host / vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as pCR 1, pBR322, pMB9, and derivatives thereof from Escherichia coli, and wider host range plasmids such as M13 and filamentous single-stranded DNA phage.

[0288] Suitable host cells for expressing polypeptides include prokaryotes, yeasts, insects or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram-negative or gram-positive organisms, such as Escherichia coli (E. coli) or bacilli. Higher eukaryotic cells include established mammalian cell lines. Cell-free translation systems can also be utilized. Suitable cloning and expression vectors for use with bacteria, fungi, yeast and mammalian cell hosts are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985).

[0289] Various mammalian or insect cell culture systems are also advantageously utilized to express recombinant proteins. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include the COS-7 monkey kidney cell line described by Gluzman (Cell 23:175, 1981), and 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 non-transcriptional elements such as an origin of replication, a suitable promoter and enhancer connected to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. The baculovirus system for producing heterologous proteins in insect cells is summarized by Luckow and Summers, Bio / Technology 6:47 (1988).

[0290] The protein produced by the transformed host can be purified according to any suitable method. Such standard methods include chromatography (for example, ion exchange, affinity and size fractionation column chromatography, etc.), centrifugation, differential solubility or by any other standard techniques for protein purification. Affinity tags such as hexa-histidine, maltose binding domain, influenza coating sequence, glutathione-S-transferase etc. can be attached to the protein to allow easy purification by passing through a suitable affinity column. The technology such as proteolysis, nuclear magnetic resonance and X-ray crystallography can also be used to characterize the separated protein with physical methods.

[0291] For example, a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration device) can be used to first concentrate the supernatant from the system in which the recombinant protein is secreted into the culture medium. After the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin can be utilized, such as a matrix or substrate with a pendant diethylaminoethyl (DEAE) group. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation exchange step can be utilized. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps utilizing hydrophobic RP-HPLC media (e.g., silica gel with pendant methyl or other aliphatic groups) can be used to further purify the cancer stem cell protein-Fc composition. Some or all of the aforementioned purification steps can also be used to provide a uniform recombinant protein in various combinations. Recombinant proteins produced in bacterial cultures can be isolated, for example, 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 utilized in expressing recombinant proteins can be destroyed by any conventional method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysing agents.

[0292] Thus, in certain embodiments, the present invention relates to personalized strategies for treating conditions (e.g., neoplasias), and more particularly tumors, by administering to a subject (e.g., a mammal, such as a human) a therapeutically effective amount of a composition (as described herein) comprising an agent (e.g., DAMPs / PAMPs) capable of stimulating an innate immune response in the subject upon administration to the subject, and one or more neoplasia / tumor-specific neoantigens (e.g., a vaccine composition capable of eliciting a specific T cell response). In some embodiments, such a composition is further associated with a nanoparticle. Indeed, in certain embodiments, whole genome / exome sequencing can be used to identify all or nearly all of the mutant neoantigens uniquely present in an individual patient's neoplasia / tumor, and a collection of these mutant neoantigens can be analyzed to identify a specific, optimized subset of neoantigens for use as a personalized cancer vaccine for treating the patient's neoplasia / tumor. For example, in some embodiments, a population of neoplasia / tumor-specific neoantigens can be identified by sequencing each patient's neoplasia / tumor and normal DNA to identify tumor-specific mutations and determining the patient's HLA allotype. This population of neoplasia / tumor-specific neoantigens and their cognate natural antigens can then be subjected to bioinformatics analysis using validation algorithms to predict which tumor-specific mutations produce epitopes that can bind to the patient's HLA allotypes, and in particular, which tumor-specific mutations produce epitopes that can bind to the patient's HLA allotype more effectively than the cognate natural antigens. Based on this analysis, one or more peptides corresponding to a subset of these mutations can be designed and synthesized for each patient and pooled together for use as a cancer vaccine in immunizing the patient. The neoantigen peptides can be combined with another anti-tumor agent. In some embodiments, such neoantigens are expected to bypass central thymic tolerance (thereby allowing a stronger anti-tumor T cell response) while reducing the potential for autoimmunity (e.g., by avoiding targeting normal self-antigens).

[0293] The present invention further provides a method for inducing a neoplasia / tumor-specific immune response in a subject, immunizing against a neoplasia / tumor, treating cancer in a subject, and / or alleviating cancer symptoms in a subject, by administering to the subject a neoantigenic peptide or vaccine composition of the present invention.

[0294] According to the present invention, the cancer vaccine can be used for patients who have been diagnosed with cancer or are at risk of developing cancer. In one embodiment, the patient can have a solid tumor, such as breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, melanoma, as well as other tumors of tissues and organs and blood tumors, such as lymphomas and leukemias, including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma.

[0295] 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 may be administered alone or in combination with other therapeutic agents. The therapeutic agent is, for example, a chemotherapeutic or biotherapeutic agent, radiation, or immunotherapy. Any suitable therapeutic treatment for a particular cancer may be administered. Examples of chemotherapeutic agents and biotherapeutic agents include, but are not limited to, aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), actinomycin D, docetaxel, doxorubicin, dronabinol, epoetin alfa, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alfa, irinotecan, lansoprazole, levamisole, leucovorin, megestrol acetate, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel Pilocarpine, prochlorperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate. For prostate cancer treatment, the preferred chemotherapeutic agent that can be combined with anti-CTLA-4 is paclitaxel.

[0296] In addition, the subject can be further administered anti-immunosuppressive or immunostimulant. For example, the subject is further administered anti-CTLA-4 antibodies, 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 inhibitors. Blocking CTLA-4 or PD-1 / PD-L1 by antibodies can enhance the immune response to cancerous cells in the patient. Specifically, when following the vaccination regimen, CTLA-4 blocking has been shown to be effective.

[0297] Those skilled in the art can determine the optimal amount and optimal dosage regimen for each peptide to be included in the vaccine composition without undue experimentation. For example, the peptide or its variant can be prepared for intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, intramuscular (im) injection. Preferred methods for peptide injection include sc, id, ip, im and iv. Preferred methods for DNA injection include id, im, sc, ip and iv. For example, a dose of between 1 and 500 mg, between 50 μg and 1.5 mg, preferably 10 μg to 500 μg of peptide or DNA can be given and will depend on the corresponding peptide or DNA. This range of doses has been successfully used in previous 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 the vaccine composition are known to those skilled in the art.

[0298] The vaccines of the present invention can be formulated so that the selection, number and / or amount of peptides present in the composition are tissue-, cancer- and / or patient-specific. For example, the precise selection of peptides can be guided by the expression pattern of the parent protein in a given tissue to avoid side effects. The selection can depend on the specific type of cancer, the state of the disease, previous treatment regimens, the patient's immune status and, of course, the patient's HLA-haplotype. In addition, vaccines according to the present invention can include personalized components according to the individual needs of a specific patient. Examples include varying the amount of peptides according to the expression of relevant neoantigens in a specific patient, unwanted side effects due to personal allergies or other treatments, and adjusting secondary treatments after a first round of treatment or a first regimen of treatment.

[0299] 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 sufficient to cure or at least partially prevent symptoms and / or complications. An amount sufficient to achieve this purpose is defined as a "therapeutically effective dose." The amount effective for this purpose will depend, for example, on the peptide composition, the route of administration, the stage and severity of the disease being treated, the patient's weight and general health, and the discretion of the prescribing physician, but a typical range for the first immunization of a 70 kg patient (which is for therapeutic or prophylactic administration) is from about 1.0 μg to about 50,000 μg of peptide, followed by increasing doses, or according to a booster regimen of from about 1.0 μg to about 10,000 μg of peptide for several weeks to several months, depending on the patient's response and condition and possibly by measuring specific CTL activity in the patient's blood. It should be remembered that the peptides and compositions of the present invention can generally be used in serious disease states, i.e., life-threatening or potentially life-threatening situations, especially when the cancer has metastasized. For therapeutic use, administration should be started as soon as possible after detection or surgical resection of the tumor. The dosage is then increased until at least the symptoms are substantially alleviated and thereafter for a period of time. Pharmaceutical compositions (e.g., vaccine compositions) for therapeutic treatment are intended for parenteral, topical, nasal, oral, or local administration. Preferably, these pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. These compositions can be administered at the site of surgical resection to induce a local immune response against the tumor.

[0300] Such embodiments are not limited to specific types of adjuvants. Generally, adjuvants are any substances that are incorporated into the vaccine composition to increase or otherwise alter the immune response to the mutant peptide. The carrier is a scaffold structure, such as a polypeptide or polysaccharide, to which these antigenic peptides (e.g., neoantigenic peptides) can be bound. Optionally, the adjuvant is covalently or non-covalently conjugated to the peptide or polypeptide of the present invention.

[0301] The ability of an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies produced against the antigen, and an increase in T cell activity is typically manifested in an increase in cell proliferation or cytotoxicity or cytokine secretion. Adjuvants can also, for example, alter the immune response by changing a primarily humoral or Th2 response to a primarily cellular or Th1 response.

[0302] Suitable adjuvants include, but are not limited to, 1018ISS, 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 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM. vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulator derived from saponin (Aquila Biotech, Worcester, Massachusetts, USA), mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox.Quil or Superfos. Several immunoadjuvants specific for dendritic cells (e.g., MF59) and their preparation have been described previously (Dupuis M, et al., Cell Immunol. 1998; 186(1): 18-27; Allison AC; Dev Biol Stand. 1998; 92: 3-11). Cytokines can also be used. Several cytokines have been directly linked to influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-α), accelerating the maturation of dendritic cells into potent antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Patent No. 5,849,589, specifically incorporated 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) can also be used as adjuvants and are important members of the pattern recognition receptor (PRR) family that recognize conserved motifs shared by many microorganisms, termed "pathogen-associated molecular patterns (PAMPS)."

[0303] Recognition of these "danger signals" activates multiple elements 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 cells and B cells, mast cells and granulocytes, and are located in different cellular compartments, such as the plasma membrane, lysosomes, endosomes and endolysosomes. Different TLRs recognize different PAMPS. For example, TLR4 is activated by LPS contained in the bacterial cell wall, 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 signal transduction pathways, which ultimately leads to the production of many key molecules associated with inflammation and immunity (especially transcription factors NF-κβ and type I interferons). TLR-mediated DC activation leads to enhanced DC activation, phagocytosis, upregulation of activation and co-stimulatory markers such as CD80, CD83, and CD86, expression of CCR7, thereby allowing DC to migrate to draining lymph nodes and facilitate antigen presentation to T cells, and increased secretion of cytokines such as type I interferons, IL-12, and IL-6. All of these downstream events are critical for the induction of adaptive immune responses.

[0304] Other receptors that may be targeted include toll-like receptors (TLRs).TLR identification and binding to pathogen-associated molecular patterns (PAMPs).PAMP targets TLR on dendritic cell surfaces and internally signals, thereby potentially increasing DC antigen uptake, maturation and T cell stimulation ability.PAMP conjugated to particle surface or co-encapsulated include unmethylated CpG DNA (bacterial), double-stranded RNA (virus), lipopolysaccharide (bacterial), peptidoglycan (bacterial), lipoarabinomannan (bacterial), zymosan (yeast), mycoplasma lipoproteins such as MALP-2 (bacterial), flagellin (bacterial), poly (inosine cytosine) acid (bacterial), lipoteichoic acid (bacterial) or imidazoquinoline (synthesis).

[0305] 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 when compared with LPS and CpG, as it induces proinflammatory cytokines and lacks IL-10 stimulation and maintains high levels of co-stimulatory molecules in DCs. In addition, poly-ICLC was recently directly compared with CpG as an adjuvant for a protein vaccine composed of human papillomavirus (HPV) 16 capsid in non-human primates (rhesus macaques) (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)).

[0306] In some embodiments, the adjuvant is a dendritic cell targeting molecule (DC). DC is effective and is responsible for eliciting an antigen-specific immune response. A biological characteristic of DC is its ability to sense the conditions of encountering antigens, which triggers the "DC maturation" process. Using receptors for various microorganisms and inflammatory products, DC responds to antigen exposure in different ways according to the properties of the pathogens (viruses, bacteria, protozoa) encountered. This information is transmitted to T cells by the cytokine release pattern that changes during antigen presentation in the lymph nodes, thereby changing the type of T cell response caused. Therefore, targeting DC not only provides the opportunity to generally quantitatively enhance the delivery of antigens and antigenic responses, but also provides the opportunity to qualitatively control the properties of the immune response according to the desired vaccination results.

[0307] Dendritic cells express numerous cell surface receptors that mediate endocytosis of bound antigens. Targeting exogenous antigens to internalizing surface molecules on systemically distributed antigen-presenting cells facilitates antigen uptake and thus overcomes a major rate-limiting step in immunity and, therefore, vaccination.

[0308] Dendritic cell targeting molecules include monoclonal or polyclonal antibodies or fragments thereof that recognize and bind to epitopes displayed on the dendritic cell surface. Dendritic cell targeting molecules also include ligands that bind to cell surface receptors on dendritic cells. One such receptor (lectin DEC-205) has been used in vitro and in mice to enhance both humoral (antibody-based) and cellular (CD8 T cell) responses by 2-4 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)).

[0309] A variety of other endocytic receptors have also been targeted in this manner, including mannose-specific lectin (mannose receptor) and IgG Fc receptor, 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 receptors, and scavenger receptors.

[0310] In some embodiments, adjuvant is CpG. It has also been reported that CpG immunostimulatory oligonucleotides can enhance the effect of adjuvants in vaccine environments. Without being bound by theory, CpG oligonucleotides work by activating innate (non-adaptive) immune system via Toll-like receptors (TLR) (mainly TLR9). The TLR9 activation of CpG triggers enhances antigen-specific humoral and cellular responses for a variety of antigens, including peptides or protein antigens, live viruses or dead viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in both preventive and therapeutic vaccines. More importantly, it enhances dendritic cell maturation and differentiation, thereby causing the activation enhancement of Th1 cells and strong cytotoxic T lymphocytes (CTL) to produce, even in the absence of CD4 T cells. Even in the presence of vaccine adjuvants (such as alum or incomplete Freund's adjuvant (IFA)) that usually promote Th2 bias, the Th1 bias induced by TLR9 stimulation is also maintained. When formulated with other adjuvants or co-administered or in preparations such as microparticles, nanoparticles, lipid emulsions or similar formulations, CpG oligonucleotides illustrate even greater adjuvant activity, and when antigen is relatively weak, these are especially necessary for inducing strong responses. They also promote immune response and allow antigen dosage to be reduced by approximately two orders of magnitude, while in some experiments, comparable antibody responses (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, Jun. 2006, 471-484) are produced to full dose vaccines in the absence of CpG. U.S. Patent number 6,406,705B1 describes the use of CpG oligonucleotides, non-nucleic acid adjuvants and antigens in combination to induce antigen-specific immune responses. Commercially available CpG TLR9 antagonists are dSLIM (double stem loop immunomodulators) from Mologen (Berlin, Germany), which are preferred components of pharmaceutical compositions of the present invention. Other TLR binding molecules can also be used, such as TLR 7, TLR 8 and / or TLR 9 in conjunction with RNA.

[0311] Xanthone derivatives such as, for example, Vadimezan or AsA404 (also known as 5,6-dimethylxanthenone-4-acetic acid (DMXAA)) can also be used as adjuvants according to embodiments of the present invention. Alternatively, such derivatives can also be administered in parallel with the vaccines of the present invention, for example, via systemic or intratumoral delivery, to stimulate immunity at the tumor site. Without being bound by theory, it is believed that such xanthone derivatives act by stimulating interferon (IFN) production via the stimulator of IFN genes (ISTING) receptor (see, for example, 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., poly i:CI2U), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies that can act therapeutically and / or as adjuvants, such as cyclophosphamide, sunitinib, bevacizumab, celecoxib, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175. One skilled in the art can readily determine the amounts and concentrations of adjuvants and additives useful in the context of the present invention without undue experimentation. Additional adjuvants include colony stimulating factors, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim).

[0312] Poly-ICLC is a synthetically prepared double-stranded RNA composed of poly I and poly C chains with an average length of approximately 5000 nucleotides, which has been stabilized against heat denaturation and hydrolysis by serum nucleases by the addition of polylysine and carboxymethyl cellulose. This compound activates the RNA helicase domains of TLR3 and MDA5 (two members of the PAMP family), leading to the activation of DCs and natural killer (NK) cells and the production of a "natural cocktail" of type I interferons, cytokines, and chemokines. In addition, poly-ICLC exerts more direct, broad-host-targeted anti-infective and possibly anti-tumor effects mediated by two IFN-inducible ribozyme systems: 2'5'-OAS and P1 / eIF2a kinase, also known as PKR (4-6), as well as RIG-I helicase and MDA5.

[0313] Such methods are not limited to producing sHDL nanoparticles that are combined with a composition comprising an agent (e.g., DAMP / PAMP) capable of stimulating an innate immune response in a subject upon administration to the subject, an antigen, and an adjuvant (e.g., a dendritic cell targeting molecule). In some embodiments, the antigen and adjuvant are conjugated to the outer surface of the sHDL nanoparticle.

[0314] In some embodiments, sHDL nanoparticles are synthesized with thiol-reactive phospholipids that allow reduction-sensitive attachment of antigens and / or adjuvants. In some embodiments, the loading of DCs within sHDL nanoparticles is facilitated by cholesterol modification of DC molecules. In some embodiments, a lyophilization method is used to prepare uniform sHDL. In some embodiments, phospholipids and ApoA mimetic peptides are dissolved in glacial acetic acid and lyophilized. In some embodiments, the antigenic peptide is incubated with sHDL in a buffer (e.g., sodium phosphate buffer (pH 7.4)) (e.g., 3 hours at room temperature) to allow conjugation of the antigenic peptide. In some embodiments, a desalting column (MWCO = 7000 Da) is used to remove unconjugated antigenic peptides. In some embodiments, incorporating cholesterol-modified DCs (Cho-DCs) into sHDL involves incubating with sHDL at room temperature for approximately 30 minutes.

[0315] Such embodiments are not limited to a specific manner of characterizing sHDL conjugated to antigens 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 GPC determination.

[0316] sHDL nanoparticles (e.g., sHDL-αGalCer) (e.g., Ag / DC-sHDL) configured to activate an immune response can be used to activate T cells in a subject for prophylactic and therapeutic applications. Activating T cells by nanoparticle vaccine compositions increases their proliferation, cytokine production, differentiation, effector function, and / or survival. Methods for measuring these are well known to those skilled in the art. The T cells activated by the nanoparticle vaccine compositions can be any cells expressing T cell receptors (including α / β and γ / δ T cell receptors). T cells include all cells expressing CD3, including T cell subsets that also express CD4 and CD8. T cells include both naive and memory cells and 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 unique categories of T cell lineages. In some embodiments, the activated T cells are CD8 + T cells.

[0317] Typically, compositions comprising sHDL nanoparticles configured to activate an immune response (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL) are used to treat a subject having or susceptible to any disease or condition in which the subject's immune system mounts an immune response. The compositions can be used as prophylactic vaccines that confer resistance to subsequent exposure to an infectious agent in a subject. The compositions can also be used as therapeutic vaccines that can be used to initiate or enhance a subject's immune response to a pre-existing antigen, such as a tumor antigen in a subject with cancer, or a viral antigen in a subject infected with a virus. The compositions can also be used as desensitization vaccines that act to "tolerate" an individual to environmental antigens, such as allergens.

[0318] The ability to target these compositions to professional antigen-presenting cells such as dendritic cells, and the ability of these compositions to elicit T cell-mediated immune responses by causing cross-presentation of antigens, makes these compositions particularly useful for eliciting cell-mediated responses to disease-associated antigens, thereby attacking the disease. Thus, in some embodiments, the type of disease to be treated or prevented is a malignancy or chronic infectious disease caused by bacteria, viruses, protozoa, helminths, or other microbial pathogens that enter cells and are attacked by cytotoxic T lymphocytes.

[0319] According to principles well known in the art, the desired outcome of a prophylactic, therapeutic or desensitizing immune response can vary depending on the disease. For example, an immune response to an infectious agent can completely prevent the colonization and replication of the infectious agent, thereby affecting "sterile immunity" and the absence of any disease symptoms. However, if a vaccine against an infectious agent reduces the number, severity or duration of symptoms; if a vaccine against an infectious agent reduces the number of individuals in a population with symptoms; or reduces the spread of the infectious agent; it can be considered effective. Similarly, an immune response to a cancer, allergen or infectious agent can completely treat the disease, can alleviate symptoms, or can be one aspect of an overall therapeutic intervention for the disease. For example, stimulation of an immune response to cancer can be combined with surgery, chemotherapy, radiology, hormones and other immunological methods to affect treatment.

[0320] A subject having an infectious agent or exposed to an infectious agent 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, a subject can be treated prophylactically, such as when there may be a risk of developing a disease from an infectious agent. Individuals who have visited or reside in areas where infectious diseases are endemic may be considered at risk and are candidates for prophylactic vaccination against a specific infectious agent. Prophylactic treatment can be applied to any number of diseases where there is a known relationship between a specific disease and a specific risk factor (such as geographic location or work environment).

[0321] A subject having a malignant tumor or at risk of developing a malignant tumor can be treated therapeutically or prophylactically with sHDL nanoparticles configured to activate an immune response as disclosed herein (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL). In mature animals, a balance is normally maintained between cell renewal and cell death in most organs and tissues. Each type of mature cell in the body has a given lifespan; as these cells die, new cells are generated through 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 emerge that no longer respond to normal growth control mechanisms. These cells give rise to cell clones that can expand to considerable numbers, thereby giving rise to tumors or neoplasms. Tumors that do not grow indefinitely and do not extensively invade healthy surrounding tissue are benign. Tumors that continue to grow and become increasingly invasive are malignant. The term cancer refers specifically to malignant tumors. In addition to uncontrolled growth, malignant tumors also exhibit metastasis. During this process, small clusters of cancerous cells are expelled from the tumor, invade the blood or lymphatic vessels, and are carried to other tissues where they continue to proliferate. 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-STIN agonist-sHDL) as disclosed herein can be used to treat subjects with malignant tumors.

[0322] Treatable malignancies are classified herein according to the embryonic origin of the tissue from which the tumor originates. Carcinomas are tumors arising from endodermal or ectodermal tissues, such as the skin or the epithelial layer of internal organs and glands. Melanoma is a type of skin cancer for which the present invention is particularly useful. Less frequently occurring sarcomas originate from mesodermal connective tissues, such as bone, fat, and cartilage. Leukemias and lymphomas are malignant tumors of the hematopoietic cells of the bone marrow. Leukemias proliferate as a single cell, while lymphomas tend to grow as a tumor mass. Malignancies may arise in many organs or tissues of the body to establish cancer.

[0323] Cancer types that can be treated with provided sHDL nanoparticles (e.g., sHDL-STING agonist-αGalCer) configured to activate an immune response (e.g., Ag / DC-STING agonist-sHDL) include, but are not limited to, the following: bladder, brain, breast, cervix, colorectal, esophageal, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, etc. Administration is not limited to the treatment of existing tumors or infectious diseases, but can also be used to prevent or reduce the risk of developing such diseases in an individual, i.e., for prophylactic use. Potential candidates for prophylactic vaccination include individuals at high risk of developing cancer, i.e., individuals with a personal or family history of certain types of cancer.

[0324] A subject who has an allergen or is at risk of being exposed to an allergen 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 a subject for the purpose of preventing and / or allergic reactions, such as those that lead to anaphylaxis. An allergic reaction can be characterized by T cells that are resistant to an antigen. H 2 response, leading to the presence of IgE antibodies. Stimulation of T H 1 Immune response and production of IgG antibodies can reduce allergic diseases. Therefore, sHDL nanoparticles configured to activate an immune response (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL) as disclosed herein can be used to produce antibodies that prevent and / or reduce allergic reactions in subjects exposed to allergens.

[0325] A subject having an immunosuppressive condition or at risk of an immunosuppressive condition 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. The sHDL nanoparticle vaccines disclosed herein can be used to treat disease conditions characterized by immunosuppression, including but not limited to AIDS or AIDS-related complex, idiopathic immunosuppression, drug-induced immunosuppression, other viral or environmentally induced conditions, and certain innate immune deficiencies. Such sHDL nanoparticle vaccine compositions can also be used to increase immune function that has been impaired by radiation therapy with immunosuppressive drugs (e.g., certain chemotherapeutic agents), and thus can be particularly useful when used in combination with such drugs or radiation therapy.

[0326] In general, methods of administering a vaccine as disclosed herein (e.g., sHDL nanoparticles configured to activate an immune response (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL)) are well known in the art. Any acceptable method known to one of ordinary skill in the art can be used to administer the formulation to a subject. Administration can be local (i.e., to a specific area, physiological system, tissue, organ, or cell type) or systemic. The vaccine can be administered by a variety of routes, including, but not limited to, oral, inhalation (nasal or pulmonary), intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal. Injections can be, for example, intravenous, intradermal, subcutaneous, intramuscular, or intraperitoneal. In some embodiments, injections can be given at multiple locations.

[0327] The administration of the preparation can be completed by any acceptable method that allows the vaccine of effective dose to reach its target.The specific mode of selection will depend on the following factors, such as the severity of the state of the experimenter being treated and the dosage required for inducing an effective immune response.As generally used herein, "effective dose" is the amount that can induce an immune response in the experimenter being treated.The actual effective dose of the vaccine can vary according to the following: the specific antigen used or its combination, the specific composition prepared, the mode of administration, and the age, body weight, condition, and route of administration and disease or illness of the individual to be vaccinated.

[0328] In certain embodiments, glycolipids encapsulated in sHDL nanoparticles serve as stimulators of natural killer T cell-mediated immune responses.

[0329] Natural killer T (NKT) cells are a heterogeneous population of T cells that share properties of both T cells and natural killer cells. Many of these cells recognize non-polymorphic CD1d molecules, which are antigen-presenting molecules that bind to their own and foreign lipids and glycolipids. NKT cells constitute only about 0.1% of all peripheral blood T cells. NKT cells are a subpopulation of T cells that co-express αβ T cell receptors, but also express a variety of molecular markers commonly associated with NK cells (such as NK1.1). The most famous NKT cells differ from conventional αβ T cells in that the diversity of their T cell receptors is much more restricted ('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 complexes (MHC). NKT cells include NK1.1 + and NK1.1 - Both, as well as CD4 + 、CD4 - 、CD8 + and CD8- cell.

[0330] In certain embodiments, the composition comprising an agent (e.g., DAMP / PAMP) capable of stimulating an innate immune response in a subject upon administration to the subject is further associated with (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, incorporated into) one or more therapeutic agents. Such embodiments are not limited to a particular type or class of therapeutic agents.

[0331] 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-carcinogenic agents, anti-angiogenic agents, tumor suppressor agents, antimicrobial agents, and the like.

[0332] In some embodiments, the therapeutic agent is configured to treat and / or prevent autoimmune diseases and / or inflammatory diseases. 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, pegseroma, etanercept, golimumab, infliximab), IL-1 inhibitors, and metalloproteinase inhibitors. In some embodiments, the therapeutic agent includes but is not limited to infliximab, adalimumab, etanercept, parenteral gold, or oral gold.

[0333] In some embodiments, the therapeutic agent is configured to treat and / or prevent cardiovascular-related diseases (e.g., atherosclerosis, heart failure, arrhythmia, atrial fibrillation, hypertension, coronary artery disease, angina pectoris, etc.). Examples of known therapeutic agents useful in treating and / or preventing cardiovascular-related diseases 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 blockers (ARBs) (e.g., candesartan, irbesartan, olmesartan medoxomil, telmisartan, eprosartan, losartan, tasosartan, valsartan), anticoagulants (e.g., heparin, fondaparinux, valsartan), aparinol, adeparinol, enoxaparin, reviparin, dalteparin, nadroparin, tinzaparin), antiplatelet agents (e.g., abciximab, clopidogrel, eptifibatide, ticlopidine, cilostazol, dipyridamole, sulfinpyrazone, tirofiban), beta-blockers (e.g., acebutolol, betaxolol, carteolol, metoprolol, penbutolol, propranolol, atenolol, bisoprolol, esmolol, nadolol, pindolol, timolol), calcium channel blockers (e.g., amlodipine, felodipine, isradipine, nifedipine, verapamil, diltiazem, nicardipine, nimodipine, nisoldipine), Diuretics, aldosterone blockers, loop diuretics (e.g., bumetanide, furosemide, ethacrynic acid, torsemide), potassium-sparing diuretics, thiazide diuretics (e.g., chlorothiazide, chlorthalidone, hydrochlorothiazide, hydroflumethiazide, methylclothiazide, metolazone, polythiazide, quinethazone, trichlorothiazide), inoptropics, bile acid sequestrants (e.g., cholestyramine, colestipol, colesevelam), benzodiazepines (e.g., clofibrate, gemfibrozil, fenofibrate), statins (e.g., atorvastatin, lovastatin, simvastatin, statin, fluvastatin, pravastatin), selective cholesterol absorption inhibitors (e.g., ezetimibe), potassium channel blockers (e.g., amiodarone, ibutilide, dofetilide), sodium channel blockers (e.g., disopyramide, mexiletine, procainamide, quinidine, flecainide, moricizine, propafenone), thrombolytics (e.g., alteplase, reteplase, tenecteplase, anistreplase, streptokinase, urokinase), vasoconstrictors, vasodilators (e.g., hydralazine, minoxidil, mecamylamine, isorbide dintrate, isorbide mononitrate, nitroglycerin).

[0334] Typically, 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 produce a more uniform formulation.

[0335] In some embodiments, the nanoparticles combined with such compositions as described herein are further combined (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, blended) with reagents that can be used to determine the location of the particles being administered. Useful reagents for this purpose include fluorescent labels, radionuclides, and contrast agents.

[0336] Suitable imaging agents include, but are not limited to, fluorescent molecules such as those described by Molecular Probes (Handbook of fluorescent probes and research products), such as 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, Fluo4, 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, LysoSensorBlue DND-167, LysoSensor Green, LysoSensor Yellow / Blu, Lysotracker Green FM, Magnesium Green, Marina Blue, Mitotracker Green FM, Mitotracker Orange CMTMRos, MitoTracker Red CMXRos, Monobromobimane, NBD amine, NeruoTrace 500 / 525green, Nile Red, Oregon Green, Pacific Blue. POP-1, propidium iodide, rhodamine 110, rhodamine red, R-phycoerythrin, Resorfin, RH414, Rhod-2, rhodamine green, rhodamine 123, ROX dyes, 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, YOYO-3. In some embodiments, ceramides are provided as imaging agents. In some embodiments, S1P agonists are provided as imaging agents.

[0337] In addition, radionuclides can be used as imaging agents. Suitable radionuclides include, but are not limited to, radioactive substances of Fe (III), Fe (II), Cu (II), Mg (II), Ca (II) and Zn (II) indium, gallium and technetium. Other suitable contrast agents include metal ions that are commonly used for chelation in paramagnetic T1-type MIR contrast agents, and include 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. In addition, metal ions known to be useful for neutron capture radiotherapy include boron and other metals with large nuclear cross-sections. Also suitable are metal ions and X-ray contrast compositions that can be used for ultrasound contrast imaging.

[0338] Examples of other suitable contrast agents include radiopaque gases or gas-emitting compounds.

[0339] In some embodiments, the nanoparticles combined with such compositions as described herein are further combined with a targeting agent (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, blended). In some embodiments, the targeting agent is used to assist in delivering the nanoparticles combined with such compositions as described herein to a desired body area (e.g., a body area affected by cardiovascular-related diseases). Examples of targeting agents include, but are not limited to, antibodies, receptor ligands, hormones, vitamins, and antigens, however, the present invention is not limited by the properties of the targeting agent. In some embodiments, antibodies are specific to disease-specific antigens. In some embodiments, receptor ligands include, but are not limited to, ligands of CFTR, EGFR, estrogen receptor, FGR2, folate receptor, IL-2 receptor, glycoprotein, and VEGFR. In some embodiments, the receptor ligand is folic acid.

[0340] In some embodiments, nanoparticles combined with such compositions as described herein can be delivered to a localized site in a patient via a medical device. Medical devices suitable for use in the present invention include known devices for local 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, perfusion catheters, and the like, which are, for example, coated with a therapeutic agent or through which an agent is administered; needle injection devices such as hypodermic needles and needle injection catheters; needle-free injection devices such as jet injectors; coated stents, bifurcated stents, vascular grafts, stent grafts, and the like; and coated vascular occlusion devices such as coils.

[0341] 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; 5,800,519; 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 RADIUS (SCIMED LIFE SYSTEMS, Inc.), SYMPHONY (Boston Scientific Corporation), Wallstent (Schneider Inc.), PRECEDENT II (Boston Scientific Corporation), and NIR (Medinol Inc.) Such devices are delivered to and / or implanted at a target location in the body by known techniques.

[0342] In some embodiments, the present invention also provides kits comprising the compositions described herein. In some embodiments, the kits include one or more reagents and tools necessary to produce such compositions and methods of using such compositions.

[0343] The size and uniformity of the nanoparticles associated with such compositions as described herein can be characterized by any suitable analytical technique. These include, but are not limited to, atomic force microscopy (AFM), electrospray ionization mass spectrometry, MALDI-TOF mass spectrometry, 13 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 homogeneity of the sHDL nanoparticle population and are important for final production quality control for in vivo applications.

[0344] In some embodiments, sHDL-TA nanoparticles are analyzed using gel permeation chromatography (GPC), which can separate sHDL nanoparticles from liposomes and free ApoA-I mimetic peptides. In some embodiments, size distribution and zeta potential are determined by dynamic light scattering (DLS) using, for example, a Malven Nanosizer instrument.

[0345] In some embodiments of the present invention, sHDL nanoparticles are prepared as part of a pharmaceutical composition in a form suitable for the intended application, with clinical applications in mind. Typically, this requires preparing a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals. However, in some embodiments of the present invention, pure sHDL nanoparticle formulations can be administered using one or more of the routes described herein.

[0346] In a preferred embodiment, the nanoparticles associated with such compositions as described herein are used in conjunction with appropriate salts and buffers so that the composition is delivered in a stable manner to allow uptake by target cells. Buffers are also used when the sHDL nanoparticles are introduced into a patient. Aqueous compositions include 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 an animal or a human. 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. Unless any conventional media or agents are incompatible with the vectors or cells of the present invention, their use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

[0347] In some embodiments of the present invention, the active composition comprises a classical pharmaceutical formulation. Administration of these compositions according to the present invention is carried out by any common route, as long as the target tissue is accessible by that route. This includes oral, nasal, buccal, rectal, vaginal or topical administration. Alternatively, administration can be by in situ, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.

[0348] Active nanoparticles combined with such compositions as described herein can also be administered parenterally or intraperitoneally or intratumorally. Solutions of the active compound as a free base or a pharmacologically acceptable salt are prepared in water by mixing with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and oils. Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0349] The pharmaceutical form suitable for injectable use includes sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium comprising the following: for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using a surfactant. The prevention of microbial action can be achieved by various antibacterial agents, antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal (thimerosal), etc. In many cases, isotonic agents, such as sugar or sodium chloride, can be preferably included. The absorption of the injectable composition can be prolonged by using an agent that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0350] Sterile injectable solutions are prepared by mixing the active nanoparticles combined with such compositions as described herein in the desired amount with various other ingredients listed above (as needed) in an appropriate solvent, followed by filtration sterilization. Generally speaking, dispersions are prepared by mixing various sterilized active ingredients into a sterile carrier comprising an alkaline dispersion medium and the desired other ingredients from those listed above. With regard to sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying techniques, which produce powders of the active ingredient and any additional desired ingredients from its previously sterile filtered solution.

[0351] After preparation, the nanoparticles combined with such compositions as described herein are administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. Easily with various dosage forms such as injectable solutions, drug release capsules and other dosage formulations. For parenteral administration in aqueous solutions, for example, if necessary, the solution is appropriately buffered and the liquid diluent is first made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. For example, a dosage can be dissolved in 1ml isotonic NaCl solution and or added to 1000ml subcutaneous perfusion fluid, or injected at the infusion site of suggestion (see, for example, " Remington's Pharmaceutical Sciences " 15th Edition, pages 1035-1038 and 1570-1580). In some embodiments of the present invention, active particles or reagents are formulated in the treatment mixture to include about 0.0001 to 1.0 milligrams per dose, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams, etc. Multiple doses may be administered.

[0352] 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 pharmaceutically formulated, for insertion into the rectum, vagina, or urethra. After insertion, the suppository softens, melts, or dissolves in the cavity fluid. Typically, for suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from a mixture containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Vaginal suppositories or pessaries are typically spherical or ovoid and weigh approximately 5 g each. Vaginal medications are available in a variety of physical forms, such as creams, gels, or liquids, which deviate from the classic suppository concept. sHDL nanoparticles can also be formulated as an inhaler.

[0353] The present invention also includes methods involving co-administration of nanoparticles in combination with such compositions as described herein with one or more additional active agents. Indeed, another aspect of the present invention provides methods for enhancing prior art therapies and / or pharmaceutical compositions by co-administering the sHDL nanoparticles of the present invention. In co-administration procedures, these agents can be administered simultaneously or sequentially. In some embodiments, the sHDL nanoparticles described herein are administered prior to the one or more other active agents. The agent or agents to be co-administered will depend on the type of condition being treated.

[0354] The present disclosure further provides kits comprising compositions comprising nanoparticles combined with such compositions as described herein or the necessary ingredients for synthesizing nanoparticles as described herein. In some embodiments, the kit includes all components necessary, sufficient, or useful for administering such nanoparticles combined with such compositions as described herein.

[0355] experiment

[0356] 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 the scope of the invention.

[0357] Example 1.

[0358] This example describes the synthesis and characterization of CDN / Zn, CDN / Zn@liposome NPs, and CDN@CaP / PEI-PEG.

[0359] like Figure 1As shown in Figure A, CDN-Zn NPs were prepared by simple coordination assembly. It was assumed that Zn with a pyramidal coordination geometry could coordinate with adenine and phosphate simultaneously. To further increase the stability of the resulting particles, CDNs / Zn nanoparticles were modified with liposomes. There are several different MOF surface modification methods, such as coordination adjustment during MOF synthesis, and post-synthesis modification by ligand exchange and silica or polymer shell coating. Since DOPA has been widely used to modify Zn-based MOFs during synthesis, it is not recommended to use DOPA for the surface modification of MOFs. 2+ Since MOFs are capped, coordination regulation is applied here to synthesize CDN / Zn@DOPA with lipid tails on its surface, which allows for another lipid layer coating.

[0360] TEM images show the morphology of the obtained CDNs-Zn and CDN-Zn@liposome NPs ( Figure 2 ).like Figure 2 As shown in a, cdAMP-Zn NPs exhibit a spherical shape with high TEM contrast on the surface. It is suspected that the rapid nucleation of cdAMP-Zn in methanol leads to the formation of Zn in the core. 2+ The coordination is insufficient, and the particle surface has Zn 2+ The saturated coordination of cdAMP-Zn increases the surface contrast, resulting in a "core-shell" like structure. It was also found that a uniform spherical structure was obtained when the synthesis was carried out in an aqueous medium because slower nucleation occurred in water (not shown). Consistent with the TEM images, DLS and zeta potential data indicated that the size of cdAMP-Zn was about 150 nm and the surface charge was neutral. Figure 2 As shown in Figure b, under the same synthesis conditions, cd-GMP NPs showed a uniform irregular spherical structure with a size of approximately 100 nm and a neutral surface charge. Compared with cdAMP-Zn and cdGMP-Zn, the morphology and charge of cGAMP-Zn were different ( Figure 2 c) The spherical nanoparticles are composed of several accumulated smaller clusters and have a small amount of positive charge on the surface. In order to increase the stability of CDN-Zn NPs, we modified CDN-Zn with liposomes. Figure 2 (d) shows a representative CDN-Zn@liposome structure of cdAMP-Zn@liposomes. TEM images demonstrate that the CDN-Zn@liposomes exhibit increased uniformity and smaller size due to the DOPA capping effect. Furthermore, after liposome-PEG modification, they exhibit a small amount of negative surface charge.

[0361] For the CaP / PEI-PEG formulation, experiments were started with the clinically used adjuvant CaP hydrogel. 2+ and PO4 3-CaP hydrogel was prepared and formed into needle-like nanostructures. In order to increase the loading of CDN on CaP hydrogel, PEI-PEG was added to increase the charge attraction to CDN, which can also increase the colloidal stability ( Figure 1 B) Unlike conventional CaP hydrogels that tend to aggregate into gels, CaP / PEI-PEG disperses well in water. Figure 2 As shown in Figure e, CDN@CaP / PEI-PEG NPs showed a uniform needle cluster structure with a size of approximately 70 nm and a surface charge of approximately +15 mV. Based on the morphology, size, and surface properties, all formulations here may have great potential in drug delivery applications.

[0362] Example II.

[0363] This example demonstrates the release profiles and in vitro STING activation of CDN-Zn and CDNs@CaP / PEI-PEG.

[0364] As two key parameters of the drug delivery system, the experiments further determined the drug loading and release characteristics of the CDN nanoformulations. For the CDN-Zn formulation, the CDN loading efficiency in the nanoformulation exceeded 90%, while for the CDN / CaP-PEI-PEG formulation, the CDN loading efficiency in the nanoformulation was greater than 80% ( Figure 3 A). As for drug release, cdAMP / Zn and cdGMP / Zn showed very similar release profiles ( Figure 3 B). During the first 18 h, release was close to zero-order, after which a slightly slower release phase was observed. It is speculated that the zero-order drug release from cdAMP / Zn and cdGMP / Zn may be due to the stable and constant dissociation of the backbone. However, further investigation under physiological conditions with different biomolecular interactions is needed. For cGAMP / Zn NPs, there was a rapid release phase during the first 8 h of incubation, followed by a slower release phase ( Figure 3 B). The overall release of cGAMP / Zn was faster than that of cdAMP / Zn and cdGMP / Zn, which may be related to its unique nanoparticle structure. For CDN@CAP / PEI-PEG, there was a significant burst release of drug, followed by another phase of constant release ( Figure 3B) This distribution can be attributed to a portion of the CDN being attached to the CAP / PEI-PEG surface through charge interactions and readily released under conditions of high ionic strength and high pH. The release profile of the CDN-Zn@liposomes is not shown here because we have not yet developed a reliable method to quantify drug loading after liposome coating on CDN-Zn. It is expected that liposomes coated on the CDN-Zn surface will significantly increase particle stability and delay drug release. Prolonged drug release will contribute to increased in situ drug exposure and immune stimulation.

[0365] The experiments tested whether the CDN delivery system could effectively activate the STING pathway and trigger an immune response in vitro. TM ISG (interferon stimulated gene) cells were used in experiments to monitor STING activation by CDN preparations. Figure 3 As shown in C, at 0.25-2 μg / ml cdAMP, activation of the IFN signaling pathway was much higher for the cdAMP / Zn formulation than for the soluble form of free cdAMP. Similar stimulation improvements were also observed for the CDN@CaP / PEI-PEG formulation compared to the free form ( Figure 3 D). These in vitro evaluation results demonstrate that CDN-Zn and CDN@CaP / PEI-PEG possess favorable properties for in vivo therapeutic applications.

[0366] Example III.

[0367] This example describes the therapeutic effects of CDN-Zn and CDNs@CaP / PEI-PEG.

[0368] Finally, the therapeutic effect of CDN formulations on tumor-bearing mice was investigated. cdAMP(ps)2 was used as a representative CDN for demonstration. 3 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 on day 17 for tetramer staining and ELISPOT analysis with AH1 antigen peptide was performed on day 22. Figure 4As shown in A, the average tumor growth of mice treated with free CDN, CDN-Zn, and CDNs@CaP / PEI-PEG was significantly delayed compared to the untreated group. Although CDN-Zn appeared to inhibit tumor growth better, there was no statistical difference between them compared to CDN and CDNs@CaP / PEI-PEG. Regarding the survival of treated mice, the median survival time of the untreated, CDN, CDN-Zn, and CDNs@CaP / PEI-PEG groups was 23 days, 42 days, 64 days, and not reached ( Figure 4 B). From the individual tumor growth curve ( Figure 4 In C), complete tumor regression was observed in: 0 out of 5 mice in the untreated group; 2 out of 5 mice in the free CDN and CDN-Zn groups; and 3 out of 5 mice in the CDN@CaP / PEI-PEG group.

[0369] For PBMC tetramer staining assay, no significant differences were observed between the groups ( Figure 4 D). PBMC tetramer staining may not be sensitive enough to show antigen-specific T cell responses after nonspecific intratumoral CDN stimulation, or the time point may not be optimal. In contrast, ELISPOT assessment on day 22 showed a significant antigen-specific immune response ( Figure 4 EF). Seven days after the second dose of CDN treatment, significant AH1 antigen-specific T cell responses were observed in the free CDN, CDN-Zn, and CDNs@CaP / PEI-PEG groups. The responses of CDN-Zn and CDNs@CaP / PEI-PEG were also higher than those of free CDN, with statistical differences observed between free CDN and CDNs@CaP / PEI-PEG. Overall, these results suggest that the therapeutic activities of both CDN-Zn and CDN@CaP / PEI-PEG are as high as or even greater than those of free CDN. The therapeutic benefits of the formulations may arise from the combined effects of slow release and increased cellular uptake. Based on this, CDN-Zn@liposomes exert improved therapeutic efficacy due to more sustained release and improved in vivo stability.

[0370] Example IV.

[0371] This example describes the materials and methods of Examples I, II, and III.

[0372] Synthesis of CDN-Zn nanoparticles (NPs)

[0373] cGAMP, cdAMP, and cdGMP were obtained from Invivogen, while cdAMP(ps)2 was obtained from MedchemExpress. CDN was dissolved in methanol before use. Simultaneously, ZnCl2 (Sigma-Aldrich) was dissolved in methanol to prepare a 100 mM stock solution. In a typical synthesis reaction, a 10:1 (n / n) Zn2+ solution was added to a 1 mg / ml CDN working solution under vigorous stirring. The solution was stirred at room temperature for an additional 24 h. The resulting CDN-Zn NPs were centrifuged at 20,000 × g for 15 min to remove free CDN and Zn 2+ , and then washed again with methanol.

[0374] Synthesis of CDN-Zn@liposomes

[0375] Two steps were used to synthesize CDN-Zn@liposomes. First, CDN-Zn@DOPANPs were synthesized by a coordination-modulation method. Briefly, a 10 molar ratio of Zn 2+ The solution was added to a mixture of CDN / DOPA (Avanti Lipids) in chloroform. After 24 hours of incubation, CDN-Zn@DOPA NPs were isolated by centrifugation at 20,000 x g for 15 minutes. 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 30% (v / v) ethanol / H2O solution at 60°C. Finally, empty liposomes were removed by evaporating the THF under reduced pressure, cooling the final solution to room temperature, and centrifuging at 20,000 x g for 20 minutes to obtain CDN-Zn@liposomes. The resulting CDN-Zn@liposomes were then resuspended in PBS for further use.

[0376] Synthesis of CDNs@CaP / PEI-PEG NPs

[0377] CDN@CaP / PEI-PEG NPs were prepared using a single-step precipitation method. Briefly, CaCl2 (Sigma-Aldrich) and Na2HPO4 (Sigma-Aldrich) solutions were simultaneously injected into a mixed solution of PEI-PEG and CDN under continuous stirring. After overnight incubation, the CDN@CaP / PEI-PEG NPs were isolated by centrifugation at 18,000 x g for 15 minutes. The resulting NPs were washed twice with histidine buffer (pH 7.4).

[0378] In vitro release analysis

[0379] Release profiles of CDN-Zn and CDN-Zn@liposomes were analyzed by Slide-A-Lyzer TM A MINI dialysis device, 3.5KMWCO (Thermo Scientific) was used for the study. Briefly, 0.5 ml of CDN-Zn or CDN-Zn@liposome solution was filled in a cup with a regenerated cellulose membrane, and 14 ml of release buffer (PBS) was placed in the tube. After the dialysis cup was inserted into the conical tube and capped, the device was incubated at 37°C with continuous shaking (200 rpm). At the indicated time points, 300 μl of release medium was collected and refilled with an equal amount of fresh PBS. The CDN concentration in the release medium was analyzed by HPLC (GPC). Finally, the release percentage was calculated based on the CDN concentration in the release buffer, the buffer volume, and the total CDN loading.

[0380] Assessing activation of interferon-stimulated genes

[0381] THP1-Blue purchased from Invivogen TM ISG (interferon stimulated gene) cells were treated and cultured according to the manufacturer's instructions. Briefly, cells were thawed immediately upon receipt and transferred to a 25 cm 2 After the first passage, cells were maintained in growth medium and passaged every 3 days with a starting cell concentration of 7 x 10 5 cells / ml, with selective antibiotics added every other passage. To assess the bioactivity of CDN preparations, 20 μl of pre-warmed solution of indicator preparation was added to a 96-well flat-bottom plate. 180 μl of cell suspension (~100,000 cells / well) was then mixed with the CDN sample. After incubation at 37°C, 5% CO₂ for 18 h, 20 μl of supernatant was collected and incubated with 180 μl of QUANTI-blue solution (Invivogen) for a colorimetric reaction. THP1 activation was quantified by measuring absorbance at 620-655 nm.

[0382] Animal studies

[0383] All animals were cared for in accordance with federal, state, and local guidelines. All work performed on animals was in accordance with and approved by the University Committee on the Use and Care of Animals (UCUCA) at the University of Michigan, Ann Arbor. Female Balb / c mice (Jackson Laboratories) aged 6–8 weeks were inoculated with 1×10 5 CT26 colon cancer cells. When the tumor size reaches ~100mm 3Two doses of 25 μg cdAMP(ps)2 in different formulations were administered via the 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 following equation: Volume = length × width 2 × 0.5. Animals were euthanized when tumors reached 1.5 cm in diameter or when they became moribund (with severe weight loss or severe ulceration). On day 17, the percentage of tumor antigen-specific CD8α+ T cells in PBMCs was analyzed using a tetramer staining assay with peptide-MHC tetramers (AH1 restricted by H-2Kb) as described previously (NIH Tetramer Core Facility, Atlanta, Georgia). On day 22, ELISPOT assays were performed using PBMCs from treated mice as described previously.

[0384] Example V

[0385] This example provides the materials and methods used in Examples VI-XI.

[0386] Screening of metal ions for modulation of innate immune stimulants in vitro

[0387] Mouse bone marrow-derived dendritic cells (BMDCs) were isolated and cultured. Briefly, bone marrow stem cells were harvested and plated on bacteriological culture dishes with GM-CSF-containing culture medium. The cell culture medium was renewed 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 stimulants, we first seeded 100,000 BMDCs / 100ul per well in a 96-well plate. Various metal ions at different concentrations were then added together with various innate immune stimulants at various concentrations. At the same time, the same concentrations of free metal ions alone or free innate immune stimulants alone were used as controls. After incubation at 37°C, 5% CO2 for 24 hours, the supernatant was collected for ELISA assays of various cytokines.

[0388] Preparations of cyclic innate immune stimulator-metal ion combinations

[0389] CDNs-metal ion coordination polymers:cGAMP, cdAMP, and cdGMP were obtained from Invivogen, while cdAMP(ps)2 was obtained from MedchemExpress. Before use, CDNs were dissolved in methanol or endotoxin-free water. Simultaneously, 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 under vigorous stirring. The solution was stirred at room temperature for an additional 24 h. The resulting CDN-metal combination was centrifuged at 20,000 x g for 15 min to remove free CDN and metal ions, and then washed again with methanol.

[0390] CDNs-metal ions@liposomes: Two steps were used to synthesize CDN-metal@liposomes.

[0391] Here, we take CDN-Zn@liposome as an example. First, Zn-CDN / H11-DOPE NPs were synthesized by the coordination-regulation method. Briefly, a 10 molar ratio of Zn 2+ The solution was added to a mixture of CDN / H11-DOPE (AvantiLipids) in chloroform. After incubation for 24 hours, the Zn-CDN / H11-DOPE NPs were isolated by centrifugation at 20,000 x g for 15 minutes. 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 50% (v / v) ethanol / H2O solution. Finally, CDN-Zn@liposomes were obtained by evaporating the THF under reduced pressure, cooling the final solution to room temperature, and removing the empty liposomes by centrifugation at 20,000 x g for 20 minutes. The resulting CDN-Zn@liposomes were then resuspended in PBS for further use.

[0392] Metal ion-CDN / polyhistidine-PEG nanocoordination polymer (NCP): Metal ion-CDN / polyhistidine-PEG NCP was prepared by a one-step precipitation method. 2+ -CDN / polyhistidine-PEG as an example. Briefly, CoCl2 (Sigma-Aldrich) solution, CDN, polyhistidine-PEG and HEPES buffer were added dropwise to the mixed solution at a fixed ratio under continuous stirring. After incubation for 24 h, Co was separated using a 10 kD centrifugal ultrafiltration filter. 2+ -CDN / polyhistidine-PEG nanoparticles (NPs) to remove free metal ions and CDNs.

[0393] CDNs@CaP / PEI-PEG NPs:CDN@CaP / PEI-PEG NPs were prepared using a single-step precipitation method. Briefly, CaCl2 (Sigma-Aldrich) and Na2HPO4 (Sigma-Aldrich) solutions were simultaneously injected into a mixed solution of PEI-PEG and CDN under continuous stirring. After overnight incubation, the CDN@CaP / PEI-PEG NPs were isolated by centrifugation at 18,000 x g for 15 minutes. The resulting NPs were washed twice with histidine buffer (pH 7.4).

[0394] Innate immune stimulant-metal mineral@anionic peptide-PEG: The innate immune stimulator—metal mineral@anionic peptide-PEG—was prepared via a single-step precipitation method. Using MnP@PGA-PEG NPs as an example, MnCl2 (Sigma-Aldrich) and Na2HPO4 (Sigma-Aldrich) solutions were simultaneously injected into a mixed solution of PGA-PEG and the innate immune stimulator under continuous stirring. After overnight incubation, the innate immune stimulator-MnP@PGA-PEG NPs were isolated by centrifugation at 18,000 x g for 15 minutes. The resulting NPs were washed twice with histidine buffer (pH 7.4).

[0395] In vitro release analysis

[0396] The release profile of the formulation was analyzed by Slide-A-Lyzer TM A MINI dialysis device, 3.5K MWCO (ThermoScientific) was used for the study. Briefly, 0.5 ml of the formulation solution was filled in a cup with a regenerated cellulose membrane, and 14 ml of release buffer (PBS) was placed in the tube. After the dialysis cup was inserted into the conical tube and capped, the device was incubated at 37 ° C with continuous shaking (200 rpm). At the indicated time points, 300 μl of release medium was collected and refilled with an equal amount of fresh PBS. The concentration of CDN in the release medium was analyzed by HPLC (GPC). Finally, the percentage release was calculated based on the CDN concentration in the release buffer, the buffer volume, and the total CDN loading.

[0397] Animal studies

[0398] All animals were cared for in accordance with federal, state, and local guidelines. All work performed on animals was in accordance with and approved by the University Committee on the Use and Care of Animals (UCUCA) at the University of Michigan, Ann Arbor. Female Balb / c mice (Jackson Laboratories) aged 6–8 weeks were inoculated with 1×10 5 CT26 colon cancer cells. When the tumor size reaches ~50mm3 The designated drug or formulation was administered by the designated route. Tumor size and survival were monitored every 2 or 3 days. Tumor size was calculated based on the following equation: Volume = Length × Width 2 × 0.5. Animals were euthanized when tumors reached 1.5 cm in diameter or when they became moribund (with severe weight loss or unhealed ulcers). On day 17, the percentage of tumor antigen-specific CD8α+ T cells in PBMCs was analyzed using a tetramer staining assay with peptide-MHC tetramers (AH1 restricted by H-2Kb) as described previously (NIHTetramer Core Facility, Atlanta, Georgia). On day 22, ELISPOT assays were performed using PBMCs from treated mice as described previously.

[0399] Example VI.

[0400] This example describes the identification of metal ions that enhance STING activation by STING agonists.

[0401] like Figure 5 As shown in A and 5B, mouse bone marrow-derived dendritic cells (BMDCs) were treated with different metal ions or co-treated with different metal ions and STING agonists. We selected metal ions from essential minerals and trace mineral elements in biological systems. Mn alone 2+ can activate BMDCs at high toxic doses. 2+ In combination with STING agonists, this resulted in a significant enhancement of STING activation at much lower concentrations. 2+ However, when 125uM or 250uM Co 2+ When combined with 5uM cGAMP, the combination greatly enhanced the activation of the STING pathway. Both concentrations were well tolerated. To further confirm whether this phenomenon still applies to human cells, we repeated the same experiment using the human monocytic cell line THP1 ( Figure 5 C) A similar trend was observed in human THP1 cells, and we verified that this phenomenon was independent of the type of STING agonist.

[0402] Example VII.

[0403] This example demonstrates that Co 2+ and Mn 2+ Enhanced STING activation and anti-cancer therapeutic efficacy.

[0404] We examined whether enhanced type I IFN responses in vitro could benefit cancer therapy in vivo. We evaluated combinations of metal ions and STING agonists in a murine tumor model. Figure 6 As shown in a and 6c, Co 2+ -CDA and Mn 2+ -CDA delayed tumor growth. In particular, there were significantly more tumor-free mice in the metal-CDA group than in the free CDA group, as evidenced by the 80% survival rate in the metal-CDA group versus the 20% survival rate in the free CDA group ( Figure 6 d) In addition, compared with the free CDA treatment, Co 2+ -CDA treatment resulted in significantly higher serum IFNβ levels 8 hr after injection ( Figure 6 b) However, for Mn 2+ -CDA combination, we did not observe the same phenomenon.

[0405] Example VIII.

[0406] This example demonstrates an improved in vivo immune response to a STING agonist-metal combination.

[0407] To investigate the mechanism of action for the improved efficacy of cancer therapy, we evaluated the antigen-specific T cell responses of treated animals and performed tumor re-challenge studies 81 days after initial treatment. As shown by ELISPOT results on day 22 of the experiment, CDA-Mn 2+ showed better T cell-specific responses, while CDA-Co 2+ The T cell ELISPOT results were similar between the free CDA group ( Figure 7 b) For tumor rechallenge studies, from CDA-Co 2+ and CDA-Mn 2+ Survivors of the treatment group completely stopped the growth of the second CT26 tumor. 2+ The treated group showed a significant increase in antigen-specific T cell responses.

[0408] Example IX.

[0409] This example demonstrates the identification of metal ions that can modulate other innate immune stimulants.

[0410] Based on our results on the STING pathway, we also examined whether metal ions could modulate other innate immune stimulants. We treated mouse BMDCs with different metal ions or combinations of different metal ions and innate immune stimulants. We observed similar metal ion-innate immune stimulant synergy; however, different metal ions synergized with different DAMPs or PAMPs, including TLR3 / 4 / 7 / 8 / 9 ligands, NOD1 / 2 ligands, TLR 7 / 8 ligands, RIG-I & CDS agonists, and inflammasome inducers. For example, Co 3+ Significantly increased the production of IFNb, TNFa, IL6, and IL2 by poly-IC, while Mn 2+ Only IFNb production by poly-IC was increased (Fig. 8a-d). 2+ MPLA increased the production of IFNb and TNFa, while Ni 2+ Increased TNFa production by MPLA (Fig. 8e-f). 2+ Increased IFNb and TNFa production in R848, while Ni 2+ Increased TNFa production in R848 ( Figure 7 gh). Ni 2+ and Mn 2+ CpG increased the production of IFNβ and TNFa (Figure 8i-j). Mn2+, Co2+, Al3+, Cu2+, Fe3+, and Ni2+ can also regulate the cytokine distribution of NOD1 / 2 ligands, TLR 7 / 8 ligands, RIG-I & CDS agonists, and inflammasome inducers ( Figure 9-12 ). These results show that our method based on metal ions is a simple but effective way to regulate the cytokine distribution of a wide range of immunostimulants. Based on this discovery, we expect that pharmaceutically acceptable formulations can be developed to make better and stronger vaccine adjuvants or cancer immunotherapeutics. For example, specific metal salts of DAMP / PAMP may perform better than the original form. Coordination polymers consisting of selected metal ions and DAMP / PAMP (with or without pharmaceutically acceptable coordination molecules) can lead to optimized metal ion-DAMP / PAMP combinations. Other pharmaceutically acceptable formulations for the co-delivery of metal ions and DAMP / PAMPs, including but not limited to metal-hydroxide / carbonate / phosphate minerals, liposomes, lipid nanoparticles, PLGA particles, hydrogels, emulsions, etc. are also possible.

[0411] Example X

[0412] This example describes representative formulations of metal-innate immune stimulators.

[0413] In order to co-deliver metal ions and innate immune stimulants to the correct target tissue with an ideal release profile, appropriate formulations based on physical and chemical properties can be designed, such as specific metal salts of DAMPs / PAMPs, coordination formulations, and other pharmaceutically acceptable formulations (hydroxide / carbonate / phosphate minerals, liposomes, lipid nanoparticles, PLGA, hydrogels, emulsions, etc.). Here, we provide several representative examples of coordination formulations: manganese-CDA-H11-DOPE@liposome nanoparticles (Mn-CDA / H11@liposomes, Figure 13 ), Co-CDA / H33-PEG coordinated nanoparticles (Co-CDA / H33-PEG, Figure 14 ) and CDA@Co2+-4-arm-PEG-His11 hydrogel (CDA@4aH11-Co hydrogel, Figure 15 ). CDA itself can bind to the nitrogen of the purine ring and the Co 2+ and Mn 2+ coordination, which can be further stabilized by polyhistidine. Nanoparticle structure ( Figure 13-14 ) or hydrogel ( Figure 15 ) are generated by designing different building blocks and can be optimized by Co 2+ / Mn 2+ The ratio and concentration of :CDA:poly-histidine-PEG, reaction time and pH were adjusted. 2+ / Mn 2+ The loading efficiency of was about 30%, while that of CDA exceeded 70%. We further tested those complex formulations in the murine CT26 colon tumor model. Figure 13-15 As shown, those nanoparticle structures or hydrogel formulations can greatly enhance STING activation in vivo compared with free CDA or free CDA + metal ions. In particular, liposome-coated nanoparticles CDA-Mn-His11-DOPE@liposomes (Mn-CDA / H11@lip) can be used for systemic delivery of STING agonists and eradicate 60% of established CT26 colon tumors ( Figure 13 ); Co-CDA / His33-PEG can significantly prolong the production of IFNb, which can be detected even 4 days after injection ( Figure 14 ); and the injectable CDA@4aH11-Co hydrogel induced a strong local ablative immune response and formed significant ulcers after the first dose ( Figure 15 f) These improved therapeutic effects were also characterized by elevated antigen-specific T cell responses, type I IFN responses, and pro-inflammatory cytokine release.

[0414] In addition to the formulations mentioned above, there are many other formulations that can be synthesized to deliver metal-innate immune stimulators. Here, we have provided some examples, where their morphology is shown in TEM images ( Figure 16 ).like Figure 16 As shown in a, CDA-Zn NPs exhibit a spherical shape with high TEM contrast on the surface, resulting in a "core-shell" like structure. We also found that a uniform spherical structure was obtained when the synthesis was carried out in aqueous medium because nucleation occurs more slowly in water. Consistent with the TEM images, DLS and zeta potential data indicate that the size of cdAMP-Zn is about 150 nm and the surface charge is neutral. Under the same synthesis conditions, CDA-Co 2+ NP shows cross-linked nanoparticle clusters; CDG-Zn 2+ Showing uniform irregular spherical structures with a size of approximately 100 nm and neutral surface charge; cGAMP-Zn 2+ The nanoparticles are shown to be spherical in shape, consisting of accumulated smaller clusters, and have a small amount of positive charge on the surface. 2+ To improve the stability of NPs, we also added other multivalent ligands, such as liposomes ( Figure 16 b), polyhistidine ( Figure 16 c) and polyhistidine-PEG ( Figure 16 d). In addition, innate immune stimulators loaded in nano-sized metal minerals can also be prepared to deliver metal ion-innate immune stimulator combinations ( Figure 16 To increase the stability of nanoparticles, PEI-PEG, PGA-PEG and other anionic peptide-PEG can be used for surface modification.

[0415] We also evaluated a subset of the above-mentioned formulations in tumor-bearing mice. 3 When the tumor was treated with 2 doses of the indicated formulations with 25 μg / dose of adAMP(ps)2, the tumor was treated with free CDN, CDN-Zn 2+ The tumor growth of mice treated with CDNs@CaP / PEI-PEG was significantly delayed. 2+ More effectively inhibited tumor growth than CDN and CDNs@CaP / PEI-PEG, even though there was no statistical difference among them. 2+The median survival time of the CDNs@CaP / PEI-PEG and CDNs@CaP / PEI-PEG groups was 23 days, 42 days, 64 days and not reached, respectively (Figure 17d). From the individual tumor growth curves (Figure 17e), we observed complete tumor regression in the following: 0 out of 5 mice in the untreated group; 2+ 2 out of 5 mice in the CDN@CaP / PEI-PEG group; and 3 out of 5 mice in the CDN@CaP / PEI-PEG group. No significant differences were observed between the groups for PBMC tetramer staining assays (Figure 17f). PBMC tetramer staining may not be sensitive enough to show antigen-specific T cell responses after nonspecific intratumoral CDN stimulation, or the time point may not be optimal. In contrast, ELISPOT assessment on day 22 showed significant antigen-specific immune responses (Figures 17f-g). 7 days after the second dose of CDN treatment, in the free CDN, CDN-Zn 2+ Significant AH1 antigen-specific T cell responses were observed in the CDNs@CaP / PEI-PEG and CDNs@CaP / PEI-PEG groups. 2+ The responses of CDNs@CaP / PEI-PEG were also higher than those of free CDN, and statistical differences were observed between free CDN and CDNs@CaP / PEI-PEG.

[0416] Example XI.

[0417] This example describes the chelation of metal ions to suppress the innate immune response.

[0418] Given our discovery of the interesting function of metal ions in regulating innate immune responses, we further evaluated whether chelated metal ions can inhibit the corresponding innate immune pathways, which can be used to treat autoimmune diseases such as systemic lupus erythematosus, Aicardi–Goutières syndrome, acute pancreatitis, age-dependent macular degeneration, alcoholic liver disease, liver fibrosis, metastasis, myocardial infarction, non-alcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, inflammatory bowel disease, multiple sclerosis, etc. Through unbiased screening, we identified several chelators that showed significant function in inhibiting innate immune responses (Figures 18-19). As shown in Figures 18a-b, as the structural complexity increases, the chelators exhibit higher inhibitory function. This is consistent with our hypothesis that the higher the structural complexity of the chelators, the better their chelating ability should be. Using THP 1 bi-KI-hSTING WT(R232)Using reporter cell lines, we co-incubated these chelators with challenging DNA / liposome transfection complexes that should have very high activity in activating the cGAS-STING-type I IFN pathway. The degree of inhibition can be understood by ISRE-induced luminescence. We found that the IC50s for DNA-induced type I IFN responses for punicalagin (PC) and tannic acid (TA) were as low as nanomolar levels and were well tolerated in in vitro assays (Figures 18b-d). We also confirmed the inhibitory effect on another human STING allele, HAQ, and obtained similar results (Figure 18e). To investigate which step of the cGAS-STING-type I IFN pathway the chelators affect, we investigated whether they could inhibit cGAMP-induced type I IFN (Figure 18f). We found that the inhibitory effect was abolished, suggesting that these chelators may act primarily on cGAS inhibition. Note that the chelators we present here are primarily natural polyphenols. Polyphenols are widely reported to eliminate ROS and are anti-inflammatory. However, less is known about their potent inhibitory effects on DNA-induced inflammation. For the same reason, we also found that these chelators can be used to inhibit the poly-IC-induced inflammatory response in the STING knockout THP1 reporter cell line ( Figure 19 We anticipate that many other chelating agents, especially those with polyphenolic structures (e.g. Figure 20 can be used as an innate immune suppressor of DNA- and RNA-induced inflammation.

[0419] Incorporation by reference

[0420] The entire disclosure of each patent document and scientific article mentioned herein is incorporated by reference for all purposes.

[0421] Equivalent

[0422] The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the foregoing embodiments are to be considered in all respects as illustrative rather than restrictive of the invention described herein. Therefore, the scope of the present invention is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalence of the claims are intended to be included therein. Sequence Listing <110> The Regents of the University of Michigan <120> Compositions and methods for metal-containing formulations capable of modulating immune responses <130> PPI21170055US <150> US 62 / 697,092 <151> 2018-07-12 <160> 386 <170> PatentIn version 3.5 <210> 1 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 1 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Glx 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 2 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 2 Gly Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys Lys 20 <210> 3 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 3 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Trp 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 4 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 4 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 5 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 5 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys Lys 20 <210> 6 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <222> (13) <223> Xaa = Hydroxyproline <400> 6 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 7 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 7 Pro Val Leu Asp Leu Phe Lys Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 8 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 8 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Gly Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 9 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 9 Pro Val Leu Asp Leu Phe Arg Glu Leu Gly Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 10 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 10 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Glx Lys Gln Lys Leu Lys 20 <210> 11 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 11 Pro Val Leu Asp Leu Phe Lys Glu Leu Leu Gln Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 12 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 12 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Gly Lys Gln Lys Leu Lys 20 <210> 13 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 13 Gly Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Gly Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 14 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (18) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (20)..(20) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (22)..(22) <223> Xaa can be any naturally occurring amino acid <400> 14 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Xaa Gln Xaa Leu Xaa 20 <210> 15 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 15 Pro Val Leu Asp Leu Phe Arg Glu Leu Trp Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 16 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 16 Pro Val Leu Asp Leu Leu Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 17 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 17 Pro Val Leu Glu Leu Phe Lys Glu Leu Leu Gln Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 18 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 18 Gly Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 19 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 19 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Gly Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 20 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 20 Pro Val Leu Asp Leu Phe Arg Glu Gly Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> twenty one <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty one Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> twenty two <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty two Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Gly 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> twenty three <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty three Pro Leu Leu Glu Leu Phe Lys Glu Leu Leu Gln Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> twenty four <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty four Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 <210> 25 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13) <223> Xaa can be any naturally occurring amino acid <400> 25 Pro Val Leu Asp Phe Phe Arg Glu Leu Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 26 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 26 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Leu 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 27 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 27 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Glx Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 28 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 28 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Trp Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 29 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 29 Ala Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 30 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 30 Pro Val Leu Asp Leu Pro Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 31 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13) <223> Xaa can be any naturally occurring amino acid <400> 31 Pro Val Leu Asp Leu Phe Leu Glu Leu Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 32 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (1)..(1) <223> Xaa can be any naturally occurring amino acid <400> 32 Xaa Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 33 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 33 Pro Val Leu Asp Leu Phe Arg Glu Lys Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 34 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 34 Pro Val Leu Asp Glx Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 35 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 35 Pro Val Leu Asp Trp Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 36 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 36 Pro Leu Leu Glu Leu Leu Lys Glu Leu Leu Gln Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 37 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 37 Pro Val Leu Asp Leu Phe Arg Glu Trp Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 38 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13) <223> Xaa can be any naturally occurring amino acid <400> 38 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Trp Lys Gln Lys Leu Lys 20 <210> 39 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 39 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Glu Glu Leu Leu Lys Ala 1 5 10 15 Leu Lys Lys Lys Leu Lys 20 <210> 40 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 40 Pro Val Leu Asp Leu Phe Asn Glu Leu Leu Arg Glu Leu Leu Glu Ala 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 <210> 41 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13) <223> Xaa can be any naturally occurring amino acid <400> 41 Pro Val Leu Asp Leu Trp Arg Glu Leu Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 42 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <400> 42 Pro Val Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Trp Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 43 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <400> 43 Pro Val Leu Asp Glu Phe Arg Glu Lys Leu Trp Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 44 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <400> 44 Pro Val Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 45 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <400> 45 Pro Val Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 46 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <400> 46 Pro Val Leu Asp Leu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 47 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 47 Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Gly Leu Glu Ala Leu 1 5 10 15 Lys Gln Lys Leu Lys 20 <210> 48 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 48 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 49 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 49 Pro Val Leu Asp Leu Phe Arg Asn Leu Leu Glu Lys Leu Leu Glu Ala 1 5 10 15 Leu Glu Gln Lys Leu Lys 20 <210> 50 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <400> 50 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Trp Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 51 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <400> 51 Pro Val Leu Asp Leu Phe Trp Glu Leu Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 52 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <400> 52 Pro Val Trp Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 53 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 53 Val Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 54 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 54 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Trp Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 55 <211> 19 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 55 Pro Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala Leu Lys Gln 1 5 10 15 Lys Leu Lys <210> 56 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 56 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Lys Lys 20 <210> 57 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 57 Pro Val Leu Asp Leu Phe Arg Asn Leu Leu Glu Glu Leu Leu Lys Ala 1 5 10 15 Leu Glu Gln Lys Leu Lys 20 <210> 58 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13) <223> Xaa can be any naturally occurring amino acid <400> 58 Pro Val Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu 20 <210> 59 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 59 Leu Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 60 <211> 19 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 60 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln <210> 61 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <400> 61 Pro Val Leu Asp Glu Phe Arg Trp Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 62 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <400> 62 Pro Val Leu Asp Glu Trp Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 63 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <400> 63 Pro Val Leu Asp Phe Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 64 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <400> 64 Pro Trp Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 65 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (12)..(12) <223> Xaa can be any naturally occurring amino acid <400> 65 Val Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala Leu 1 5 10 15 Lys Gln Lys Leu Lys 20 <210> 66 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 66 Pro Val Leu Asp Leu Phe Arg Asn Leu Leu Glu Glu Leu Leu Glu Ala 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 <210> 67 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 67 Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala Leu 1 5 10 15 Lys Gln Lys Leu Lys 20 <210> 68 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13) <223> Xaa can be any naturally occurring amino acid <400> 68 Pro Val Leu Asp Glu Phe Arg Glu Leu Leu Lys Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 69 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13) <223> Xaa can be any naturally occurring amino acid <400> 69 Pro Val Leu Asp Glu Phe Arg Lys Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 70 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13) <223> Xaa can be any naturally occurring amino acid <400> 70 Pro Val Leu Asp Glu Phe Arg Glu Leu Leu Tyr Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 71 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (14)..(14) <223> Xaa can be any naturally occurring amino acid <400> 71 Pro Val Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Leu Xaa Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 72 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (13)..(13) <223> Xaa can be any naturally occurring amino acid <400> 72 Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Xaa Leu Trp Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 <210> 73 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> misc_feature <223> Xaa can be any naturally occurring amino acid <220> <...

Claims

1. A nanoparticle composition comprising one or more DAMPs or PAMPs, and One or more cations selected from the group consisting of: Zn 2+ , Mn 2+ 、Co 2+ and Co 3+ and related phosphates or carbonates, in, The one or more DAMPs or PAMPs are selected from STING agonists and Toll-like receptor agonists, wherein the one or more STING agonists 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, fluorinated 3'3'-cGAMP, fluorinated c-di-AMP, 2'3'-c-di-AMP, 2'3'-c-di-AM (PS) 2 (Rp, Rp), fluorinated c-di-GMP, 2'3'-c-di-GMP, c-di-IMP, , , , , , Gemcitabine 、STING-agonist-C11 、STING agonist-1 、STING agonist G10 , cGAM(PS)2, 2'3'-cGAM(PS)2(Rp / Sp), 2'2'-cGAM(PS)2, 2'3'-cGAM(PS)2, fluorinated cGAMP, fluorinated 2'3'-cGAMP, fluorinated 2'2'-cGAMP, 2'3'-cdAMP, 2'2'-cdAMP, 3'3'-cdAMP, c-di-AM(PS)2, 2'2'-c-di-AM(PS)2, 3'3'-c-di -AM(PS)2, fluorinated 2'3'-cdAMP, fluorinated 2'2'-cdAMP, fluorinated 3'3'-cdAMP, 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, fluorinated cdGMP, fluorinated 2'3'-c dGMP, fluorinated 2'2'-cdGMP, fluorinated 3'3'-cdGMP, 2'3'-cAIMP, 2'2'-cAIMP, 3'3'-cAIMP, fluorinated 3'3'-cAIMP, fluorinated 2'3'-cAIMP, fluorinated 2'2'-cAIMP, cAIM(PS)2 difluoro, 3'3'-cAIM(PS)2 difluoro(Rp / Sp), 2'3'-cAIM(PS)2 difluoro, 2'2'-cAIM( PS)2 difluoro, 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, fluorinated c-di-IMP, fluorinated 2'3'-cdIMP, fluorinated 2'2'-cdIMP, fluorinated 3'3'-cdIMP and amidobenzimidazole-based compounds, Wherein, the Toll-like receptor agonist is selected from TLR-3 agonist, TLR-4 agonist, TLR-5 agonist, TLR-7 agonist, TLR-8 agonist, and TLR-9 agonist.

2. The composition according to claim 1, wherein The composition is capable of stimulating an innate immune response in a subject upon administration to the subject.

3. The composition according to claim 2, wherein The subject has cancer or is at risk of developing cancer.

4. The composition according to claim 2, wherein The composition is used to elicit an immune response for vaccine applications.

5. The composition according to claim 2, wherein The composition is capable of stimulating an innate immune response in at least one cancer cell upon administration to the subject, wherein the subject has cancer.

6. The composition according to claim 2, wherein Stimulating an innate immune response includes stimulating an innate cytokine response mediated by cytokines, wherein the innate cytokine response is mediated by type 1 interferon.

7. The composition according to claim 1, wherein The composition is associated with the nanoparticles, wherein the association is selected from the group consisting of complexation, conjugation, encapsulation, absorption, adsorption and incorporation.

8. The composition according to claim 7, wherein The nanoparticles are selected from the group consisting of metal-polyhistidine-PEG, 4-arm-PEG-polyhistidine-metal hydrogel and liposome.

9. The composition according to claim 8, wherein The liposome is metal-polyhistidine-1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine@liposome.

10. The composition according to claim 7, wherein The nanoparticle is further associated with an antigen, wherein the association is selected from the group consisting of complexation, conjugation, encapsulation, absorption, adsorption and incorporation.

11. The composition according to claim 10, wherein The antigen 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, 2 and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, Mage-A1,2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA, Pmel 17, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, p58, CEA, RAGE, NY-ESO, 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 antigen E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA72-4, CAM 17.1, NuMa, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3, CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733, human EGFR protein or its fragments, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, WT1 and WT1-derived peptide sequences WT1 126–134 RMFPNAPYL, WT1 122–140 SGQARMFPNAPYLPSCLES or WT1 122–144SGQARMFPNAPYLPSCLESQPTI, MUC1 and MUC1-derived peptides or glycopeptides RPAPGS, PPAHGVT or PDTRP, LMP2, EGFRvIII, GD2, Ras mutant, p53 mutant, proteinase 3, survivin, hTERT, EphA2, EphA4, LMW-PTP, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucose GM1, mesothelin, sLe, CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, XAGE 1, B7H3, legumin, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-α, PDGFR-β, MAD-CT-2, Fos-related antigen 1, ERBB2, folate receptor 1, IDH1, IDO, LY6K, fms-related tyrosine kinase 1, KDR, PADRE, TA-CIN, SOX2, neoantigens, and aldehyde dehydrogenase.

12. The composition according to claim 10, wherein The antigen is derived from an autologous antigen.

13. The composition according to claim 10, wherein The antigen is conjugated to the outer surface of the nanoparticle.

14. The composition according to claim 1, wherein The composition is combined with an adjuvant, wherein the combination is selected from the group consisting of complexation, conjugation, encapsulation, absorption, adsorption and incorporation.

15. The composition according to claim 14, wherein The adjuvant is selected from the group consisting of: CPG, poly IC, poly-ICLC, 1018 ISS, aluminum salt, Amplivax, BCG, CP-870,893, CpG7909, CyaA, dSLIM, cytokines, IC30, IC31, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, PLGA microparticles, imiquimod, resiquimod, gademod, 3M-052, SRL172, virus-like particles, YF-17D, VEGF trap, β-glucan, Pam3Cys, Aquila's QS21 stimulator, vadimezan, AsA404, 3M MEDI9197, glucopyranosyl lipid adjuvants, GLA-SE, CD1d ligands, STING agonists, CL401, CL413, CL429, Flagellin, RC529, E6020, imidazoquinoline-based small molecule TLR-7 / 8a, virosomes, AS01, AS02, AS03, AS04, AS15, IC31, CAF01, ISCOM, bacterial toxins, and combinations thereof.

16. The composition according to claim 7, wherein The nanoparticles are associated with an adjuvant, wherein the association is selected from the group consisting of complexation, conjugation, encapsulation, absorption, adsorption and incorporation.

17. The composition according to claim 16, wherein The adjuvant is selected from the group consisting of: CPG, poly IC, poly-ICLC, 1018 ISS, aluminum salt, Amplivax, BCG, CP-870,893, CpG7909, CyaA, dSLIM, cytokines, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, PLGA microparticles, resiquimod, gademod, 3M-052, SRL172, virus-like particles, YF-17D, VEGF trap, β-glucan, Pam3Cys, Aquila's QS21 stimulator, vadimezan, AsA404, 3M MEDI9197, glucopyranosyl lipid adjuvants, GLA-SE, CD1d ligands, STING agonists, CL401, CL413, CL429, Flagellin, RC529, E6020, imidazoquinoline-based small molecule TLR-7 / 8a, virosomes, AS01, AS02, AS03, AS04, AS15, IC31, CAF01, ISCOM, bacterial toxins, and combinations thereof.

18. The composition according to claim 7, wherein The average particle size of the nanoparticles is between 6 and 500 nm.

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