Compositions and methods for metal-containing formulations that can modulate immune responses

Abstract

To provide compositions capable of stimulating the innate immune response.SOLUTION: A composition comprises nanoparticles comprising: one or more DAMPs or PAMPs; and one or more cations selected from the group consisting of Zn2+, Mn2+, Ca2+, Fe2+, Fe3+, Cu2+, Ni2+, Co2+, Pb2+, Sn2+, Ru2+, Au2+, Mg2+, VO2+, Al3+, Co3+, Cr3+, Ga3+, Tl3+, Ln3+, MoO3+, Cu+, Au+, Tl+, Ag+, Hg2+, Pt2+, Pb2+, Hg2+, Cd2+, Pd2+, Pt4+, Na+ and K+.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 697,092, filed on 12 July 2018, the entire contents of which are incorporated herein by reference. Description of research and development funded by the federal government. This invention was made with government support under CA210273, granted by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0002] This disclosure provides compositions and methods for stimulating the innate immune response of a subject by means of agents that, when administered to the subject, can stimulate the subject's innate immune response (e.g., damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs)). In particular, the disclosure relates to compositions of DAMP / PAMPs and metal ions, as well as systems and methods for utilizing such nanoparticles (e.g., in diagnostic and / or therapeutic settings). [Background technology]

[0003] The innate immune system is the first line of defense in humans, and its activation can induce the secretion of pro-inflammatory cytokines and modulate the adaptive immune system. DAMPs and PAMPs are two major innate immune stimulants. DAMPs are endogenous host biomolecules released upon tissue injury and include heat shock proteins and HMGB1 (High Mobility Group Box 1), ATP, uric acid, hyaluronic acid fragments, heparin sulfate, and tumor-derived DNA. PAMPs are conserved pathogen components recognized by various pathogen recognition receptors (PRRs) and induce anti-pathogenic inflammation. PAMPs include Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), cytosolic DNA sensors (CDSs), IFN gene (STING) agonist stimulants, purine-containing or purine-derived agents, and C-type lectin receptors (CLRs).

[0004] DAMP and PAMP were able to induce the production of pro-inflammatory cytokines and changes in the pro-inflammatory phenotype of immune cells, which are important in both cancer and autoimmune diseases. On the other hand, changes in the pro-inflammatory phenotype were able to disrupt the immunosuppressive tumor microenvironment and adjust the "cold tumor" to a "hot tumor." Therefore, TLR-3, TLR4, TLR7, TLR9, NLRP3, and STING agonists are currently undergoing clinical trials for cancer immunotherapy. In particular, the tumor-derived DNA-cGAS-STING pathway has recently been found to be extremely important for observing tumor immunity, and the dramatic effects shown in preclinical cancer immunotherapy studies have led to the implementation of numerous Phase I clinical trials of STING agonists. Meanwhile, DAMP and PAMP are widely involved in the development and progression of autoimmune diseases. Inhibiting abnormal innate immune responses is becoming increasingly clear as an effective treatment for many incurable autoimmune diseases. By modulating the immune response mediated by DAMP and PAMP, novel therapies for a variety of human diseases, including cancer and autoimmune diseases, may be provided.

[0005] This invention addresses this need. [Overview of the project]

[0006] Immune checkpoint inhibitors allow the patient's own immune system to fight cancer. However, the current average response rate to immune checkpoint inhibition is only about 30%. This is thought to be because some tumors have characteristics of "low-temperature tumors," which are not easily recognized by the immune system. Characteristics of such tumors include low inflammatory response, low mutational burden, and lack of tumor infiltration of T cells and other pro-inflammatory immune cells. On the other hand, "high-temperature tumors," which have many inflammatory characteristics that the immune system can recognize, have a good therapeutic response rate to cancer immunotherapy. Therefore, it is important to understand how to transform "low-temperature tumors" into "high-temperature tumors."

[0007] Accumulated evidence indicates that the observation of tumor immunity mediated by the innate immune system recognizes the presence of tumors by sensing tumor cell-derived DNA via the STING pathway. Activation of the STING pathway triggers an innate immune cascade, including type I interferon response and other pro-inflammatory phenotypic changes, which further trigger adaptive antitumor responses. Therefore, STING is considered a "trigger" for the return from "low-temperature tumors" to "high-temperature tumors." For example, intratumoral administration of STING agonists can induce antitumor immune responses against both local and metastatic tumors. Type I interferon response, as well as antigen-specific T cell infiltration, has been clinically demonstrated to be a favorable characteristic of cancer treatment outcomes. Therefore, developing STING agonists with high in vivo stability, favorable pharmacokinetic properties, and an acceptable safe profile is of paramount importance and highly translatable value.

[0008] However, current evaluations of most human STING agonists are based on cyclic dinucleotides and their derivatives. Their low molecular weight, poor pharmacokinetic parameters, and serious side effects severely limit their systemic administration.

[0009] Experiments conducted in the process of developing embodiments of the present invention showed that Zn 2+ We demonstrated that CDN containing cdi-AMP, cGAMP, and cGMP assembles into uniform nanoparticles in the presence of Zn. 2+ It was also shown that such CDNs, assembled into homogeneous nanoparticles in the presence of calcium phosphate, can be further stabilized in lipid vesicles. Further experiments demonstrated that CDNs can be formulated into nanoparticles in the presence of calcium phosphate, as well as copolymers of cationic poly(ethyleneimine) (PEI) and polyethylene glycol (PEG). Such CDN-nanoparticle assemblies (e.g., copolymers of CDN and PEI-PEG formulated into nanoparticles in the presence of calcium phosphate) (e.g., Zn 2+ It has been further shown that the CDN formulated into nanoparticles in the presence of liposomes enables increased STING agonist delivery efficacy and reduced STING agonist toxicity by increasing cancer cell uptake and providing more precise targeting to the tumor microenvironment (e.g., TME).

[0010] These results indicate that the CDN-Zn embodiment exhibits the following unique characteristics compared to previous drug delivery systems: 1) a reversible assembly suitable for sustained-release drugs released without loss of biological activity, 2) high loading efficiency and capacity, 3) increased cellular uptake, 4) pH-sensitive release at low pH, 5) good biocompatibility, 6) flexible surface chemistry suitable for surface modification and functionalization, and 7) low cost and ease of scale-up.

[0011] The CDN@CaP / PEI-PEG embodiment exhibits the following unique characteristics compared to previous drug delivery systems: 1) increased cellular uptake, 2) high loading efficiency, 3) pH-sensitive release at low pH, 4) biocompatibility, and 5) low cost and ease of scale-up.

[0012] These nanoparticles related to CDN can induce a specific immune response against tumors through systemic administration, thereby avoiding the need for direct local injection into the tumors. Such results are of great clinical importance.

[0013] Further experiments conducted during the development of embodiments of the present invention have discovered that certain metal ions can significantly enhance the STING activation and type I IFN response of STING agonists. For example, in the optimized state, Mn 2+ or Co 2+ has been shown to enhance the STING activity of cGAMP by more than 60-fold. Furthermore, the administration of STING agonists combined with Mn 2+ or Co 2+ to mouse tumors has been shown to significantly improve the therapeutic effect, characterized by an increase in serum type I IFN concentration, a higher tumor eradication effect, and longer survival of the animals. After treatment, in 80% of the tumor-bearing mice, the resulting tumors were eradicated, remained resistant to difficult secondary tumors even 80 days later, and long-term immunity against tumor recurrence was shown. Furthermore, this phenomenon has been found to be generalizable in various other innate immune pathways, such as TLR3 / 4 / 7 / 8 / 9 ligands, NOD1 / 2 ligands, TLR 7 / 8 ligands, RIG-I&CDS agonists, inflammasome-inducible types, etc. For example, Co 3+ dramatically increased the dendritic cell-mediated production of IFNβ, TNFα, IL6, and IL2 via cell IC, while Mn 2+ increased the production of IFNβ via cell IC. Mn 2+ increased the production of IFNβ and TNFα via MPLA, and Ni 2+ increased the production of TNFα via MPLA. Mn 2+ increased the R848-mediated production of IFNβ and TNFα, and Ni 2+ increased the production of TNFα via R848. In addition, Ni 2+ and Mn 2+ increased the production of IFNβ and TNFα via CpG.

[0014] Based on these results, several pharmaceutically acceptable formulations were developed to precisely deliver combinations of metal-containing innate immunostimulants to desired targets and promote immune activation. For example, liposome-coated nanoparticles, CDA-Mn-His11-DOPE@liposome (Mn-CDA / H11@lip), can be used for systemic delivery of STING agonists and can eradicate 60% of resulting CT26 colorectal tumors. Co-CDA / His33-PEG can significantly extend IFNb production, which was detectable even 4 days after injection. Furthermore, experiments were conducted to test whether chelated intracellular metal ions inhibit innate immune responses. Through equitable screening, several chelating agents were identified that can effectively inhibit DNA-induced cGAS-STING-TypeI IFN / NFκB responses and poly-IC-induced TLR3-cGAS-STING-Type-I IFNs, suggesting their potential usefulness in the treatment of autoimmune diseases. Overall, these results represent a simple but effective approach to addressing several unresolved medical challenges, including improving the efficacy of vaccine adjuvants, developing cancer immunotherapies, and controlling autoimmune diseases.

[0015] Therefore, such results and embodiments represent a novel class of drug delivery systems for both local and systemic delivery of drugs that can stimulate an innate immune response in a subject upon administration to that subject.

[0016] Therefore, the present invention provides compositions and methods for stimulating the innate immune response of a subject at the time of administration, by administering a drug capable of stimulating the innate immune response of the subject. In particular, the invention relates to such compositions comprising a drug capable of stimulating the innate immune response of a subject at the time of administration, methods for synthesizing such compositions, and systems and methods utilizing such compositions (e.g., in diagnostic and / or therapeutic settings).

[0017] Therefore, in certain embodiments, the present invention relates to one or more DAMPs or PAMPs and either a) or b) below: a) calcium phosphate and a copolymer 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 b) Zn 2+ Mn 2+ Ca 2+ Fe 2+ Fe 3+ Cu 2+ Ni 2+ Co 2+ Pb 2+ Sn 2+ , Ru 2+ Au 2+ Mg 2+ , VO 2+ , Al 3+ Co 3+ , Cr 3+ , Ga 3+ , Tl 3+ , Ln 3+ MoO 3+ Cu + Au + , Tl + Ag + Hg 2+ Pt 2+ Pb 2+ Hg 2+ , Cd 2+ , Pd 2+ Pt 4+ kaNa + , K + The present invention provides a composition comprising one or more cations selected from the group consisting of phosphates and carbonates related thereto.

[0018] In some embodiments, the composition has the ability to stimulate an innate immune response in a subject upon administration. In some embodiments, the subject has cancer or is at risk of developing cancer. In some embodiments, the composition is used to induce an immune response to vaccine application. In some embodiments, the subject has cancer, and the composition has the ability to stimulate an innate immune response in at least one cancer cell upon administration to the subject. In some embodiments, stimulating an innate immune response includes stimulating an innate cytokine response mediated by cytokines, the innate cytokine response mediated via type 1 interferon.

[0019] Accordingly, in certain embodiments, the present invention provides a method for treating cancer in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a composition comprising an agent (e.g., DAMP / PAMP) capable of stimulating an innate immune response in the subject upon administration to the subject. In some embodiments, the innate immune response is an innate cytokine response mediated by cytokines in the subject. In some embodiments, the innate cytokine response is mediated by type 1 interferon in the subject.

[0020] Such methods are not limited to a specific mode of administration. In some embodiments, administration is systemic. In some embodiments, administration is local.

[0021] In some embodiments, the composition is administered together with a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is aldesleukin, altretamine, amiphostine, asparaginase, bleomycin, capecitabine, carboplatin, carmastine, cladribine, cisapplied, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin-α, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, or hydroxyurea. These include one or more of the following: idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisol, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (TAXOL), pilocarpine, prochlorperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate.

[0022] Such compositions are not limited to specific DAMP or PAMP agonists. In some embodiments, DAMP and PAMP agonists are selected from STING agonists, purine-containing factors or purine derivative factors, Toll-like receptor (TLR) agonists, NOD-like receptor (NLR) agonists, RIG-I-like receptor (RLR) agonists, cytoplasmic DNA sensor (CDS) agonists, C-type lectin receptor (CLR) agonists, and inflammasome inducers. In some embodiments, DAMP and PAMP agonists are selected from TLR-3 agonists, TLR-4 agonists, TLR-5 agonists, TLR-7 agonists (e.g., imiquimod), TLR-8 agonists (e.g., regiquimod), TLR-9 agonists, and NLRP3 agonists.

[0023] Such compositions are not limited to specific purine-containing factors or purine-derived factors. In some embodiments, the purine-containing factors or purine-derived factors are 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluoro, cAIM(PS)2, difluoro(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluoride, c-di-AMP fluoride, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluoride, 2'3'-c-di-GMP c-di-IMP, cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, 3'3'-cGAMP, cGAM(PS)2, 2'3'-cGAM(PS)2(Rp / Sp), 2'2'-cGAM(PS)2, 2'3'-cGAM(PS)2, cGAMP fluoride, 3'3'-cGAMP fluoride, 2'3'-cGAMP fluoride, 2'2'-cGAMP fluoride, c-di-AMP, 2'3'-cdAMP, 2'2'-cdAMP, 3'3'-cdAMP, c-di-AM(PS)2, 2'3'-c-di-AM(PS)2(Rp,Rp), 2'2'-c-di-AM(PS)2, 3'3'-c-di-AM(PS)2, c-di-AMP fluoride, 2'3'-cdAMP fluoride, 2'2'-cdAMP fluoride fluoride, 3'3'-cdAMP fluoride, cdGMP, 2'3'-cdGMP, 2'2'-cdGMP, 3'3'-cdGMP, c-di-GM(PS)2, 2'3'-c-di- GM(PS)2, 2'2'-c-di-GM(PS)2, 3'3'-c-di-GM(PS)2, cdGMP fluoride, 2'3'-cdGMP fluoride, 2'2'-cdGMP fluoride Fluorinated, 3'3'-cdGMP fluorinated, cAIMP, 2'3'-cAIMP, 2'2'-cAIMP, 3'3'-cAIMP, cAIMP difluoro (3'3'-cAIMP fluorinated) Fluorinated compound, 2'3'-cAIMP fluorinated compound, 2'2'-cAIMP fluorinated compound, cAIM(PS)2 difluoro, 3'3'-cAIM(PS)2 difluoro(Rp / Sp), 2'3 '-cAIM(PS)2 difluoro, 2'2'-cAIM(PS)2 difluoro, c-di-IMP, 2'3'-cdIMP, 2'2'-cdIMP, 3'3'-cdIMP, c-d i-IM(PS)2, 2'3'-c-di-IM(PS)2, 2'2'-c-di-IM(PS)2, 3'3'-c-di-IM(PS)2, c-di-IMP fluoride, 2'3'-cdIMP fluoride, 2'2'-cdIMP fluoride, 3'3'-cdIMP fluoride, imiquimod, resikimod, 6-(4-amino-imidazoquinolyl)-norleucine,

[0024] [ka]

[0025] The inhibitors are selected from RNA, siRNA, microRNA, interfering RNA, mRNA, replicon mRNA, RNA analogs, DNA, and purine-based PI3K inhibitors.

[0026] Such compositions are not limited to a specific type or kind of STING agonist. In some embodiments, the STING agonist is a small molecule agonist of STING. In some embodiments, the small molecule agonist of STING is a cyclic dinucleotide. For example, in some embodiments, the cyclic dinucleotides include cGAMP, cdiAMP, cdiGMP, and cAIMP. Further examples of cyclic pruritic dinucleotides are described in some detail, for example, U.S. Patent No. 7,709,4587,592,326; WO2007 / 054279; and Yan et al., Bioorg. Med. Chem Lett. 18: 5631 (2008). Each of these documents is incorporated herein by reference. In some embodiments, the additional STING agonist is selected from 5,6-dimethylxanthenon-4-acetic acid (DMXAA), methoxybone, 6,4'-dimethoxyflavone, 4'-methoxyflavone, 3',6'-dihydroxyflavone, 7,2'-dihydroxyflavone, daidzein, formononetin, and letusin 7-methyl ether, or any derivative thereof. In some embodiments, STING small molecule agonists include, but are not limited to, 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluor, cAIM(PS)2, difluor(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluoride, c-di-AMP fluoride, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluoride, 2'3'-c-di-GMP, c-di-IMP, SB11285, STING-agonist-C11, STING-agonist-1, STING-agonist-G10, and gemcitabine.

[0027] In some embodiments, STING small molecule agonists are

[0028] [ka]

[0029] , 2’3’-cGAMP, 3’3’-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluoro, cAIMP(PS)2, difluoro (Rp / Sp), 2’2’-cGAMP, 2’3’-cGAM(PS)2(Rp / Sp), 3’3’-cGAMP fluoride, c-di-AMP fluoride, 2’3’-c-di-AMP, 2’3’-c-di-AM(PS)2(Rp, Rp), c-di-GMP fluoride, 2’3’-c-di-GMP, c-di-IMP, cGAMP, 2’3’-cGAMP, 2’2’-cGAMP, 3’3’-cGAMP, cGAM(PS)2, 2’3’-cGAM(PS)2(Rp / Sp), 2'2’-cGAM(PS)2, 2’3’-cGAM(PS)2, cGAMP fluoride, 3’3’-cGAMP fluoride, 2’3’-cGAMP fluoride, 2’2’-cGAMP fluoride, c-di-AMP, 2’3’-cdAMP, 2’2’-cdAMP, 3’3’-cdAMP, c-di-AM(PS)2, 2’3’-c-di-AM(PS)2(Rp, Rp), 2’2’-c-di-AM(PS)2, 3’3’-c-di-AM(PS)2, c-di-AMP fluoride, 2’3’-cdAMP fluoride, 2’2’-cdAMP fluoride, 3’3’-cdAMP fluoride, cdGMP, 2’3’-cdGMP, 2’2’-cdGMP, 3’3’-cdGMP, c-di-GM(PS)2, 2’3’-c-di-GM(PS)2, 2’2’-c-di-GM(PS)2, 3’3’-c-di-GM(PS)2, cdGMP fluoride, 2’3’-cdGMP fluoride, 2’2’-cdGMP fluoride, 3’3’-cdGMP fluoride, cAIMP, 2’3’-cAIMP, 2’2’-cAIMP, 3’3’-cAIMP, cAIMP difluoro (3’3’-cAIMP fluoride, 2’3’-cAIMP fluoride, 2’2’-cAIMP fluoride, cAIM(PS)2 difluoro, 3’-3’-cAIM(PS)2 difluoro (Rp / Sp), 2’3’-cAIM(PS)2 difluoro, 2’2’-cAIM(PS)2 difluoro, c-di-IMP, 2’3’-cdIMP, 2’2’-cdIMP, 3’3’-cdIMP, c-di-IM(PS)2, 2’3’-c-di-IM(PS)2, 2’2’-c-di-IM(PS)2,Selected from 3'3'-c-di-IM(PS)2, c-di-IMP fluoride, 2'3'-cdIMP fluoride, 2'2'-cdIMP fluoride, 3'3'-cdIMP fluoride, and amidebenzimidazole-based compounds.

[0030] As already mentioned, there are two significant limitations to using CDN as a cancer treatment: 1) poor pharmacokinetics and 2) serious and inappropriate side effects. Regarding poor pharmacokinetics, when administered via intratumoral infusion, CDN diffuses easily due to its low molecular weight and high hydrophilicity, and when administered via intravenous infusion, CDN may exhibit low bioavailability to tumor tissue due to its in vivo instability, low lipophilicity, and rapid efflux. Regarding serious and inappropriate side effects, as an immunological sensor for viral infections, STING is widely distributed throughout the body. Therefore, high doses of STING agonists or systemically administered STING agonists may nonspecifically activate the innate immune system and cause cytokine storms. The present invention addresses these limitations by providing prodrugs of such small molecule agonists of DAMP and / or PAMP (including STING agonists).

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

[0032] In some embodiments, CDNs are modified with cleavable lipid moieties to produce CDN prodrugs. For example, three synthetic pathways for lipid-CDN prodrugs are intended, as shown in the following scheme. Each pathway is activated by a different mechanism: estrase-based activation for pathway 1, phosphoramidase-based activation for pathway 2, and reduction of environmental sensitivity activation for pathway 3.

[0033] Scheme 1: Synthetic pathway of lipid-CDN prodrugs

[0034] [ka]

[0035] Scheme 2: Synthetic pathway of lipid-CDN prodrugs

[0036] [ka]

[0037] Scheme 3: Synthetic pathway of lipid-CDN prodrugs

[0038] [ka]

[0039] After modification, the lipid-CDN prodrug is intended to be administered either in free or liposomal form. Such embodiments significantly improve pharmacokinetics and reduce the side effects of CDN. For example, an injected lipid-CDN prodrug is intended to be retained at the injection site, slowly releasing CDN into the tumor, conferring high bioavailability and reducing side effects on normal tissue. For example, a liposomal lipid-CDN prodrug can be administered either intravenously or topically. Such a liposomal lipid-CDN can significantly prolong drug circulation in the blood and increase tumor accumulation and lymph node drainage. More importantly, CDN is inactive after lipid modification and can only be reactivated when cleaved by esterase. Furthermore, previous studies have shown that metastatic lymph nodes can be distinguished from non-tumor lymph nodes by their high esterase levels, enabling selective activation of lipid-CDN prodrugs at tumor sites.

[0040] In some embodiments, STING-activating compounds are provided (see, for example, WO2017011920, WO2017027646, WO2017011622, U.S. Patent Application Publication No. 20160287623, WO2016100261, U.S. Patent Application Publication No. 20160074507, and WO2015161762).

[0041] In some embodiments, cGAS-modulating compounds are provided (see, for example, WO2014179335).

[0042] In some embodiments, STING inhibitory compounds are provided (see, for example, U.S. Patent Application Publication No. 20170037400).

[0043] In some embodiments, compounds are provided that can kill STING-deficient and / or cGAS-deficient cancer cells (see, for example, WO2016201450).

[0044] In some embodiments, STING pathway agonists are provided in combination with pharmaceutically active components (see, for example, STING activation / chemotherapy (WO2016096577), STING activation / selected vaccine formulations that stimulate an immune response (see U.S. Patent Applications Publications 20150056224 and 20140205653), and STING activation / cytokine generation (WO2013185052)).

[0045] In some embodiments, such compositions comprising a drug (e.g., DAMP / PAMP) that can stimulate an innate immune response in a subject upon administration to the subject, work in conjunction with nanoparticles (e.g., by complexation, binding, encapsulation, absorption, adsorption, or mixing).

[0046] In some embodiments, such compositions that interact with nanoparticles further interact with calcium phosphate and copolymers of PEI / PEG, PH-PEG, PK-PEG, or PGA-PEG (e.g., complexation, binding, encapsulation, absorption, adsorption, mixing). In fact, in some embodiments, the interaction between a drug that can stimulate an innate immune response in a subject and nanoparticles occurs in the presence of calcium phosphate and copolymers of PEI / PEG, PH-PEG, PK-PEG, or PGA-PEG.

[0047] In some embodiments, such compositions that work in conjunction with nanoparticles include 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+ , Tl3+ , 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 further cooperate with (e.g., complex formation, binding, encapsulation, absorption, adsorption, mixing) one or more cations selected from the group consisting of these phosphates or carbonates. In fact, in some embodiments, the cooperation between the agent that can stimulate the innate immune response in the subject and the nanoparticles is in the presence of such cations (e.g., Zn 2+ , Co 2+ , or Mn 2+ ).

[0048] In some embodiments, such compositions that cooperate with the nanoparticles and one or more cations (e.g., Zn 2+ , Co 2+ , or Mn 2+ ) or calcium phosphate further cooperate with hydrophobic molecules (e.g., complex formation, binding, encapsulation, absorption, adsorption, mixing).

[0049] 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.

[0050] Examples of lipid molecules applicable to embodiments of the present invention include lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidyl This includes, but is not limited to, phatidylethanolamine (POPE), palmitoyloleoylphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dieryloylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl group in these lipids is preferably C 10 ~C 24 These are acyl groups derived from fatty acids having carbon chains, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0051] Other non-exclusive examples of lipid molecules include sterols such as cholesterol, their derivatives such as cholestanol, cholestanone, cholestane, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

[0052] Other examples of lipid molecules suitable for use in the present invention include, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkylaryl sulfate polyethyl oxylate fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and non-phosphorus-containing lipids such as sphingomyelin.

[0053] Other examples of lipid molecules suitable for use in the present invention include fatty acids and their derivatives or analogues. These include oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linolenic acid, linolenic acid, dicaprate, monoolein (1-monoleoyl-rac-glycerol), dilauric acid, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, and their C 1-10This includes alkyl esters (e.g., methyl, isopropyl, and t-butyl), as well as their mono- and di-glycerides (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).

[0054] Other examples of lipid molecules suitable for use in the present invention include lipid molecules modified with PEG (PEG-lipids). Examples of PEG-lipids include, for example, PEG-DAA, which is linked to dialkyloxypropyl as described in PCT publication no. WO05 / 026372; PEG-DAG, which is linked to diacylglycerol as described in U.S. Patent Publication no. 20030077829 and 2005008689; PEG-PE, which is linked to phospholipids such as phosphatidylethanolamine; PEG-PE, which is linked to ceramide as described in U.S. Patent No. 5,885,613; PEG-PE, which is linked to cholesterol or its derivatives; and mixtures thereof. The disclosures of these patent documents are incorporated herein by reference in their entirety for all purposes. Additional PEG-lipids include, but are not limited to, PEG-C-DOMG, 2KPEG-DMG, and mixtures thereof.

[0055] PEG is a linear, water-soluble polymer of ethylene-PEG repeat units having two terminal hydroxyl groups. PEGs are classified by their molecular weight; for example, PEG2,000 has an average molecular weight of approximately 2,000 daltons, and PEG5,000 has an average molecular weight of approximately 5,000 daltons. PEGs are commercially available from Sigma Chemical Co. and other companies and include, for example, monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol succinate (MePEG-S), monomethoxypolyethylene glycol succinidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol amine (MePEG-NH2), monomethoxypolyethylene glycol torecylate (MePEG-TRES), and monomethoxypolyethylene glycol imidazolyl carbonyl (MePEG-IM). Other PEGs (e.g., mPEG(20kDa)amine), such as those described in U.S. Patent Nos. 6,774,180 and 7,053,150, are also useful for preparing the PEG-lipid complexes of the present invention. These patent disclosures are incorporated herein by reference in their entirety for all purposes. Furthermore, monomethoxypolyethylene glycol acetate (MePEG-CH2COOH) is particularly effective in preparing PEG-lipid complexes, for example, PEG-DAA complexes.

[0056] The PEG portion of the PEG-lipid complexes described herein may have an average molecular weight in the range of about 550 daltons to about 10,000 daltons. In certain examples, 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 preferred embodiments, the PEG portion has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0057] In certain examples, PEG may be substituted with alkyl groups, alkoxy groups, acyl groups, or aryl groups. PEG can be directly bonded to lipids or linked to lipids via a linker moiety. Any linker moiety suitable for linking PEG to lipids may include, for example, non-ester-containing linker moieties and ester-containing linker moieties. In a preferred embodiment, the linker moiety is a non-ester-containing linker moiety. As used herein, the term "non-ester-containing linker moiety" means a linker moiety that does not contain a carboxylic acid ester bond (-OC(O)-). Suitable non-ester-containing linker moieties include, but are not limited to, amides (-C(O)NH-), aminos (-NR-), carbonyls (-C(O)-), carbamates (-NHC(O)O-), ureas (-NHC(O)NH-), disulfides (-SS-), ethers (-O-), succinyls (-(O)CCH2CH2C(O)-), succinamidyls (-NHC(O)CH2CH2C(O)NH-), ethers, disulfides, and combinations thereof (such as linkers containing both carbamate and amide linker moieties). In preferred embodiments, a carbamate linker is used to link PEG to a lipid.

[0058] In other embodiments, an ester-containing linker moiety is used to bind PEG to lipids. Suitable ester-containing linker moieties include, for example, carbonates (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof.

[0059] Phosphatidylethanolamines having acyl chain groups of various chain lengths and saturations can be bound to PEG to form lipid complexes. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. 10 ~C 20Phosphatidylethanolamines containing saturated or unsaturated fatty acids having a carbon chain length of are preferred. Phosphatidylethanolamines containing mono- or diunsaturated fatty acids, as well as mixtures of saturated and unsaturated fatty acids, can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0060] In some embodiments, nanoparticles interacting with a composition containing a drug (e.g., DAMP / PAMP) that can stimulate an innate immune response in a subject upon administration to the subject further interact with one or more drugs configured to bind to target cancer cells (e.g., complexation, binding, encapsulation, absorption, adsorption, mixing).

[0061] In some embodiments, the agents that bind to target cancer cells include α-actinin-4, Bcr-Abl fusion protein, Casp-8, β-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, Mum-2, and Mum-3, neo-PAP, myosin class I, OS-9, and pml-RARα fusion protein. Citrate, PTPRK, K-ras, N-ras, triose phosphate isomerase, Bage-1, Gage3, Gage4, Gage5, Gage6, Gage7, GnTV, Herv-K-mel, Lage-1, Mage-A1, Mage-A2, Mage-A3, Mage-A4, Mage-A6, Mage-A10, Mage-A12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP- 1, 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, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA125, CA15-3 (CA27.29\BCAA), CA195, CA242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM),Human EGFR proteins or their fragments, such as human EGFR residues 306-325 (SCVRACGADSYEMEEDGVRK (SEQ ID NO: 374)) and residues 897-915 (VWSYGVTVWELMTFGSKPY (SEQ ID NO: 375)), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophyllin 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 variant, p53 variant, proteinase 3 (PR1), survivorbin, 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, fucosyl GM1, mesothelin, sLe (animal), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, A KAP-4, XAGE1, B7H3, Regmine, Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-α, PDGFR-β, MAD-CT-2, Fos-related antibody 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,The tumor antigen is selected from the group consisting of aldehyde dehydrogenases and any derivative thereof.

[0062] In some embodiments, one or more drugs configured to bind to target cancer cells are bound to the outer surface of the nanoparticles. In some embodiments, one or more drugs configured to bind to target cancer cells are encapsulated within the nanoparticles.

[0063] In some embodiments, nanoparticles acting in conjunction with such compositions containing a drug (e.g., DAMP / PAMP) that can stimulate an innate immune response in a subject upon administration to the subject, further act in conjunction with an adjuvant (e.g., complexation, binding, encapsulation, absorption, adsorption, mixing).

[0064] In some embodiments, the adjuvant is CPG, polyIC, polyICLC, 1018 ISS, aluminum salts (e.g., aluminum hydroxide, aluminum phosphate), Amplivax, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, cytokines (GM-CSF, IL-2, IFN-α, Flt-3L, etc.), IC30, IC31, imiquimod, ImuFact IMP321, IS patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, montanide IMS1312, montanide ISA206, montanide ISA50V, montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTelRTM, vector system, PLGA microparticles, imiquimod, regiquimod, gardiquimod, 3M-052, SRL172, virosom and other virus-like particles, YF-17D, VEGF trap, β-glucan, Pam3Cys, Aquila's QS21 stimulon, bajimezan, AsA404 (DMXAA), 3M MEDI9197, glucopyranosyllipid adjuvants (GLA), GLA-SE, CD1d ligands (C20:2, OCH, AH04-2, α-galatosylceramide, α-C-galatosylceramide, α-mannosylceramide, α-fructosylceramide, β-galatosylceramide, β-mannosylceramide, etc.), STING agonists (e.g., cyclic [G(3',5')pA(3',5')p], cyclic [G(2',5')pA(3',5')p], cyclic [G(2',5')pA(2',5')p], cyclic di The following are selected from the group consisting of adenylate monophosphate (cyclic dinucleotides including cyclic diguanylate monophosphate), CL401, CL413, CL429, flagellin, RC529, E6020, imidazoquinoline small molecules TLR-7 / 8a (including its lipid-derived analogs), visomes, AS01, AS02, AS03, AS04, AS15, IC31, CAF01, ISCOM, cytokines (GM-CSF, IL-2, IFN-a, Flt-3L, etc.), and bacterial toxins (CT, LT, etc.).In some embodiments, the adjuvant is any derivative of the adjuvant (e.g., cholesterol-modified CpG) or any combination thereof. In some embodiments, the adjuvant is a dendritic cell targeting molecule.

[0065] Such compositions, which include a drug (e.g., DAMP / PAMP) capable of stimulating an innate immune response in a subject upon administration and which work in conjunction with nanoparticles, are not limited to specific types of nanoparticles.

[0066] In some embodiments, the nanoparticles are sHDL nanoparticles. In some embodiments, the nanoparticles are sHDL nanoparticles, meta-polyhistidine-DOPE liposomes, metal-polyhistidine-PEG, 4-arm-PEG-polyhistidine-metal hydrogels, sHDL-polyhistidine, fullerenes, encapsulated metallofullerene buckyballs, trimetal nitride templated encapsulated 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, and carbon The following are selected from the group consisting of nanohorn peapods, liposomes, nanoshells, dendrimers, any nanostructures, microstructures, derivatives thereof formed using layer-by-layer processing or self-assembly processing, polymer electrolytes, nanoparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, microspheres and nanospheres of glass and polymers, biodegradable PLGA microspheres and nanospheres, gold nanoparticles, silver nanoparticles, carbon nanoparticles, iron nanoparticles, modified micelles, and organometallic framework (MOF) coordination polymers (CP).

[0067] In some embodiments, the average size of the nanoparticles is 6 to 500 nm.

[0068] In some embodiments, the nanoparticles are sHDL nanoparticles. In some embodiments, the sHDL nanoparticles comprise a mixture of at least one phospholipid and at least one HDL apolipoprotein or apolipoprotein mimetic. In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein AI (apoA-I), apolipoprotein A-II (apoA-II), apolipoprotein A4 (apoA4), apolipoprotein Cs (apoCs), and apolipoprotein E (apo E). In some embodiments, the phospholipids are dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butylamide], 1,2-dihex The following are selected from the group consisting of sadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof. In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic.

[0069] In some embodiments, the ApoA-I mimetic is sequence number 1-336, WDRVKDLATVYVDVLKDSGRDYVSQF (sequence number: 341), LKLLDNWDSVTSTFSKLREOL (sequence number: 342), PVTOEFWDNLEKETEGLROEMS (sequence number: 343), KDLEEVKAKVQ (sequence number: 344), KDLEEVKAKVO (sequence number: 345), PYLDDFQKKWQEEMELYRQKVE (sequence number: 346), PLRAELQEGARQKLHELOEKLS (sequence number: 347), PLGEEMRDRARAHVDALRTHLA (sequence number: 348), PYSDELRQRLAARLEALKENGG (sequence number: 349), ARLAEYHAKATEHLSTLSEKAK (sequence number: 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), VLTLALVAVAGARAEVSADOVAT (SEQ ID NO: 357), NNAKEAVEHLOKSELTOOLNA (SEQ ID NO: 358), LPVLVWLSIVLEGPAPAOGTPDVSS (SEQ ID NO: 359), LPVLVVVLSIVLEGPAPAQGTPDVSS (SEQ ID NO: 360), ALDKLKEFGNTLEDKARELIS (SEQ ID NO: 361), VVALLALLASARASEAEDASL (Sequence ID: 362), HLRKLRKRLLRDADDLQKRLAVYOA (Sequence ID: 363), AQAWGERLRARMEEMGSRTRDR (Sequence ID: 364), LDEVKEQVAEVRAKLEEQAQ (Sequence ID: 365), DWLKAFYDKVAEKLKEAF (Sequence ID: 236), DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA (Sequence ID: 366), PVLDLFRELLNELLEALKQKL (Sequence ID: 367), PVLDLFRELLNELLEALKQKLA (Sequence ID: 368), PVLDLFRELLNELLEALKQKLK (Sequence ID: 4), PVLDLFRELLNELLEALKQKLA (Sequence ID: 369), PVLDLFRELLNELLEALKKLLK It is represented by one of the following: (Sequence ID: 370), PVLDLFRELLNELLEALKKLLA (Sequence ID: 371), PLLDLFRELLNELLEALKKLLA (Sequence ID: 372), and EVRSKLEEWFAAFREFAEEFLARLKS (Sequence ID: 373).

[0070] In some embodiments, the average particle size of sHDL nanoparticles is 6 to 70 nanometers.

[0071] In some embodiments, nanoparticles acting in conjunction with a composition containing a drug (e.g., DAMP / PAMP) capable of stimulating an innate immune response in a subject upon administration to the subject, further acting in conjunction with one or more novel antigenic peptides (e.g., complexation, binding, encapsulation, absorption, adsorption, mixing), where each of the one or more novel antigenic peptides is specific to a novel antigenic mutation identified from a tumorigenic biological sample obtained from the subject. In some embodiments, the subject is human.

[0072] In some embodiments, one or more novel antigen peptides are in the range of about 5 to about 50 amino acids in length. In some embodiments, one or more novel antigen peptides are in the range of about 15 to about 35 amino acids in length. In some embodiments, one or more novel antigen peptides are in the range of about 18 to about 30 amino acids in length. In some embodiments, one or more novel antigen peptides are in the range of about 6 to about 15 amino acids in length.

[0073] In some embodiments, nanoparticles interacting with a composition containing a drug (e.g., DAMP / PAMP) that can stimulate an innate immune response in a subject upon administration to that subject may further interact with (e.g., complex, bind, encapsulate, absorb, adsorb, or mix) one or more biomolecular drugs.

[0074] Such compositions are not limited to specific biomolecular drugs.

[0075] In some embodiments, the biomolecular drug is a nucleic acid. Such embodiments include, but are not limited to, RNA, siRNA, microRNA, interfering RNA, mRNA, replicon mRNA, RNA analogs, and DNA, encompassing any type of nucleic acid molecule.

[0076] In some embodiments, the biomolecular drug is a peptide.

[0077] In some embodiments, the peptides include adrenocorticotropic hormone (ACTH), growth hormone peptide, melanocyte-stimulating hormone (MSH), oxytocin, vasopressin, corticotropin-releasing factor (CRF), CRF-related peptide, gonadotropin-releasing peptide (GAP), growth hormone-releasing factor (GRF), luteinizing hormone-releasing hormone (LH-RH), orexin, prolactin-releasing peptide (PRP), somatostatin, thyrotropin-releasing hormone (THR), THR analog, calcitonin (CT), CT precursor peptide, and cal Citonin gene-related peptide (CGRP), parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), amyrin, 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 peptide, opioid peptide, thymic peptide, adrenomedullin peptide, allostatin peptide, amyloid-beta protein fragment, antimicrobial peptide, antioxidant peptide, apoptosis-related peptide, cystocyte peptide (BCP), bombesin, bone Gla protein peptide, cocaine and amphetamine-related transcript (CART) peptide, cell adhesion peptide, chemotactic peptide Tide, complement inhibitors, cortisutin peptide, fibronectin fragment, fibrin-related peptide, FMRF, FMRF amide-related peptide (FaRP), galanin, galanin-related peptide, growth factors, growth factor-related peptide, G-2 therapeutic peptide-binding protein fragment, guarilin, urogalilin, inhibin peptide, interleukin (IL), interleukin receptor protein, laminin fragment, leptin fragment peptide, leukokinin, pituitary adenylate cyclase-activating polypeptide (PAPCAP), pancreastatin, polypeptide repeat chain,Signaling factors, thrombin inhibitors, toxins, trypsin inhibitors, virus-related peptides, adjuvant peptide analogs, α-conjugation factors, antiarrhythmic peptides, appetite suppressant peptides, α-1 antitrypsin, bovine pineal gland anti-reproductive peptide, brucine, C3 peptide P16, cadherin peptide, chromogranin A fragment, contraceptive tetrapeptide, conanttokin G, conanttokin T, crustacean cardioactive peptide, C-telopeptide, cytochrome b588 peptide, decorsin, delicious peptide peptide), delta sleep-inducing peptide, diazepam binding inhibitor fragment, nitric oxide synthase inhibitor peptide, OVA peptide, platelet calpain inhibitor (P1), plasminogen activator inhibitor 1, ligin, schizophrenia-related peptide, sodium potassium A therapeutic peptidase inhibitor-1, speract, sperm-activating peptide, cystemin, thrombin receptor agonist, tuftosin, lipid-mobilizing hormone, uremic pentapeptide, antifreeze polypeptide, tumor necrosis factor (TNF), Leech[Des Asp10]decorcin, L-ornityl taurine hydrochloride, P-aminophenylacetyl tuftosin, Ac-Glu-Glu-Val-Val-Ala-Cys-pNA, Ac-Ser-Asp-Lys-Pro, Ac-rfwink-NH2, Cys-Gly-Tyr-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-Gly, D-Ala-Leu, DDDDD, DDDDDD, NPNANPNA, VAITVLVK, VGVRVR, VIHS, VPDPR Val-Thr-Cys-Gly, RSR, sea urchin sperm activating peptide, SHU-9119 antagonist, MC3-R antagonist, MC4-R antagonist, Glaspimod, HP-228, α2-plasmin inhibitor, APC tumor suppressor, early pregnancy factor, γ interferon, glandular kallikrein N-1, placental ribonuclease inhibitor, sarcolesin-binding protein, surfactant protein D, Wilms tumor suppressor, GABAB 1b receptor peptide, prion-related peptide (iPRP13), choline-binding protein fragment, telomerase inhibitor,These include cardiostatin peptides, endostatin-derived peptides, prion inhibitor peptides, N-methyl D-aspartate receptor antagonists, and C-peptide analogs.

[0078] In some embodiments, the peptides are 177Lu-DOTA0-Tyr3-octreotate, Abarelix acetate, ADH-1, Afamelanotidec, Melanotan I, CUV1647, Albiglutide, Aprotinin, Argipressin, Atosiban acetate, Bacitracin, Benthromide, BH3 domain, Bivalirudin, Bivalirudin trifluoroacetate hydrate hydrate), bricibimod, bortezomib, buserelin, buserelin acetate, calcitonin, carbetocin, carbetocin acetate, cecropine A and B, ceruretide, ceruretide diethylamine, cetrorelix, cetrorelix acetate, cyclosporine, silengitide (Cilengitidec), EMD121974, corticocholin acetate injector, hCRF, corticocholin sheep triflutate, corticocholin trifluoroacetate, corticotropin, cosyntropin, ACTH 1-24, Tetracosactide hexaacetate, Darbavancin, Daptomycin, Degarelix acetate, Trifluoroacetate Deptreotide (with sodium pertechnetate), Desmopressin acetate, Desmopressin DDAVP, Dulaglutide, Ecalantide, Edtreotide (with Yttrium-90), Elcatonin acetate, Enalapril maleate (or 2-butanediate), Enfuvirtide, Epfihibatide, Exenatide, Ganirelix acetate, Glatiramer acetate, Glutathione, Gonadrelin, Gonadrelin acetate, GnRH, LHRH, Goserelin, Goserelin acetate, Gramicidin, Histrelin acetate, Human calcitonin, Icatibant, Icatibant acetate, IM862, Ogluphanide disodium, KLAKLAK, Lanreotide acetate, Repiridine, Leuprolide, Leuprolide acetate, Leuprorelin, Liraglutide, Lisinopril, Lixisenatide, Lypressin, Magenin 2, MALP-2Sc, Macrophage-activating lipopeptide-2 compoundSynthetic), Nafarelin acetate, Nesiritide, NGR-hTNF, Octreotide acetate, Oritabancin, Oxytocin, Pasireotide, Peginesatide, Pentagastrin, Pentetreotide (with Indium-111), Phenipressin, Pleurocidin, Plumrintide, Protirelin, Tyroleverine, TRH, TRF, Salmon Calcitonin, Salaracin acetate, Secretin (human), Secretin (porcine), Semaglutide, Seractide acetate, ACTH, Corticotropin, Sermorelin acetate, GRF 1-29, Synaplutide, KL4 in Lusynactant, Syncalid, Somatrelin acetate, GHRH, GHRF, GRF, Somatostatin acetate, Spaglumat magnesium (or sodium) salt Selected from (salt), substance P, taltirelin hydrate, teduglutide, teicoplanin, teravancin, teriparatide, terlipressin acetate, tetracosactide, thymalfacin, thymosin α-1, thymopentin, trebananib, triptorelin, triptorelin pamoate, tyroserulotide, uralitide, vancomycin, bapreotide acetate, vasoactive intestinal peptide acetate, Vx-001c, TERT572Y, diconotide acetate, α5-α6Bax peptide, and β-defensin.

[0079] In some embodiments, the peptide is any peptide that helps achieve a desired objective using the composition. For example, in some embodiments, the peptide is any peptide that facilitates the treatment of any type of disease and / or disorder.

[0080] In some embodiments, the peptide is an antigen.

[0081] In some embodiments, the antigen is selected from the group consisting of peptide-based antigens, protein-based antigens, polysaccharide-based antigens, sugar-based antigens, lipid-based antigens, glycolipid-based antigens, nucleic acid-based antigens, inactive organism-based antigens, attenuated organism-based antigens, viral antigens, bacterial antigens, parasitic antigens, allergen-derived antigens, and tumor antigens.

[0082] In some embodiments, the antigen is a tumor antigen as described herein.

[0083] In some embodiments, the antigens include, but are not limited to, FimH for urinary tract infections; soluble F protein from respiratory syncytial virus (RSV); NEF, GAG, and ENV proteins from HIV; Streptococcus pneumoniae proteins; HMGB1 protein; hemagglutinin and neuroamidase proteins for influenza; viral antigens from HPV types 16 and 18; gL2, ICP4, gD2ΔTMR, or ICP4.2 from HSV-2; antigens from S. pneumoniae such as pneumolysoid; choline-binding protein A (CbpA) or pneumococcal surface protein A (PspA), SP1912, SP1912L, SP0148 with or without a signal sequence, SP2108 with or without a signal sequence; antigens from Chlamydia trachomatis such as CT209 polypeptide antigen, CT253 polypeptide antigen, CT425 polypeptide antigen, CT497 polypeptide antigen, and CT843 polypeptide antigen; and amyloid-β peptides.

[0084] In some embodiments, the antigen is bound to the outer surface of the nanoparticle. In some embodiments, the antigen is encapsulated within the nanoparticle.

[0085] In certain embodiments, the present invention provides a composition capable of inhibiting cGAS-STING activation and type I IFN response, comprising one or more cell-permeable chelating agents or derivatives thereof, which can prevent intracellular metal ions from being utilized for cGAS-STING-type I IFN activation.

[0086] In a particular embodiment, the present invention provides a composition comprising one or more cell-permeable chelating agents (e.g., metal ion chelating agents) that can modulate innate immune activation by preventing intracellular metal ions from being utilized in the innate immune pathway.

[0087] In some embodiments, such cell-permeable chelating agents (e.g., metal ion chelating agents) include, but are not limited to, the polyphenol chelating agents (-)-epigallocatechin gallate (EGCG), punicalagin, (-)-catechin gallate, (-)-catechin, tannic acid, tannin, punicalin, vescalagin, procyanidin C1, geraniin, theaflavin 3,3'-digalate, lipid-modified NTA, porphyrin, EDTA, NOTA, DOTA, TPEN, and clofelemer.

[0088] In some embodiments, compositions capable of inhibiting cGAS-STING activation and type I IFN response are used in the treatment of subjects who have or are at risk of developing autoimmune disorders.

[0089] Thus, the present invention provides a method for treating autoimmune disorders by administering a composition capable of modulating innate immune activation, comprising one or more cell-permeable chelating agents (e.g., metal ion chelating agents), to a subject (e.g., a human subject) to prevent intracellular metal ions from being utilized in the innate immune pathway. In such embodiments, such cell-permeable chelating agents (e.g., metal ion chelating agents) include, but are not limited to, the polyphenol chelating agents (-)-epigallocatechin gallate (EGCG), punicalagin, (-)-catechin gallate, (-)-catechin, tannic acid, tannin, punicalin, vescalagin, procyanidin C1, geraniin, theaflavin 3,3'-digalate, lipid-modified NTA, porphyrin, EDTA, NOTA, DOTA, TPEN, and clofelemer.

[0090] Examples of autoimmune disorders include, but are not limited to, systemic lupus erythematosus, Ecardi-Goutier syndrome, acute pancreatitis, age-related macular degeneration, alcoholic liver disease, hepatic fibrosis, metastasis, myocardial infarction, non-alcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, inflammatory bowel disease, and multiple sclerosis.

[0091] In some embodiments, additional therapeutic agents are administered with such compositions. Such additional therapeutic agents include, but are not limited to, disease-modifying antirheumatic drugs (e.g., leflunomide, methotrexate, sulfasalazine, hydroxychloroquine), biological agents (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), glucorticoids (e.g., prednisone, methylprednisone), TNF-α inhibitors (e.g., adalimumab, certolizumab pegol, etanercept, golimumab, infliximab), IL-1 inhibitors, and metalloproteinase inhibitors. In some embodiments, the additional therapeutic agent includes, but is not limited to, infliximab, adalimumab, etanercept, parenteral gold, or oral gold.

[0092] In certain embodiments, the present invention provides a method for treating cancer in a subject, comprising administering to the subject one or more compositions described herein (e.g., compositions comprising one or more DAMPs and / or PAMPs) and one or more adjuvants (as described herein), chemotherapeutic agents, immunosuppressants, immunostimulants, and antigens (as described herein). In some embodiments, the subject is a human subject.

[0093] In some embodiments, the immunostimulant is selected from an anti-CTLA-4 antibody, an anti-PD-1, anti-PD-L, anti-TIM-3, anti-BTLA, anti-VISTA, anti-LAG3, anti-CD25, anti-CD27, anti-CD28, anti-CD137, anti-OX40, anti-GITR, anti-ICOS, anti-TIGIT, and an IDO inhibitor.

[0094] In some embodiments, the chemotherapeutic agent is aldesleukin, altretamine, amiphostine, asparaginase, bleomycin, capecitabine, carboplatin, carmastine, cladribine, cisapplied, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin-α, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, The following are selected: idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisol, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (TAXOL), pilocarpine, prochlorperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate.

[0095] In some embodiments, the cancer is one or more selected from bladder cancer, brain cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, abdominal cancer, head and neck cancer, testicular cancer, melanoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, B-cell lymphoma, and uterine cancer.

[0096] Additional embodiments will be apparent to those skilled in the art based on the teachings contained herein. [Brief explanation of the drawing]

[0097] [Figure 1]This is a schematic diagram of the synthesis of CDN-Zn, CDN-Zn@liposomes, and CDN@CaP / PEI-PEG. (A) Coordination crosslinking between Zn2+ and CDN enables the assembly of CND-ZnNPs, which are then further modified by liposomes. (B) Due to charge interactions between the CDN and PEI-PEG backbone, CDN can be packed into CaP / PEI-PEG NPs during synthesis. [Figure 2] Characterization of CDN-Zn, CDN-Zn@liposomes, and CDN@CaP / PEI-PEG. TEM images (top panel), size (center panel), and zeta potential (bottom panel) of cdAMP-Zn(a), cdGMP-Zn(b), cGAMP-Zn(c), CDN-Zn@liposome(d), and CDN@CaP / PEI-PEG(e). [Figure 3] Release graphs of different CDN formulations and in vitro STING activity. (A) Filling effect of CDN on formulations. The red line shows the absorbance of CDN before filling, while the blue line shows the absorbance of unfilled free CDN in the supernatant after filling. (B) Release kinetics of CDN from nano formulations. (C) Representative THP1 activity evaluation with different concentrations of free CDN and CDN-Zn. The CDN used was cdAMP. (D) Representative THP1 activity with free CDN and CDN@CaP / PEI-PEG. The CDN used was cdAMP(ps)2. [Figure 4] Therapeutic efficacy of CDN preparations in a CT26 tumor model. (AC) Balb / C mice were inoculated with 1.5 × 10⁵ CT26 tumor cells on day 0 for 6-7 weeks. On days 10 and 15, tumor-bearing mice were treated with the indicated preparation containing 25 ug / dose of adAMP(ps)2 intratumorally. (A) Mean tumor growth curve of tumor-bearing mice, (B) Survival of mice after different treatments. (C) Tumor growth curves of individual mice in different groups. (DE) PBMCs were collected 7 days after the second dose of CDN treatment for (D) tetramer staining and (E) ELISPOT analysis with AH1 peptide. (F) PBMCs were collected 7 days after the first dose of CDN treatment for ELISPOT analysis with AH1 peptide. [Figure 5]In vitro enhancement of cGAS-STING-Type-I IFN activity with metal ions. (ac) Bone marrow-derived dendritic cells (BMDC) (ab) and human monocyte cell line THP1 (c) were incubated with various concentrations of metal ions, with or without STING agonists. STING activity was quantified by interferon-β (IFN-β) release in cell culture medium. [Figure 6] Enhancement of STING activity and cancer therapeutic effect in vivo by CO2+ and Mn2+. (a) Individual tumor growth curves after three intratumoral injections of the indicated formulation on days 9, 12, and 15 after tumor inoculation. (b) Serum IFN-β concentration 8 hours after the first dose of the indicated formulation. (c) Tumor growth (c) and survival (d) of individual tumor-bearing mice after treatment with the indicated formulation. [Figure 7] Enhancement of STING activity by CO2+ and Mn2+ resulted in improved antigen-specific immune response after in vivo. (a) Percentage of AH1-specific CD8+ T cells in PBMCs at day 16; (b) Number of IFN-y secreting cells per 5E4 PBMC after stimulation with AH1 peptide at day 22; (ce) Timeline (c), tumor growth curve (d), and percentage of AH1-specific CD8+ T cells in splenic CD8+ T cells; (e) Tumor re-induction study initiated at day 81. [Figure 8] Changes in cytokine graphs in vitro due to metal ions of representative PAMPs. (a-d) Bone marrow-derived dendritic cells (BMDCs) were incubated with various concentrations of metal ions, with or without the TLR3 agonist polyIC. (ef) 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 cell culture media were quantified by ELISA assay. [Figure 9]In vitro, the immune response to representative NOD-like receptor (NLR) ligands was regulated by metal ions. (a-f) Bone marrow-derived dendritic cells (BMDCs) were incubated with various concentrations of metal ions, with or without the NOD1 agonist C12-iE-DAP. (g-1) BMDCs were incubated with various concentrations of metal ions, with or without the NOD2 agonist C18-MDP. Cytokine levels in cell culture media were quantified by ELISA assay. Control: Relative PAMP in physiological saline. [Figure 10] In vitro, the immune response to representative RIG-I-like receptor-(RLR) ligands was regulated by metal ions. (a-f) Bone marrow-derived dendritic cells (BMDCs) were incubated with different concentrations of metal ions, with or without the RLR ligand Poly(dA:dT) / LyoVec™ (Invivogen). Cytokine levels in the cell culture medium were quantified by ELISA assay. Control: Relative PAMP in physiological saline. [Figure 11] In vitro, the immune response to typical pro-inflammatory substances was regulated by metal ions. (a-f) Bone marrow-derived dendritic cells (BMDCs) were pretreated with 300 ng / ml phorbol 12-myristate 13-acetic acid (PMA) for 3 hours, washed twice, and then treated with 10-200 mg / ml alum crystals. NLRP3 inflammasome formation could be characterized by IL-1β secretion. (gk) BMDCs were incubated with non-canonical inflammasome inducers from E. coli outer membrane vesicles and various metal ions at different concentrations. Cytokine levels in cell culture media were quantified by ELISA assay. Control: Relative PAMP in physiological saline. [Figure 12] In vitro immunological effects of metal ions alone. (a-f) Bone marrow-derived dendritic cells (BMDCs) were treated with different concentrations of metal ions. Cytokine levels in cell culture media were quantified by ELISA assay. Control: Relative PAMP in physiological saline. [Figure 13]Representative formulations composed of innate immunostimulants and metal ions. (a) Outline of the composition of metal ion-polyHis-DOPE@liposome nanoparticles. (b) TEM image of manganese-CDA-H11-DOPE@liposome nanoparticles (Mn-CDA / H11@liposome). (ce) Tumor growth curves of CT26 colon tumor models treated with the indicated formulations, and the number of tumor-free treated mice out of 5 mice: (c) 5 μg of free CDA / Mn2+ or Mn-CDA / H11@lipsome containing 5 μg of CDA was administered three times. (d) Mn-CDA / H11@lipsome containing 1 μg of free CDA / Mn2+ or 1 μg of CDA was administered three times by intratumoral (IT) injection on days 9, 12, and 15 after tumor inoculation; (e) Mn-CDA / H11@liposome containing 20 μg of free CDA / Mn2+ or 20 μg of CDA was administered three times by intravenous (IV) injection on days 9, 12, and 15 after tumor inoculation. (f) Antigen-specific T cell ratio in PBMCs 7 days after initial AH-1 administration. (g) ELISPOT count per 100,000 PBMCs 14 days after initial administration. (hj) Serum IFN-β, IP10, and TNF-α levels 4 hours after adaptive infusion injection. [Figure 14] Representative formulations composed of innate immunostimulants and metal ions. (a) Overview of the metal ion-poly-His-PEG nanoparticle composition. (b) TEM image of Co-CDA / H33-PEG nanoparticles. (c) In vitro STING activity of BMDCs treated with the indicated formulation. (d) Serum IFN-β after a single intratumoral injection of the indicated formulation in a B16F10 melanoma model. (ef) Tumor growth (e) and individual tumor growth (f) of mice treated with the indicated formulation. Mn-CDA-H33-PEG containing 5ug of free CDA / Mn2+ or 5ug of CDA was administered three times by CT16 tumor, IT injection, on days 9, 12, and 15 after tumor inoculation. (gh) AH-1 antigen-specific T cell ratio (g) in PBMCs 7 days after initial administration and ELISPOT measurement per 100,000 PBMCs 14 days after initial administration. [Figure 15]Representative formulations composed of innate immunostimulants and metal ions 3. (a) Schematic composition of a metal ion-4arm-PEG-polyHis coordinated hydrogel. Shows CDA@Co2+-4arm-PEG-His11 hydrogel (CDA@4aH11-Co hydrogel). (b) Retention of trypan Blue@4aH11-Co hydrogel for injection at the injection site 6 hours after injection. (ce) Individual tumor growth in mice treated with the indicated formulation. Hydrogels containing 20 ug of free CDA / Mn2+ or 20 ug of CDA were injected intratumor (IT) three times on days 9, 12, and 15 after tumor inoculation. (f) Representative tumor images after treatment with CDA@4aH11-Co hydrogel. [Figure 16] Metal ions and PAMPs can be delivered using several other representative formulations: (a) self-assembly of metal ions and CDN; (b) liposome-coated CDN-metal ion coordination nanoparticles; (c) polyhistidine-coated nanoparticles; (de) polymer-stabilized metal-CDN coordination nanoparticles or metal mineral nanoparticles. Poly(histidine) polyethylene glycol copolymer: PH-PEG or pHis-PEG, poly(ethyleneimine)-polyethylene glycol: PEI-PEG, poly(lysine) polyethylene glycol PEG: PK-PEG, anionic poly(glutamic acid) polyethylene glycol: PGA-PEG. [Figure 17]Therapeutic effects of the selected formulations in the CT26 colorectal tumor model, as shown in Figure 12. (a) Representative THP1 activity evaluation by different concentrations of free CDN and CDN-Zn. The CDN used here is cdAMP. (b) Representative THP1 activity by free CDN and CDN@CaP / PEI-PEG. The CDN used here is cdAMP(ps)2. (be) 6-7 week old Balb / c mice were inoculated with 1.5 × 10⁵ CT26 tumor cells on day 0. On days 10 and 15, tumor-carrying mice were treated with the indicated formulations containing 25 ug / dose of adAMP(ps)2 in the tumor. (c) Mean tumor growth curve of tumor-carrying mice, (d) Survival of mice after different treatments, (e) Tumor growth curves of individual mice in different groups. (fg) Tetramer staining (f) 7 days after the first dose of treatment, ELISPOT analysis (g) 7 days after the second dose of treatment. [Figure 18] Chelated metal ions for inhibiting the cGAS-STING-I type IFN pathway. (a) Molecular structures of representative chelating agents that can inhibit the cGAS-STING-I type IFN pathway. (bc) Dose-inhibition curves of the IFN-I response. (b) NF-κB inflammatory response. (c) DNA / Lipofectamine 2000 (ThermoFisher, 11668027) treated THP 1 dual KI-hSTINGWT (R232) reporter cells (Invivogen, thpd-r232). (d) Cell viability of (bc) indicated compounds in the indicated compounds. (e) Dose-inhibition curves of the IFN-I response by indicator compounds in DNA / Lipofectamine 2000 (ThermoFisher, 11668027) treated THP 1-ISG hSTINGHAQ reporter cells (Invivogen, thp-isg). (f) Dose inhibition curves of the IFN-I response by the indicated compounds in cGAMP-treated THP1 dual KI-hSTINGWT(R232) reporter cells (Invivogen, thpd-r232). [Figure 19] Chelated metal ions for inhibiting the TLR3-I type IFN pathway. Dose inhibition curves of the IFN-I response by the indicated compounds in poly-IC / lipofectamine 2000 (ThermoFisher) treated THP-1 dual STING KO reporter cells (Invivogen). [Figure 20] Molecular structures of other representative powerful polyphenol chelating agents. [Modes for carrying out the invention]

[0098] [Definition] To facilitate understanding of the present invention, several terms and expressions are defined below: As used herein, the term “lipid” or “lipid molecule” refers to fatty substances that are insoluble in water and include fats, oils, waxes, and related compounds. They may be produced in the blood (endogenous) or ingested through diet (exogenous). Lipids are essential for normal bodily functions and, whether produced from exogenous or endogenous sources, must be transported for use by cells and then released for use by cells. The production, transport, and release of lipids for use by cells is called lipid metabolism. There are several classes of lipids, but the two main classes are cholesterol and triglycerides. Cholesterol is ingested through diet and may be produced by cells in most organs and tissues in the body, primarily the liver. Cholesterol can be found in free form, or more frequently, as cholesterol esters bound to fatty acids. As used herein, “lipid” or “lipid molecule” refers to any lipid-soluble compound. Non-limiting examples of lipid compounds include fatty acids, cholesterol, phospholipids, complex lipids, and their derivatives or analogues, which are typically classified into at least three classes: (1) "simple lipids" including fats and oils, as well as waxes; (2) "lipid compounds" including phospholipids and glycolipids; and (3) "lipid derivatives" such as steroids. Lipids or lipid molecules suitable for use in the present invention include both membrane-forming lipids and non-membrane-forming lipids.

[0099] As used herein, the term “lipoprotein” refers to a spherical compound structured such that water-insoluble lipids are partially contained within a water-soluble outer shell. Depending on the type of lipoprotein, the contents may include varying amounts of free and esterified cholesterol, triglycerides, and apoproteins, or apoproteins. There are five main types of lipoproteins, classified according to their function and lipid content, as well as their apoprotein content, and increasing concentration: (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 in conjunction with lipoproteins.

[0100] As used herein, the terms “HDL” or “high-density lipoprotein” refer to high-density lipoprotein. HDL consists of nearly equal amounts of lipid and protein complexes that function as transporters of cholesterol in the blood. HDL is primarily synthesized and secreted by epithelial cells of the liver and small intestine. Immediately after secretion, HDL is in the form of disc-shaped particles containing apolipoprotein AI (also called apoA-I) and phospholipids as its main components; this is also called nascent HDL. In the blood, this nascent HDL receives free cholesterol from the cell membranes of peripheral cells or is produced during the hydrolysis of other lipoproteins, and forms mature spherical HDL, holding cholesterol esters converted from the aforementioned cholesterol at its hydrophobic center by the action of LCAT (lecithin cholesterol acyltransferase). HDL plays a crucial role in a lipid metabolic process called “reverse cholesterol transport,” taking up cholesterol from peripheral tissues into the blood and transporting it to the liver. Since reverse cholesterol transport is considered one of the main mechanisms by which HDL has a protective effect against atherosclerosis, high levels of HDL are associated with a reduced risk of atherosclerosis and coronary heart disease (CHD).

[0101] As used herein, the terms “synthetic HDL,” “sHDL,” “reconstituted HDL,” or “rHDL” refer to particles structurally similar to natural HDL, composed of lipids, preferably ApoA-I or its mimics, that associate with at least one HDL protein. Typically, the components of sHDL may be derived from blood or produced by recombinant technology.

[0102] As used herein, the term "complexing" refers to non-covalent interactions between biomolecular drugs (e.g., antigens, adjuvants, etc.) and nanoparticles and / or microparticles.

[0103] As used herein, the term “binding” refers to a covalent bond between a biomolecular agent (e.g., an antigen, an adjuvant, etc.) and nanoparticles and / or microparticles.

[0104] As used herein, the term “encapsulation” refers to the arrangement of a biomolecular agent (e.g., antigen, adjuvant, etc.) that is encapsulated or completely contained within nanoparticles and / or microparticles.

[0105] As used herein, the term "absorption" refers to a biopolymer (e.g., antigen, adjuvant, etc.) that is incorporated into the interior of nanoparticles and / or microparticles, i.e., inside their outer surface, and stably retained therein.

[0106] As used herein, the term “adsorption” refers to the attachment of biomacromolecules (e.g., antigens, adjuvants, etc.) to the outer surface of nanoparticles and / or fine particles. Such adsorption is preferably caused by electrostatic attraction. Electrostatic attraction is an attractive force or bond generated between two or more opposite charges or ionic chemical groups. Generally, adsorption is typically reversible.

[0107] As used herein, the term “mixture” refers to a biopolymer (e.g., antigen, adjuvant, etc.) dissolved, dispersed, or suspended in nanoparticles and / or microparticles. In some cases, the biopolymer can be uniformly mixed in the nanoparticles and / or microparticles.

[0108] As used herein, the terms “biological biopolymer” or “biomolecule” or “biomolecule agent” mean, as used herein, molecules having a molecular weight greater than 1 kDa that can be isolated from living organisms or cell cultures, e.g., eukaryotic (e.g., mammalian) cell cultures or prokaryotic (e.g., bacterial) cell cultures. In some embodiments, the use of the terms means polymers, e.g., nucleic acids (including, but not limited to, RNA, siRNA, microRNA, interfering RNA, mRNA, replicon mRNA, RNA analogs, DNA, etc.), polypeptides (e.g., proteins), carbohydrates, and lipids. In some embodiments, the term “biomolecule” means proteins. In some embodiments, the term “biomolecule” means recombinant proteins or fusion proteins. In some embodiments, proteins are soluble. In some embodiments, biopolymers are antibodies, e.g., monoclonal antibodies. In some embodiments, biopolymers are adjuvants, antigens, therapeutic agents, contrast agents, etc.

[0109] As used herein, the term “antigen” is defined herein as a molecule containing one or more epitopes that stimulate the host immune system to obtain an antigen-specific immune response and / or humoral antibody response in a cell. Antigens can be peptides, proteins, polysaccharides, sugars, lipids, nucleic acids, and combinations thereof. Antigens may originate from viruses, bacteria, parasites, plants, protozoa, fungi, tissues, or transformed cells such as cancer or leukemia cells, and may be whole cells or their immune components, such as cell wall components. Antigens may be oligonucleotides or polynucleotides that express the antigen. Antigens can be natural or synthetic antigens, such as haptens, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens (see, for example, 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)).

[0110] As used herein, the term “neoantigen” or “neoantigenicity” means a class of tumor antigens resulting from tumor-specific mutations that alter the amino acid sequence of a genome-coding protein.

[0111] As used herein, "tumor-specific antigen" is defined herein as an antigen that is unique to tumor cells and does not occur in or on other cells of the body.

[0112] As used herein, the term "tumor-associated antigen" is defined herein as an antigen that is not specific to tumor cells and occurs inside or on normal cells under conditions that do not induce an immune response to the antigen.

[0113] As used herein, the term “adjuvant” is defined herein as a substance that, when administered together with other antigens, increases the immune response to those other antigens. Adjuvants are also referred to herein as “immunostimulants” and “immunomodulators.”

[0114] As used herein, the term "antigen-presenting cell" is defined herein as a highly specialized cell capable of processing antigens and presenting peptide fragments on its cell surface along with molecules required for lymphocyte activation. The primary antigen-presenting cells for T cells are dendritic cells, macrophages, and B cells. The primary antigen-presenting cell for B cells is follicular dendritic cells.

[0115] 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 on CD8 T cells (cytotoxic T cells) with MHC class I molecules. This process induces cellular immunity against most tumors and viruses that do not infect antigen-presenting cells. Cross-presentation is also necessary for inducing cytotoxic immunity in protein antigen-based vaccinations, such as tumor vaccines.

[0116] As used herein, the terms “immune,” “immunological,” or “immune” response refer to the occurrence of a humoral and / or cellular response to an antigen.

[0117] As used herein, the term “kit” means any delivery system for delivering materials. In relation to the sHDL nanoparticles described herein (e.g., compositions comprising sHDL nanoparticles encapsulating siRNA) (e.g., compositions comprising sHDL nanoparticles configured to activate an immune response), such a delivery system includes a system that enables the storage, transport, or delivery of such compositions and / or adjuvants (e.g., written instructions for use of the materials) from one place to another. For example, a kit includes one or more enclosures (e.g., boxes) containing the required drugs and / or adjuvants. As used herein, the term “fragmentation kit” means a delivery system comprising two or more separate containers, each containing a portion of the overall kit components. The containers may be delivered together or separately to the recipient of interest. For example, a first container may contain a composition comprising sHDL nanoparticles, or components necessary to synthesize such sHDL nanoparticles, while a second container contains a second drug (e.g., siRNA, antigen, adjuvant) (e.g., antibiotic or spray applicator). In practice, any delivery system containing two or more separate containers, each containing a portion of the overall kit components, falls under the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all the components necessary to synthesize and utilize any of the sHDL nanoparticles described (e.g., in a single box containing each of the desired components). The term “kit” includes both fragmented and combined kits.

[0118] As used herein, the term "subject" refers to any animal (e.g., mammal) that is to be the recipient of a particular treatment, including but not limited to humans, non-human primates, and rodents. Typically, the terms "subject" and "patient" are used synonymously in this specification with respect to human subjects.

[0119] As used herein, the term “sample” is used in its broadest sense. In a sense, it means specimens or cultures obtained from any source, as well as biological samples and environmental samples. Biological samples may be obtained from animals (including humans) and include fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma and serum. Environmental samples include environmental materials such as surface materials, soil, water, quartz, and industrial samples. However, such examples should not be construed as limiting the types of samples to which the present invention is applicable.

[0120] As used herein, the term "in vitro" refers to an artificial environment and the processes or reactions that occur within it. An in vitro environment may, but is not limited to, a test tube and a cell culture. The term "in vivo" refers to a natural environment (e.g., an animal or a cell) and the processes or reactions that occur within it.

[0121] As used herein, the terms “drug” or “therapeutic agent” mean any molecule, molecular complex or substance administered to a living organism for diagnostic or therapeutic purposes, including medical imaging, surveillance, contraception, cosmetic, nutritional supplement, pharmaceutical and preventive uses. The term “drug” further means any such molecule, molecular complex or substance that is chemically modified and / or functionally bound to a biological or biocompatible structure.

[0122] As used herein, the term “solvent” refers to the medium in which a reaction takes place. A solvent may be, but is not limited to, a liquid. Categories of solvents include, but are not limited to, nonpolar, polar, protic, and aprotic.

[0123] [Detailed description of the invention] CDN's cyclic-di-AMP (produced by Listeria monocytogenes) and its analog, cyclic-di-GMP (produced by Legionella pneumophila), are recognized by host cells as PAMP (Pathogen Associated Molecular Pattern), which binds to PRR (Pathogen Recognition Receptor) known as STING. STING is an adapter protein in the cytoplasm of host mammalian cells that activates the tank-binding kinase (TBK1)-IRF3 signaling axis, resulting in the induction of IFN-β and other IRF-3-dependent gene products that potently activate innate immunity. STING is now recognized as a component of the host cytosolic surveillance pathway, sensing infection by intracellular pathogens and in response inducing IFN-β production, leading to the expression of an adaptive defense pathogen-specific immune response consisting of both antigen-specific CD4 and CD8 T cells and pathogen-specific antibodies.

[0124] Immunotherapy is advancing cancer treatment in multiple fields. Recently, it has been discovered that a strong anti-tumor immune response can be initiated by activating the innate immune system via cyclic GAM-AMP (cGAMP), which activates the IFN gene stimulating (STING) pathway. Besides cGAMP, various other cyclic dinucleotides (CDNs), such as cdiAMP, cdiGMP, and cAIMP, can activate the STING pathway, which is recognized as an essential immune defense mechanism against tumors and exogenous pathogens. However, due to their low molecular weight, poor pharmacokinetic properties, and severe toxicity to non-target cells, STING agonists require direct local injection into the tumor. Experiments conducted in the process of developing embodiments for this invention showed that CDNs (1)Zn 2+Or (2) it was discovered that it can aggregate into uniform nanoparticles in the presence of either calcium phosphate and PEI-PEG. Based on such results, two categories of drug delivery systems for the delivery of CDN were developed. In a subcutaneous CT26 tumor model, the formulation was shown to significantly inhibit 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.

[0125] These nanoparticles related to CDN can induce a specific immune response against tumors via systemic administration, thereby avoiding the need for direct local injection into the tumors, and such results are clinically very important.

[0126] Further experiments conducted during the process of developing embodiments for the present invention showed that specific metal ions such as Mn 2+ and Co 2+ can enhance the STING activation and type I IFN response of STING agonists. In a mouse CT26 colorectal tumor model, Mn 2+ / Co 2+The combination of -STING agonists was shown to result in elevated serum type I IFN concentrations, leading to a higher tumor eradication effect and promoting longer survival in tumor-bearing mice, with 80% of mice cured and resistant to difficult secondary tumors. Furthermore, this phenomenon was found to be common for various other innate immune pathways, including, but not limited to, Toll-like receptor (TLR) 3 / 4 / 7 / 8 / 9 ligands, NOD1 / 2 ligands, TLR7 / 8 ligands, RIG-I & CDS agonists, and inflammasome inducers. Based on these findings, several pharmaceutically acceptable formulations have been developed, such as metal salts of DAMP / PAMP, coordinations, and other metal-loading formulations (hydroxyl / carbonate / phosphate minerals, liposomes, self-assembling nanoparticles, PLGA, hydrogels, emulsions, etc.), to precisely deliver the combination of metal innate immunostimulants to the desired target and release in an ideal manner. Finally, it was found that several chelating agents can effectively inhibit the DNA-induced cGAS-STING-Type-I IFN / NFκB response and the polyIC-induced TLR3-cGAS-STING-Type-I IFN response.

[0127] Therefore, such results and embodiments represent a novel class of drug delivery systems for both local and systemic delivery of drugs that can stimulate an innate immune response in a subject upon administration to that subject.

[0128] Therefore, the present invention provides compositions and methods for stimulating an innate immune response in a subject at the time of administration, by administering a drug capable of stimulating an innate immune response in a subject. In particular, the invention relates to such compositions comprising a drug capable of stimulating an innate immune response in a subject at the time of administration, methods for synthesizing such compositions, and systems and methods utilizing such compositions (e.g., in diagnostic and / or therapeutic settings).

[0129] Therefore, in a particular embodiment, the present invention provides one or more DAMPs or PAMPs with either a) or b) below: a) Calcium phosphate, and copolymers of cationic poly(ethyleneimine) (PEI) with polyethylene glycol (PEG), poly(histidine) polyethylene glycol (PH-PEG), lipid poly-histidine, poly(lysine) polyethylene glycol PEG (PK-PEG), or anionic poly(glutamic acid) polyethylene glycol (PGA-PEG); or b) Zn 2+ Mn 2+ Ca 2 +, Fe 2+ Fe 3+ Cu 2+ Ni 2+ Co 2+ Pb 2+ Sn 2+ , Ru 2+ Au 2+ Mg 2+ , VO 2+ , Al 3+ Co 3+ , Cr 3+ , Ga 3+ , Tl 3+ , Ln 3+ MoO 3+ Cu + Au + , Tl + Ag + Hg 2+ Pt 2+ Pb 2+ Hg 2+ , Cd 2+ , Pd 2+ Pt 4+ kaNa + , K + , one or more cations selected from the group consisting of related phosphates and related carbonates; The present invention provides a composition containing [a certain substance].

[0130] Such compositions are not limited to specific DAMP or PAMP agonists.

[0131] In some embodiments, the DAMP or PAMP agonist is selected from STING agonists, purine-containing factors or purine derivative factors, Toll-like receptor (TLR) agonists, NOD-like receptor (NLR) agonists, RIG-I-like receptor (RLR) agonists, cytoplasmic DNA sensor (CDS) agonists, C-type lectin receptor (CLR) agonists, and inflammasome inducers.

[0132] In some embodiments, the DAMP or PAMP agonist is selected from TLR-3 agonists, TLR-4 agonists, TLR-5 agonists, TLR-7 agonists (e.g., imiquimod), TLR-8 agonists (e.g., regiquimod), TLR-9 agonists, and NLRP3 agonists.

[0133] Such compositions are not limited to specific purine-containing factors or purine-derived factors. In some embodiments, the purine-containing factors or purine-derived factors are 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluoro, cAIM(PS)2, difluoro(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluoride, c-di-AMP fluoride, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluoride, 2'3'-c-di-GMP, c-di-IMP, cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, 3'3'-cGAMP, cGAM(PS)2, 2'3'-cGAM(PS)2(Rp / Sp), 2'2'-cGAM(PS)2, 2'3'-cGAM(PS)2, cGAMP fluoride, 3'3'-cGAMP fluoride, 2'3'-cGAMP fluoride, 2'2'-cGAMP fluoride, c-di-AMP, 2'3'-cdAMP, 2'2'-cdAMP, 3'3'-cdAMP, c-di-AM(PS)2, 2'3'-c-di-AM(PS)2(Rp,Rp), 2'2'-c-di-AM(PS)2, 3'3'-c-di-AM(PS)2, c-di-AMP fluoride, 2'3'-cdAMP fluoride, 2'2'-cdAMP fluoride fluoride, 3'3'-cdAMP fluoride, cdGMP, 2'3'-cdGMP, 2'2'-cdGMP, 3'3'-cdGMP, c-di-GM(PS)2, 2'3'-c-di- GM(PS)2, 2'2'-c-di-GM(PS)2, 3'3'-c-di-GM(PS)2, cdGMP fluoride, 2'3'-cdGMP fluoride, 2'2'-cdGMP fluoride Fluorinated, 3'3'-cdGMP fluorinated, cAIMP, 2'3'-cAIMP, 2'2'-cAIMP, 3'3'-cAIMP, cAIMP difluoro (3'3'-cAIMP fluorinated) Fluorinated compound, 2'3'-cAIMP fluorinated compound, 2'2'-cAIMP fluorinated compound, cAIM(PS)2 difluoro, 3'3'-cAIM(PS)2 difluoro(Rp / Sp), 2'3 '-cAIM(PS)2 difluoro, 2'2'-cAIM(PS)2 difluoro, c-di-IMP, 2'3'-cdIMP, 2'2'-cdIMP, 3'3'-cdIMP, c-d i-IM(PS)2, 2'3'-c-di-IM(PS)2, 2'2'-c-di-IM(PS)2, 3'3'-c-di-IM(PS)2, c-di-IMP fluoride, 2'3'-cdIMP fluoride, 2'2'-cdIMP fluoride, 3'3'-cdIMP fluoride, imiquimod, resikimod, 6-(4-amino-imidazoquinolyl)-norleucine,

[0134] [ka]

[0135] And selected from purine-based PI3K inhibitors.

[0136] 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 a derivative thereof. In some embodiments, the small molecule agonist of STING is a cyclic dinucleotide. For example, in some embodiments, the cyclic dinucleotides include, but are not limited to, 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluor, cAIM(PS)2, difluor(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluoride, c-di-AMP fluoride, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluoride, 2'3'-c-di-GMP, c-di-IMP, SB11285, STING-agonist-C11, STING-agonist-1, STING-agonist-G10, gemcitabine, and additional STING agonists described herein.

[0137] 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. Further examples of cyclic prurien dinucleotides are described in detail, for example, U.S. Patents 7,709,458 and 7,592,326; WO2007 / 054279; and Yan et al., Bioorg. Med. Chem Lett. 18: 5631 (2008). Each of these is incorporated herein by reference.

[0138] Further suitable STING agonists for use in the disclosed method include, but are not limited to, flavonoids. In some embodiments, the STING agonist may comprise a flavonoid. In other embodiments, the STING agonist may consist of a flavonoid. Suitable flavonoids include, but are not limited to, 10-(carboxymethyl)-9(10H)acridone (CMA), 5,6-dimethylxanthenone-4-acetic acid (DMXAA), methoxybone, 6,4'-dimethoxyflavone, 4'-methoxyflavone, 3',6'-dihydroxyflavone, 7,2'-dihydroxyflavone, didozein, formononetin, letsin 7-methyl ether, xanthone, or any combination thereof. In some embodiments, the STING agonist may be 10-(carboxymethyl)-9(10H)acridone (CMA). In some embodiments, the STING agonist may be 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In some embodiments, the STING agonist may be methoxybone. In some embodiments, the STING agonist may be 6,4'-dimethoxyflavone. In some embodiments, the STING agonist may be 4'-methoxyflavone. In some embodiments, the STING agonist may be 3',6'-dihydroxyflavone. In some embodiments, the STING agonist may be 7,2'-dihydroxyflavone. In some aspects, the STING agonist may be didozei. In some embodiments, the STING agonist may be formononetin. In some embodiments, the STING agonist may be letusin 7-methyl ether. In some embodiments, the STING agonist may be xanthone. In some embodiments, the STING agonist may be any combination of the above flavonoids. Therefore, for example, in some embodiments, the flavonoid includes DMXAA.

[0139] In some embodiments, STING small molecule agonists include, but are not limited to, 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP, cAIMP difluor, cAIM(PS)2, difluor(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluoride, c-di-AMP fluoride, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluoride, 2'3'-c-di-GMP, c-di-IMP, SB11285, STING-agonist-C11, STING-agonist-1, STING-agonist-G10, and gemcitabine.

[0140] In certain embodiments, the present invention provides a composition capable of inhibiting cGAS-STING activation and type I IFN response, comprising one or more cell-permeable chelating agents or derivatives thereof, which can prevent intracellular metal ions from being utilized for cGAS-STING-type I IFN activation.

[0141] In one embodiment, the present invention provides a composition that can modulate innate immune activation by comprising one or more cell-permeable chelating agents (e.g., metal ion chelating agents) and preventing intracellular metal ions from being utilized by the innate immune pathway.

[0142] In some embodiments, such cell-permeable chelating agents (e.g., metal ion chelating agents) include, but are not limited to, the polyphenol chelating agents (-)-epigallocatechin gallate (EGCG), punicalagin, (-)-catechin gallate, (-)-catechin, tannic acid, tannin, punicalin, vescalagin, procyanidin C1, geraniin, theaflavin 3,3'-digalate, lipid-modified NTA, porphyrin, EDTA, NOTA, DOTA, TPEN, and clofelemer.

[0143] In some embodiments, compositions capable of inhibiting cGAS-STING activation and type I IFN response are used in the treatment of subjects who have or are at risk of developing autoimmune disorders.

[0144] Thus, the present invention provides a method for treating autoimmune disorders by administering a composition capable of modulating innate immune activation, comprising one or more cell-permeable chelating agents (e.g., metal ion chelating agents), to a subject (e.g., a human subject) to prevent intracellular metal ions from being utilized in the innate immune pathway. In such embodiments, such cell-permeable chelating agents (e.g., metal ion chelating agents) include, but are not limited to, the polyphenol chelating agents (-)-epigallocatechin gallate (EGCG), punicalagin, (-)-catechin gallate, (-)-catechin, tannic acid, tannin, punicalin, vescalagin, procyanidin C1, geraniin, theaflavin 3,3'-digalate, lipid-modified NTA, porphyrin, EDTA, NOTA, DOTA, TPEN, and clofelemer.

[0145] Examples of autoimmune disorders include, but are not limited to, systemic lupus erythematosus, Ecardi-Goutier syndrome, acute pancreatitis, age-related macular degeneration, alcoholic liver disease, hepatic fibrosis, metastasis, myocardial infarction, non-alcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, inflammatory bowel disease, and multiple sclerosis.

[0146] In some embodiments, additional therapeutic agents are administered along with such compositions. Examples of additional therapeutic agents include disease-modifying antirheumatic drugs (e.g., leflunomide, methotrexate, sulfasalazine, hydroxychloroquine), biological agents (e.g., rituximab, infliximab, etanercept, adalimumab, golimumab), nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, celecoxib, ketoprofen, naproxen, piroxicam, diclofenac), analgesics (e.g., acetaminophen, tramadol), and immunomodulators (e.g., The additional therapeutic agent is selected from the group consisting of anakinra, abatacept, glucorticoids (e.g., prednisone, methylprednisone), TNF-α inhibitors (e.g., adalimumab, certolizumab pegol, etanercept, golimumab, infliximab), IL-1 inhibitors, and metalloproteinase inhibitors, and in some embodiments, the additional therapeutic agent includes, but is not limited to, infliximab, adalimumab, etanercept, parenteral gold, or oral gold.

[0147] In some embodiments, such compositions comprising a drug (e.g., DAMP / PAMP) that can stimulate an innate immune response in a subject upon administration to the subject, work in conjunction with nanoparticles (e.g., by complexation, binding, encapsulation, absorption, adsorption, or mixing).

[0148] In some embodiments, such compositions that interact with nanoparticles further interact with calcium phosphate and copolymers of PEI / PEG, PH-PEG, PK-PEG, or PGA-PEG (e.g., complexation, binding, encapsulation, absorption, adsorption, mixing). In fact, in some embodiments, the interaction between a drug that can stimulate an innate immune response in a subject and nanoparticles occurs in the presence of calcium phosphate and copolymers of PEI / PEG, PH-PEG, PK-PEG, or PGA-PEG.

[0149] In some embodiments, such compositions that work in conjunction with nanoparticles include Zn 2+ 、 Mn 2+ Ca2+ 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+ kaNa + , K + , and further interact with one or more cations selected from the group consisting of these phosphates or carbonates (e.g., complexation, binding, encapsulation, absorption, adsorption, mixing). In fact, in some embodiments, the interaction of a drug that can stimulate an innate immune response in a subject with nanoparticles is such as cations (e.g., Zn 2+ Co 2+ , or Mn 2+ ) exists in the presence of.

[0150] It is known in the art that STING (interferon gene stimulator) is an adapter for multiple cytoplasmic DNA receptors and is a pattern recognition receptor (PRR) that recognizes cyclic diadenosine monophosphate (c-di-AMP) and cyclic diguanosine monophosphate (c-di-GMP), which are second messengers in bacteria. Cytoplasmic DNA binds to cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) to produce cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which then binds to the adapter protein STING, is activated, and induces IFN. STING consists of five putative transmembrane regions, is mainly located in the endoplasmic reticulum, and activates both the NF-κB and IRF3 transcription pathways to induce the expression of type I interferons (IFN-α and IFN-β), which then exert a potent antiviral state after expression.

[0151] Therefore, DAMPs and PAMPs (e.g., STING agonists) can stimulate innate cytokine responses in cancer cells. Thus, in some embodiments, DAMPs and PAMPs (e.g., STING agonists) can stimulate innate cytokine responses in cancer cells.

[0152] Innate cytokine responses stimulated by DAMP or PAMP are mediated via cytokines. In some embodiments, for example, innate cytokine responses may be mediated via type 1 interferons.

[0153] As described above, the present invention provides compositions and methods for stimulating the innate immune response of cancer cells (e.g., tumor cells) using agents (e.g., DAMP / PAMP) that can stimulate the innate immune response of a target upon administration to the target, in order to suppress and / or inhibit the proliferation of cancer cells (e.g., tumor cells). In particular, the invention relates to agents (e.g., DAMP / PAMP) that can stimulate the innate immune response of a target upon administration to the target, methods for synthesizing such nanoparticles, and compositions (e.g., complexation, binding, encapsulation, absorption, adsorption, mixing) relating to systems and methods utilizing such nanoparticles (e.g., in diagnostic and / or therapeutic settings).

[0154] In fact, experiments conducted during the development of embodiments of the present invention showed that Zn 2+ We demonstrated that CDN containing cdi-AMP, cGAMP, and cGMP assembles into uniform nanoparticles in the presence of Zn. 2+ It was also shown that such CDNs, assembled into homogeneous nanoparticles in the presence of calcium phosphate, can be further stabilized in lipid vesicles. Further experiments demonstrated that CDNs can be formulated into nanoparticles in the presence of calcium phosphate, as well as copolymers of cationic poly(ethyleneimine) (PEI) and polyethylene glycol (PEG). Such CDN-nanoparticle assemblies (e.g., copolymers of CDN and PEI-PEG formulated into nanoparticles in the presence of calcium phosphate) (e.g., Zn 2+ It has been further shown that the CDN formulated into nanoparticles in the presence of liposomes enables increased STING agonist delivery efficacy and reduced STING agonist toxicity by increasing cancer cell uptake and providing more precise targeting to the tumor microenvironment (e.g., TME).

[0155] The present invention relates to a composition containing a drug (e.g., DAMP / PAMP) that can stimulate an innate immune response in a subject upon administration to that subject, and is not limited to specific types or kinds of nanoparticles (e.g., complexed, conjugated, encapsulated, absorbed, adsorbed, mixed).

[0156] Examples of nanoparticles, though not limited to these, include metal-polyhistidine-DOPE@liposome, metal-polyhistidine-PEG, 4-arm-PEG-polyhistidine-metal hydrogel, and sHDL-polyhistidine, fullerene (mainly C 60 , C 70 , C 76 , C 80 , C 84 Examples include internally-bodied metallic fullerenes (TNTs), internally-bodied metallic fullerenes (TNTs) containing additional atoms, ions, or clusters within the fullerene cage, internally-bodied trimetallic nitride molecular clusters within carbon cages, single-walled and multi-walled carbon nanotubes, silver nanorods, single-walled and multi-walled boron / nitrate nanotubes, carbon nanotubes (nanotubes with internal metallic fullerenes and / or other internal chemical structures), carbon nanohorn endpods, liposomes, nanoshells, dendrimers, quantum dots, superparamagnetic nanoparticles, nanorods, and cellulose nanoparticles. Embodiments of the particles may also include microparticles having the ability to enhance efficacy or selectivity. Other non-limiting exemplary nanoparticles include glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, and gold, silver, carbon, and iron nanoparticles.

[0157] In some embodiments, the nanoparticles are modified micelles. In these embodiments, the modified micelles comprise a polyol polymer modified to contain a hydrophobic polymer block. As used in this disclosure, the term “hydrophobic polymer block” refers to a segment of a polymer that is hydrophobic itself. As used herein, the term “micelle” refers to an aggregate of molecules dispersed in a liquid, and a typical micelle in an aqueous solution forms an aggregate with a hydrophilic “head” region that comes into contact with the surrounding solvent, isolating a hydrophobic single tail region at the center of the micelle. In some embodiments, the head region may be, for example, a surface region of the polyol polymer, and the tail region may be, for example, a hydrophobic polymer block region of the polyol polymer.

[0158] The present invention further encompasses the use of micrometer-scale particles in addition to nanometer-scale particles. When microparticles are used, they are relatively small, preferably on the order of 1 to 50 micrometers. For the sake of ease of discussion, as used herein, “nanoparticles” include true nanoparticles (sizes from about 1 nm to about 1000 nm), microparticles (e.g., about 1 micrometer to about 50 micrometers), or both.

[0159] Examples of nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metallic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers, dendrimers having covalently bonded metal chelates, nanofibers, nanohorns, nanoonions, 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 two or more alloys thereof). Nanoparticles may include a core or a core and shell, such as core-shell nanoparticles.

[0160] In some embodiments, the nanoparticles are sHDL nanoparticles. Generally, sHDL nanoparticles consist of a mixture of HDL apolipoprotein and amphiphilic lipids.

[0161] The present invention is not limited to the use of a specific type or category of HDL apolipoprotein. HDL apolipoproteins include, for example, apolipoprotein AI (apo AI), apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein C (apo Cs), and apolipoprotein E (apo E). In some embodiments, HDL apolipoproteins are selected from preproapoproteins, preproApoA-I, proApoA-I, AρoA-I, preproApoA-II, proApoA-II, ApoA-II, preApoA-IV, proApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-IMilano, ρroApoA-IMilano, ApoA-IMilano, ρreproApoA-IParis, proApoA-IParis, and peptide mimes of ApoA-IParis and mixtures thereof. Preferably, the carrier particles consist of Apo AI or Apo A-II, but other lipoproteins, including apolipoprotein A4, apolipoprotein Cs, or apolipoprotein E, may be used alone or in combination to formulate carrier particle mixtures for therapeutic agent delivery. In some embodiments, mimics of such HDL apolipoproteins are used.

[0162] ApoA-I is synthesized in the liver and small intestine as a preproapolipoprotein, secreted as a proprotein that is rapidly broken down to produce a mature polypeptide with 243 amino acid residues. ApoA-I mainly consists of 6 to 8 distinct 22-amino acid repeats separated by a proline linker, and in some cases, stretches composed of several residues. ApoA-I forms three stable complexes with lipids: small, low-lipid complexes called pre-β-1HDL; flattened, disc-shaped particles containing polar lipids (phospholipids and cholesterol) called pre-β-2HDL; and spherical or mature HDL (HDL3 and HDL2) containing both polar and nonpolar lipids. Most HDL in the circulating population contains both ApoA-I and ApoA-II (the second major HDL protein).

[0163] In some embodiments, ApoA-I agonists or mimetic compounds are provided. In some embodiments, such ApoA-I mimetic compounds can form amphiphilic α-helices that mimic the activity of ApoA-I and have specific activity that approaches or exceeds the activity of the native molecule. Some ApoA-I mimetic compounds are peptides or peptide analogs that form amphiphilic helices (in the presence of lipids), bind to lipids, form pre-β-like or HDL-like complexes, activate lecithin:cholesterol acyltransferase (LCAT), increase serum levels of the HDL fraction, and promote cholesterol efflux.

[0164] The present invention is not limited to the use of a specific ApoA-I mimetic. In some embodiments, any of the ApoA-I mimetic described in Srinivasa, et al., 2014 Curr. Opinion Lipidology Vol. 25(4): 304-308 is used. In some embodiments, any of the ApoA-I mimetic described in U.S. Patent Application Publications 2011 / 0046056 and 2013 / 0231459 is used.

[0165] In some embodiments, the "22A" ApoA-I mimetic (PVLDLFRELLNELLEALKQKLK) (Sequence ID 4) is used (see, for example, U.S. Patent No. 7,566,695). In some embodiments, any of the following ApoA-I mimetics shown in Table 1, as described in U.S. Patent No. 7,566,695, are used: Table 1. ApoA-I mimics

[0166] [Table 1] JPEG0007862013000008.jpg214169 JPEG0007862013000009.jpg215169 JPEG0007862013000010.jpg211169 JPEG0007862013000011.jpg211169 JPEG0007862013000012.jpg212169 JPEG0007862013000013.jpg210169 JPEG0007862013000014.jpg162169

[0167] * indicates a peptide with N-terminal acetylation and C-terminal amidation; indicates a peptide with N-terminal dansylation; sp indicates a peptide that exhibited solubility issues under experimental conditions; X is Aib; Z is Nal; O is Orn; He(%) indicates percent helicity; mic indicates a micelle; and ~ indicates a missing amino acid.

[0168] In some embodiments, an ApoA-I mimetic having the following sequence, as described in U.S. Patent No. 6,743,778, is used: Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Lys Glu Ala Phe (SEQ ID NO: 255).

[0169] In some embodiments, one of the following ApoA-I mimics shown in Table 2, such as those described in U.S. Patent Application Publication No. 2003 / 0171277, is used:

[0170] [Table 2] JPEG0007862013000016.jpg213169 JPEG0007862013000017.jpg141169

[0171] In some embodiments, an ApoA-I mimetic having the following sequence, as described in U.S. Patent Application Publication No. 2006 / 0069030, is used: FAEKFKEAVKDYFAKFWD (Sequence ID 333).

[0172] In some embodiments, ApoA-I mimics having the following sequences, as described in U.S. Patent Application Publication 2009 / 0081293, are used: DWFKAFYDKVAEKFKEAF (SEQ ID NO: 334), DWLKAFYDKVAEKLKEAF (SEQ ID NO: 335), PALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ (SEQ ID NO: 336).

[0173] In some embodiments, an ApoA-I mimetic having one of the following sequences is used: 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: 341) 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: 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), TVLLLTICSLEGALVRRQAKEPCV (SEQ ID NO: 347), PLGEEMRDRARAHVDALRTHLA (SEQ ID NO: 348), P 354), QTVTDYGKDLME (SEQ ID NO: 355), KVKSPELOAEAKSYFEKSKE (SEQ ID NO: 356), VLTLALVAVAGARAEVSADOVATV (SEQ ID NO: 357), NNAKEAVEHLOKSELTOOLNAL (SEQ ID NO: 358), LPVLVWLSIVLEGPAPAOGTPDVSS (SEQ ID NO: 359), LPVLVVVLSIVLEGPAPAQGTPDVSS (SEQ ID NO: 360), ALDKLKEFGNTLEDKARELIS (SEQ ID NO: 361), VVALLALLASARASEAEDASL L (SEQ ID NO: 362), HLRKLRKRLLRDADDLQKRLAVYOA (SEQ ID NO: 363), AQAWGERLRARMEEMGSRTRDR (SEQ ID NO: 364), LDEVKEQVAEVRAKLEEQAQ (SEQ ID NO: 365), DWLKAFYDKVAEKLKEAF (SEQ ID NO: 236), DWLKAFYDKVAEKLKEAFPDWAKAAYDKAAEKAKEAA (SEQ ID NO: 366), PVLDLFRELLNELLEALKQKL (SEQ ID NO: 367), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 368),PVLDLFRELLNELLEALKQKLK (SEQ ID NO: 4), PVLDLFRELLNELLEALKQKLA (SEQ ID NO: 369), PVLDLFRELLNELLEALKKLLK (SEQ ID NO: 370), PVLDLFRELLNELLEALKKLLA (SEQ ID NO: 371), PLLDLFRELLNELLEALKKLLA (SEQ ID NO: 372), and EVRSKLEEWFAAFREFAEEFLARLKS (SEQ ID NO: 373).

[0174] Amphiphilic lipids include, for example, any lipid molecule having both a hydrophobic and a hydrophilic portion. Examples include phospholipids and 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-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butylamide], and 1,2-dihexadecanoyl-sn-glycero These include -3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butylamide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof. In some embodiments, the phospholipids are complexed with imaging agents (e.g., rhodamine (Rhod)-labeled DOPE (DOPE-Rhod)). In some embodiments, the phospholipid is a thiol-reactive phospholipid such as dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dihexadecanoyl-sn-glycero-3-phosphothioethanol, or N-4-(p-maleimidophenyl)butyryl)dipalmitoylphosphatidylethanolamine (MPB-DPPE).

[0175] In some embodiments, exemplary phospholipids include small alkyl chain phospholipids, egg phosphatidylcholine, soybean phosphatidylcholine, dimyristoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl phosphatidylcholine, dilauroyl phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol such as dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dimyristoyl phosphatidic acid, dimyristoyl phosphatidylethanolamine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, brain phosphatidylserine, egg sphingomyelin, palmitoyl sphingomyelin, phytosphingomyelin, distearoyl phosphatidylglycerol salt, phosphatidic acid, galactosylcerebroside, cerebroside, dilauryl phosphatidylcholine, (1,3)-D-mannosyl-(1,3) diglyceride, aminophenyl glycoside, 3-cholesteryl-6'-(glycosylthio) hexyl ether glycolipid and its derivatives, but are not limited thereto. The phospholipid fraction containing SM and palmitoyl sphingomyelin can optionally contain small amounts of any type of lipid, including lysophospholipids, sphingomyelins other than palmitoyl sphingomyelin, galactosylcerebrosides, gangliosides, cerebrosides, glycerides, triglycerides, and cholesterol and its derivatives, but is not limited thereto.

[0176] 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).

[0177] Generally, the sHDL nanoparticles thus formed are spherical and have a diameter of about 5 nm to about 20 nm (e.g., 4 - 75 nm, 4 - 60 nm, 4 - 50 nm, 4 - 22 nm, 6 - 18 nm, 8 - 15 nm, 8 - 10 nm, etc.). In some embodiments, the sHDL nanoparticles are subjected to size exclusion chromatography to obtain a more uniform preparation.

[0178] Compared to other strategies involving conventional nanoparticle vehicles, sHDL nanoparticles have attractive biocompatibility and cargo loading capabilities. For example, their ultrasmall yet adjustable size (e.g., 10 - 20 nm) enables sHDL nanoparticles to be efficiently drained into lymph nodes and deliver cargo peptide antigens and nucleic acid - based adjuvants to dendritic cells present in the lymph nodes, thereby positioning them as an efficient platform for the co - delivery of STING agonists and adjuvants for tumor immunotherapy.

[0179] In certain embodiments, a composition comprising nanoparticles associated with such a composition that can stimulate the subject's innate immune response upon administration to the subject (e.g., DAMP / PAMP), and any type of biopolymeric agent (e.g., nucleic acid, peptide, glycolipid, etc.) is associated with the nanoparticles is provided.

[0180] In some embodiments, the biopolymeric agent is a peptide.

[0181] For example, in some embodiments, the peptide is an antigen.

[0182] For example, in some embodiments, the antigen is a tumor antigen. The antigen can be a tumor antigen including, but not limited to, the following tumor-associated antigens or tumor-specific antigens: α-actinin-4, Bcr-Abl fusion protein, Casp-8, β-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, Mum-2, and Mum-3, neo- PAP, Myosin Class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triose phosphate isomerase, Bage-1, Gage3, Gage4, Gage5, Gage6, Gage7, GnTV, Herv-K-mel, Lage-1, Mage-A1, Mage-A2, Mage-A3, Mage-A4, Mage-A6, Mage-A10, Mage-A12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, and TRP2-Int2, Mela nA(MART-I), gp100(Pmel17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO(LAGS), SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA125, CA15-3(CA27.29\BCAA), CA195, CA242, CA-50,Human EGFR proteins or their fragments, such as CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), 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 / cyclophyllin 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 variant, p53 variant, proteinase 3 (PR1), survivorbin, 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, fucosyl GM1, mesothelin, sLe (animal), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, XAGE1, B7H3, regmine, Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-α, PDGFR-β, MAD-CT-2, Fos-related antibody 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 dehydrogenases.

[0183] In some embodiments where the antigen is a biomolecule, the composition further comprises an adjuvant (as described herein).

[0184] In some embodiments, the peptides are: adrenocorticotropic hormone (ACTH), growth hormone peptide, melanocyte-stimulating hormone (MSH), oxytocin, vasopressin, corticotropin-releasing factor (CRF), CRF-related peptide, gonadotropin-releasing peptide (GAP), growth hormone-releasing factor (GRF), luteinizing hormone-releasing hormone (LH-RH), orexin, prolactin-releasing peptide (PRP), somatostatin, thyrotropin-releasing hormone (THR), THR analog, calcitonin (CT), CT-precursor peptide Butide, calcitonin gene-related peptide (CGRP), parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), amylin, glucagon, insulin, insulin-like peptide, neuropeptide Y (NPY), pancreatic polypeptide (PP), peptide YY (PYY), cholecystokinin (CCK), CCK-related peptide, gastrin-releasing peptide (GRP), gastrin, gastrin-related peptide, gastrin-inhibiting peptide, motilin, secretin, vasoactive intestinal peptide (VIP), VIP-related peptide, atrial natriuretic peptide ANP (Angiotensin Protein), Brain Natriuretic Peptide (BNP), C-type Natriuretic Peptide (CNP), Tachykinin, Angiotensin, Renin Substrate, Renin Inhibitor, Endothelin, Endothelin-related Peptides, Opioid Peptides, Thymus Peptides, Adrenomedullin Peptides, Allostatin Peptides, Amyloid-β Protein Fragments, Antimicrobial Fragment Peptides, Antioxidant Peptides, Apoptosis-related Peptides, Capsular Cell Peptides (BCP), Bombecin, Bone Gla Protein Peptides, Cocaine and Amphetamine-related Transcription (CART) Peptides, Cell Adhesion Peptides D, chemotactic peptides, complement inhibitors, cortisutin peptides, fibronectin fragments, fibrin-related peptides, FMRF, FMRF amide-related peptides (FaRP), galanin, galanin-related peptides, growth factors, growth factor-related peptides, G therapeutic peptide-binding protein fragments, guarylin, uroguanilin, inhibin peptides, interleukin (IL), interleukin receptor proteins, laminin fragments, leptin fragment peptides, leukokinin, pituitary adenylate cyclase-activating polypeptide (PAPCAP),Pancreastatin, polypeptide repeat chain, signaling factor, thrombin inhibitor, toxin, trypsin inhibitor, virus-related peptide, adjuvant peptide analog, α-conjugation factor, antiarrhythmic peptide, appetite suppressant peptide, α-1 antitrypsin, bovine pineal gland anti-reproductive peptide, brucine, C3 peptide P16, cadherin peptide, chromogranin A fragment, contraceptive tetrapeptide, conantkin G, conantkin T, crustacean cardioactive peptide, C-telopeptide, cytochrome b588 peptide, decorsin, delicious peptide peptide), delta sleep-inducing peptide, diazepam binding inhibitor fragment, nitric oxide synthase blocking peptide, OVA peptide, platelet calpain inhibitor (P1), plasminogen activator inhibitor 1, rigin, schizophrenia-related peptide, sodium potassium A therapeutic peptidase inhibitor-1, speract, sperm-activating peptide, cystemin, thrombin receptor agonist, tuftosin, lipid-mobilizing hormone, uremic pentapeptide, antifreeze polypeptide, tumor necrosis factor (TNF), Leech[Des Asp10] Decolsin, L-Ornityl Taurine Hydrochloride, P-Aminophenylacetyl Tuftosin, Ac-Glu-Glu-Val-Val-Ala-Cys-pNA, Ac-Ser-Asp-Lys-Pro, Ac-rfwink-NH2, Cys-Gly-Tyr-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-Gly, D-Ala-Leu, DDDDD, DDDDDD, NPNANPNA, VAITVLVK, VGVRVR, VIHS, VPDPR, Val-Thr-Cys-Gly, RSR, sea urchin sperm activating peptide, SHU-9119 antagonist, MC3-R antagonist, MC4-R antagonist, Glaspimod, HP-228, α2-plasmin inhibitor, APC tumor suppressor, early pregnancy factor, γ interferon, glandular kallikrein N-1, placental ribonuclease inhibitor, sarcolesin-binding protein, surfactant protein D, Wilms tumor suppressor, GABAB 1b receptor peptide, prion-related peptide (iPRP13),Choline-binding protein fragments, telomerase inhibitors, cardiostatin peptides, endostatin-derived peptides, prion inhibitor peptides, N-methyl-D-aspartate receptor antagonists, and C-peptide analogs.

[0185] In some embodiments, the peptide is selected from: 177Lu-DOTA0-Tyr3-octreotate, Abarelix acetate, ADH-1, Afamelanotidec, Melanotan I, CUV1647, Albiglutide, Aprotinin, Argipressin, Atosiban acetate, Bacitracin, Benthromide, BH3 domain, Bivalirudin, Bivalirudin trifluoroacetate hydrate hydrate), bricibimod, bortezomib, buserelin, buserelin acetate, calcitonin, carbetocin, carbetocin acetate, seclopin A and B, ceruretide, ceruretide diethylamine, cetrorelix, cetrorelix acetate, cyclosporine, silengitide (Cilengitidec), EMD121974, corticocholelin acetate injection, hCRF, corticocholelin oubaiin triflutate, corticocholelin trifluoroacetate, corticotropin, cosintropin, ACTH1-24, tetracosactide hexaacetate, dalbavancin, daptomycin, degarelix acetate, depreotide trifluoroacetate (with sodium pertechnetate), desmopressin acetate, desmopressin DDAVP, dulaglutide, ecalantide, edotreotide ( (with ttrium-90), elcatonin acetate, enalapril maleate (or 2-butanediate), enfuvirtide, eptifibatide, exenatide, ganirelix acetate, glatiramer acetate, glutathione, gonadrelin, gonadrelin acetate, GnRH, LHRH, goserelin, goserelin acetate, gramicidin, histrelin acetate, human calcitonin, incatibant, incatibant acetate, IM862, ogluphanide disodium, KLAKLAK, lanreotide acetate, repiridine, leuprolide, leuprolide acetate, leuprorelin, liraglutide, lisinopril, lixisenatide, lypressin, magainin 2, MALP-2Sc, macrophage-activating lipopeptide-2 compound (macrophage-activating lipopeptide-2Synthetic), Nafarelin acetate, Nesiritide, NGR-hTNF, Octreotide acetate, Oritabancin, Oxytocin, Pasireotide, Peginesatide, Pentagastrin, Pentetreotide (with Indium-111), Phenipressin, Pleurocidin, Plumrintide, Protirelin, Tyroleverine, TRH, TRF, Salmon Calcitonin, Salaracin acetate, Secretin (human), Secretin (porcine), Semaglutide, Seractide acetate, ACTH, Corticotropin, Sermorelin acetate, GRF 1-29, Synaplutide, KL4 in Lusynactant, Syncalid, Somatrelin acetate, GHRH, GHRF, GRF, Somatostatin acetate, Spaglumat magnesium (or sodium) salt (salt), substance P, taltirelin hydrate, teduglutide, teicoplanin, teravancin, teriparatide, terlipressin acetate, tetracosactide, thymalfacin, thymosin α-1, thymopentin, trebananib, triptorelin, triptorelin pamoate, tyroserulotide, uralitide, vancomycin, bapreotide acetate, vasoactive intestinal peptide acetate, Vx-001c, TERT572Y, diconotide acetate, α5-α6Bax peptide, and β-defensin.

[0186] In some embodiments, the peptide is any peptide that helps achieve a desired objective using the composition. For example, in some embodiments, the peptide is any peptide that facilitates the treatment of any type of disease and / or disorder.

[0187] In some embodiments, the biomolecular drug is a nucleic acid. Such embodiments include, but are not limited to, RNA, siRNA, microRNA, interfering RNA, mRNA, replicon mRNA, RNA analogs, and DNA, encompassing any type of nucleic acid molecule.

[0188] In some embodiments, nanoparticles coupled with the above composition, which includes an agent (e.g., DAMP / PAMP) and an antigen that, when administered to a target, can stimulate an innate immune response in the target, are used to induce an immune response. In some embodiments, such nanoparticles further cooperate (e.g., complex, bind, encapsulate, absorb, adsorb, mix) with an adjuvant (e.g., a dendritic cell target molecule (DC)). In some embodiments, the nanoparticles are administered co-administered with the adjuvant. In some embodiments, the antigen further cooperates (e.g., complex, bind, encapsulate, absorb, adsorb, mix) with the adjuvant. In some embodiments, the antigen does not further cooperate (e.g., complex, bind, encapsulate, absorb, adsorb, mix) with the adjuvant. In some embodiments, the antigen is bound to a hydrophobic molecule. In some embodiments, the adjuvant is bound to a hydrophobic molecule. In some embodiments, the average size of the nanoparticles is in the range of 6 nm to 500 nm.

[0189] In some embodiments, the hydrophobic molecule is a lipid molecule. In some embodiments, the lipid molecule is a film-forming lipid molecule. In some embodiments, the lipid molecule is a non-film-forming lipid molecule.

[0190] Examples of lipid molecules applicable to embodiments of the present invention include, but are not limited to, the following: lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmi Phospholipids such as toyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dieryloylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl group in these lipids is C 10 ~C 24 It is preferable that the acyl group is derived from a fatty acid having a carbon chain (for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl).

[0191] Other non-limiting examples of lipid molecules include sterols (e.g., cholesterol, and derivatives of cholesterol such as cholestanol, cholestanone, cholestane, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof).

[0192] Other examples of lipid molecules suitable for use in the present invention include, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkylaryl sulfate polyethyl oxylate fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and non-phosphorus-containing lipids such as sphingomyelin.

[0193] Other examples of lipid molecules suitable for use in the present invention include fatty acids and their derivatives or analogues. These include oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monoleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, and their C 1-10This includes alkyl esters (e.g., methyl, isopropyl, and t-butyl), as well as their monoglycerides 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. Pharmacol., 1992, 44, 651-654).

[0194] Other examples of lipid molecules suitable for use in the present invention include PEG-modified lipid molecules (PEG lipids). Examples of PEG lipids include, but are not limited to, PEG-DAA (PEG-DAA) bonded to dialkyloxypropyl, as described in PCT Publication WO05 / 026372, PEG-DAG (PEG-DAG) bonded to diacylglycerol, as described in U.S. Patent Publications 20030077829 and 2005008689, PEG bonded to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG bonded to ceramide, as described in U.S. Patent No. 5,885,613, PEG bonded to cholesterol or its derivatives, and mixtures thereof. The disclosures in these patent documents are incorporated herein by reference in their entirety for all purposes. Additional PEG lipids include, but are not limited to, PEG-C-DOMG, 2KPEG-DMG, and mixtures thereof.

[0195] 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, PEG2000 has an average molecular weight of approximately 2,000 daltons, and PEG5000 has an average molecular weight of approximately 5,000 daltons. PEG is commercially available from Sigma Chemical Co. and other companies and includes, for example, monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol succinic acid (MePEG-S), monomethoxypolyethylene glycol succinimidyl succinic acid (MePEG-S-NHS), monomethoxypolyethylene glycol amine (MePEG-NH2), monomethoxypolyethylene glycol torecylate (MePEG-TRES), and monomethoxypolyethylene glycol imidazolyl carbonyl (MePEG-IM). Other PEGs, such as those described in U.S. Patents 6,774,180 and 7,053,150 (e.g., mPEG(20kDa)amine), are also useful for preparing the PEG-lipid complexes of the present invention. The disclosures of these patents are incorporated herein by reference in their entirety for all purposes. Furthermore, monomethoxypolyethylene glycol acetate (MePEG-CH2COOH) is particularly effective for preparing PEG-lipid complexes, including, for example, PEG-DAA complexes.

[0196] The PEG portion of the PEG-lipid complex described in this specification may have an average molecular weight in the range of about 550 daltons to about 10,000 daltons. In certain examples, 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 preferred embodiments, the PEG portion has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0197] In certain examples, PEG can optionally be substituted with alkyl groups, alkoxy groups, acyl groups, or aryl groups. PEG can be directly bonded to lipids. Alternatively, PEG may be bonded to lipids via a linker moiety. Any linker moiety suitable for coupling PEG to lipids (including, for example, non-ester-containing and ester-containing linker moieties) can be used. In preferred embodiments, the linker moiety is a non-ester-containing linker moiety. As used herein, the term "non-ester-containing linker moiety" refers to a linker moiety that does not contain a carboxylic acid ester bond (-OC(O)-). Suitable non-ester-containing linker moieties include, but are not limited to, amides (-C(O)NH-), aminos (-NR-), carbonyls (-C(O)-), carbamates (-NHC(O)O-), ureas (-NHC(O)NH-), disulfides (-SS-), ethers (-O-), succinyls (-(O)CCH2CH2C(O)-), succinamidyls (-NHC(O)CH2CH2C(O)NH-), ethers, disulfides, and combinations thereof (such as linkers containing both carbamate and amide linker moieties). In a preferred embodiment, a carbamate linker is used to bind PEG to a lipid.

[0198] In other embodiments, an ester-containing linker moiety is used to bind PEG to lipids. Suitable ester-containing linker moieties include, for example, carbonates (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonic acid esters, and combinations thereof.

[0199] Lipid complexes can be formed by attaching phosphatidylethanolamines having various acyl chain groups with different chain lengths and saturations to PEG. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art.

[0200] C 10 ~C20 Phosphatidylethanolamine containing saturated or unsaturated fatty acids having a carbon chain length within the range is preferred. Phosphatidylethanolamine having a monounsaturated fatty acid or a disaturated fatty acid, as well as a mixture 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).

[0201] Such embodiments are not limited to specific antigens. In fact, the antigen can be a peptide, protein, polysaccharide, saccharide, 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 or leukemia cells, and can be the whole cell or its immunogenic components, such as cell wall components or its molecular components.

[0202] In some embodiments, the antigen is known in the art and is available from commercial, government, and scientific sources. In some embodiments, the antigen is a completely inactivated or attenuated organism. These organisms can be infectious organisms such as viruses, parasites, and bacteria. These organisms can be tumor cells. The antigen can be a purified or partially purified polypeptide derived from a tumor or virus or bacterial source. Criteria for identifying and selecting effective antigen peptides (e.g., the minimal peptide sequence capable of inducing an immune response) can be found in the art. The antigen can be a recombinant polypeptide produced by expressing DNA encoding the polypeptide antigen in a heterologous expression system. The antigen can be DNA encoding all or part of the antigen protein. The DNA can be in the form of vector DNA such as plasmid DNA.

[0203] Antigens may be supplied as a single antigen or as a combination of antigens. Antigens may be supplied as a complex mixture of polypeptides or nucleic acids.

[0204] In some embodiments, the antigen is an autoantigen. As used herein, the term “autoantigen” refers to an immunogenic antigen or epitope that is unique to mammals and may be involved in the pathogenesis of autoimmune diseases.

[0205] In some embodiments, the antigen is a viral antigen. Viral antigens can be isolated from any virus, including but not limited to viruses from the following virus families: Arenaviridae, Alterivirus, Astroviridae, Baculoviridae, Badnavirus, Varnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capylovirus, Karlavirus, Kalimovirus, Circoviridae, Crosterovirus, Comoviridae, Coronavirusidae (e.g., severe acute respiratory syndrome) Coronaviruses (such as SARS virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strains)), Flaviviridae (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, and Dengue virus 4), Hepadnaviridae, Herpesviridae (e.g., human herpesvirus) Viruses (1, 3, 4, 5, and 6, as well as cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Liposrixviridae, Microviridae, Orthomyxoviridae (e.g., influenza A, influenza B, and influenza C), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvovirus Family Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aftovirus), Poxviridae (e.g., vaccinia and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentiviruses such as human immunodeficiency virus (HIV)-1 and HIV-2), Rhabdoviridae (e.g., rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (e.g., rubella virus, dengue virus, etc.), and Totiviridae.Suitable viral antigens also include all or part of dengue protein M, dengue protein E, dengue D1NS1, dengue D1NS2, and dengue D1NS3.

[0206] Viral antigens may originate from specific strains of papillomavirus, herpesviruses, namely herpes simplex 1 and herpes simplex 2; hepatitis viruses such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis delta virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV); tick-borne encephalitis viruses; parainfluenza, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choriomyelitis.

[0207] In some embodiments, the antigen is a bacterial antigen. Bacterial antigens include Actinomyces, Anabaena, Bacillus, Bacteroides, Budelovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Haemophilus influenzae type B (HIB), Hyphomicrobium, Legionella, Leptospira, Listeria, Meningococcus A, Meningococcus B, and Meningococcus C may originate from any bacterium, including but not limited to the genera Metanovacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oschilatoria, Prochloron, Proteus, Pseudomonas, Rhodospirillum, Rickettsia, Salmonella, Sigella, Spirillum, Spirochete, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, as well as Treponema, Vibrio, and Yersinia.

[0208] In some embodiments, the antigen is a parasitic antigen. Parasitic antigens can be obtained from parasites such as, but are not limited to, antigens derived from Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia rickettsiai, Typholickettsia, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanosoma brussei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni. These include all or part of sporozoite antigens, plasmodian antigens, such as circumsporozoite protein, sporozoite surface proteins, hepatic phase antigens, apical membrane associated proteins, or merozoite surface proteins.

[0209] In some embodiments, the antigens are allergens and environmental antigens, such as, but not limited to, antigens derived from natural allergens, including: pollen allergens (tree pollen allergens, herb pollen allergens, weed pollen allergens, and grass pollen allergens), insect allergens (inhalation allergens, saliva allergens, and poison allergens), animal hair allergens and dander allergens, and food allergens. Important pollen allergens from trees, grasses, and herbs originate from the taxonomic Fagales, Oleales, Pinales, and Planales (including, in particular, birch (genus Betula), alder (genus Alnus), hazel (genus Corylus), hornbeam (genus Carpinus), and olive (genus Olea), ferns (genus Cryptomeria and Juniperus), and plane trees (genus Plana)); Poales (i.e., including grasses of the genera Rhus, Morus alba, Styrax, Orchard grass, Phragmites, Phragmites, Rye, and Sorghum); Asterales and Urticales (including, in particular, herbs of the genera Ragweed, Artemisia, and Lamiaceae). Other allergen antigens that may be used include venomous allergens, such as those derived from dust mites of the genera Dermatophagoides and Euroglyphus, allergens from flour mites (e.g., Lepidoglyphys, Glycyphagus, and Tyrophagus), allergens from cockroaches, flies, and fleas (e.g., Blatella, Periplaneta, Chironomids, and Ctenocepphalides), allergens from mammals such as cats, dogs, and horses, allergens from birds, and allergens from stinging insects such as those derived from the taxonomic order Hymenoptera, including bees (Apidae), wasps (Vespidae, and Formicidae). Other allergen antigens that may be used may include inhaled allergens derived from fungi such as Alternaria and Cladosporium.

[0210] In some embodiments, the antigen is a tumor antigen (as described herein).

[0211] One of the major obstacles to the development of therapeutic and tumor-specific immunotherapies is the identification and selection of highly specific and limited tumor antigens to evade autoimmunity. Tumor neoantigens, which arise as a result of genetic alterations within malignant cells (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.), are the most tumor-specific class of antigens.

[0212] In some embodiments, the antigen is a neoantigen. The term neoantigen is used herein to define any newly expressed antigenic determinant. Neoantigens may arise during structural changes of proteins as newly expressed determinants (particularly on the surface of transformed or infected cells) as a result of complex formation of one or more molecules or as a result of cleavage of molecules, which in turn results in the presentation of a new antigenic determinant. Thus, as used herein, the term neoantigen encompasses antigens expressed during infection (e.g., viral, protozoan, or bacterial infection), prion-borne diseases, and cell transformation (cancer), in the latter case, where neoantigens may be referred to as tumor-associated antigens.

[0213] The present invention is not limited to any particular method for identifying neoantigens. In some embodiments, neoantigen identification includes identifying all or nearly all mutations within the neoplasm / tumor at the DNA level using whole-genome sequencing of the tumor, whole-exome sequencing (e.g., only captured exons), or RNA sequencing of compatible germ cell samples derived from each patient. In some embodiments, neoantigen identification includes analyzing the identified mutations with one or more peptide-MHC binding prediction algorithms to generate multiple candidate neoantigen T cell epitopes that can be expressed within the neoplasm / tumor and bind to the patient's HLA allele. In some embodiments, neoantigen identification includes synthesizing multiple candidate neoantigen peptides selected from all sets of neoopen reading frame peptides and predictive binding peptides used in cancer vaccines.

[0214] Thus, the present invention is at least in part based 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 neoplasms / tumors. In particular, these mutations are present in the genome of the neoplasm / tumor cells of interest but not in the normal tissues from which the subject originates. Such mutations are of special interest if they result in changes that produce proteins with altered amino acid sequences specific to the patient's neoplasm / tumor (e.g., neoantigen). Useful mutations may include, for example, (1) non-synonymous mutations resulting in different amino acids in a protein; (2) read-through mutations in which a stop codon is modified or deleted, resulting in the translation of a longer protein with a novel tumor-specific sequence at the C-terminus; (3) splice site mutations that include an intron in mature mRNA, thereby resulting in a unique tumor-specific protein sequence; (4) chromosomal rearrangements (i.e., gene fusions) that produce a chimeric protein with a tumor-specific sequence at the junction of two proteins; and (5) frameshift mutations or deletions that result in a new open reading frame with a novel tumor-specific protein sequence. For example, peptides containing mutations or mutant polypeptides resulting from splice sites, frameshifts, readthroughs, or gene fusion mutations in tumor cells can be identified by sequencing tumor DNA, RNA, or proteins relative to normal cells.

[0215] Personalized neoantigen peptides derived from common tumor driver genes are within the scope of the present invention and may further include previously identified tumor-specific mutations.

[0216] Preferably, any suitable sequencing-by-synthesis platform is available for mutation identification. Four major sequencing-by-synthesis platforms are currently available: Genome Sequencers from Roche / 454 Life Sciences, HiSeq Analyzer from Illumina / Solexa, SOLiD systems from Applied BioSystems, and Heliscope systems from Helicos Biosciences. Sequencing-by-synthesis platforms are also described by Pacific Biosciences and VisiGen Biotechnologies. Each of these platforms can be used in the methods of the present invention. In some embodiments, multiple nucleic acid molecules to be sequenced are bound to a support (e.g., a solid support). To immobilize the nucleic acid on the support, a capture sequence / universal priming site can be added to the 3' and / or 5' ends of the template. The nucleic acid may be bound to the support by hybridizing the capture sequence to a complementary sequence covalently bound to the support. A capture sequence (also called a universal capture sequence) is a nucleic acid sequence that is complementary to a support-bound sequence and can serve dually as a universal primer.

[0217] Instead of a capture sequence, elements of a binding pair (e.g., an antibody / antigen, receptor / ligand, or avidin-biotin pair, as described in U.S. Patent Application No. 2006 / 0252077) may be ligated to each fragment to be captured on a surface coated with the second element of the binding pair. Following capture, the sequence may be analyzed by, for example, single-molecule detection / sequencing, including template-dependent synthesis sequencing, as described in the Examples and U.S. Patent No. 7,283,337. In the synthesis-time sequencing method, the surface-bound molecule is exposed to multiple labeled nucleotide triphosphates in the presence of polymerase. The template sequence is determined by the order of the labeled nucleotides incorporated into the 3' end of the growing chain. This can be done in real-time or step-and-repeat mode. For real-time analysis, different optical labels may be incorporated for each nucleotide, and multiple lasers may be used to stimulate the incorporated nucleotides.

[0218] Any cell type or tissue may be used to obtain nucleic acid samples for use in the sequencing methods described herein. In some embodiments, DNA or RNA samples are obtained from neoplasms / tumors or bodily fluids such as blood or saliva obtained by known techniques (e.g., venipuncture). Alternatively, nucleic acid testing may be performed on dry samples (e.g., hair or skin).

[0219] Various methods are available to detect the presence of specific mutations or alleles in an individual's DNA or RNA. Advances in this field have provided accurate, easy, and inexpensive large-scale SNP genotyping. More recently, several new techniques have been described, including, for example, dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, and various DNA "chip" technologies such as the TaqMan system and Affymetrix SNP chip. These methods typically require amplification of the target gene region by PCR. Yet another newly developed method, based on invasive cleavage followed by mass spectrometry, or the generation of small signal molecules by immobilized padlock probes and rolling circle amplification, may ultimately eliminate the need for PCR. Some of the methods known in the art for detecting specific single nucleotide polymorphisms are summarized below. The methods of the present invention are understood to include all available methods.

[0220] PCR-based detection methods may include the simultaneous multiple amplification of several markers. For example, it is well known in the art to select PCR primers to produce PCR products that do not overlap in size and can be analyzed simultaneously.

[0221] Alternatively, different markers can be amplified using primers that are labeled differently and therefore can be detected differently from each other. Naturally, hybridization-based detection methods enable the detection of differences between multiple PCR products in a sample. Other techniques that enable multiple analysis of multiple markers are known in the art.

[0222] Several methods have been developed to facilitate the analysis of single nucleotide polymorphisms (SNPs) in genomic DNA or cellular RNA. In one embodiment, SNPs can be detected by using specialized exonuclease-resistant nucleotides, such as those disclosed in U.S. Patent No. 4,656,127. This method allows a primer complementary to the allele sequence immediately 3' of the polymorphic site to be hybridized to a target molecule obtained from a specific animal or human. If the polymorphic site on the target molecule contains a nucleotide complementary to a specific exonuclease-resistant nucleotide derivative, that derivative is incorporated into the end of the hybridized primer. Such incorporation confers resistance to the exonuclease to the primer, thereby enabling its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, the finding that the primer is resistant to the exonuclease reveals that the nucleotide present in the polymorphic site of the target molecule is complementary to the nucleotide of the nucleotide derivative used in the reaction. This method has the advantage of not requiring the determination of a large amount of irrelevant sequence data.

[0223] In another embodiment of the present invention, a solution-based method is used to determine the identity of nucleotides at a polymorphic site (see, for example, French Patent No. 2,650,840; PCT Application No. WO1991 / 02087). A primer complementary to the allele sequence immediately 3' of the polymorphic site may be used, as in the method of U.S. Patent No. 4,656,127. This method determines the identity of nucleotides at the polymorphic site using a labeled dideoxynucleotide derivative, which, if complementary to the nucleotides at the polymorphic site, will be incorporated into the terminus of the primer.

[0224] An alternative method known as Genetic Bit Analysis (GBA®), described in International Publication No. WO1992 / 15712, uses a mixture of a labeled terminator and a primer complementary to the 3' sequence of the polymorphic site. Thus, the incorporated labeled terminator is determined by the nucleotide present in the polymorphic site of the target molecule being evaluated and is complementary to that nucleotide. In contrast to the method of Cohen et al. (French Patent No. 2,650,840; International Patent Application No. WO1991 / 02087), the GBA® method is preferably a heterophase assay in which the primer or target molecule is immobilized on a solid phase. Recently, several primer-induced nucleotide incorporation procedures for assaying DNA polymorphism sites have been described (e.g., Komher, J. Set al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, BP, Nucl. Acids Res. 18:3671 (1990); Syvanen, A.-C, et al., Genomics 8:684-692 (1990); Kuppuswamy, M. Net al., Proc. Natl. Acad. Sci. (USA) 88:1143-1147 (1991); Prezant, T. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. et al. See al., Analyze Biochem. 208:171-175 (1993). These methods differ from GBA® in that they all rely on the incorporation of labeled deoxynucleotides to distinguish between the bases of the polymorphic sites. In this form, the signal is proportional to the number of incorporation of deoxynucleotides, so polymorphisms occurring in the same nucleotide run can yield a signal proportional to the length of the run (see, for example, Syvanen, A.-C, et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

[0225] An alternative method for identifying tumor-specific neoantigens is direct protein sequencing. Protein sequencing of enzymatic digests using multidimensional MS techniques (MSn), including tandem mass spectrometry (MS / MS), can also be used to identify the neoantigens of the present invention. Such proteomic approaches enable rapid and highly automated analysis (see, e.g., K. Gevaert and J. Vandekerckhove, Electrophoresis 21:1145-1154 (2000)). It is further conceivable 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, which falls within the scope of the present invention. For example, meta-shotgun protein sequencing can be used to identify expressed neoantigens (see, for example, Guthals et al. (2012) Shotgun Protein Sequencing with Meta-contig Assembly, Molecular and Cellular Proteomics 11(10):1084-96).

[0226] Tumor-specific neoantigens are sometimes identified using MHC multimers to identify neoantigen-specific T cell responses. For example, high-throughput analysis of neoantigen-specific T-cell responses in patient samples may be performed using MHC tetramer-based screening techniques (see, for example, Hombrink et al. (2011) High-Throughput Identification of Potential Minor Histocompatibility Antigens by MHC Tetramer-Based Screening: Feasibility and Limitations 6(8):1-11; Hadrup et al. (2009) Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers, Nature Methods, 6(7):520-26; van Rooij et al. (2013) Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an Ipilimumab-responsive melanoma, Journal of Clinical Oncology, 31:1-4; and Heemskerk et al. (2013) The cancer antigenome, EMBO Journal, 32(2):194-203). It is considered to be within the scope of the present invention that such tetramer-based screening techniques may be used for the initial identification of tumor-specific neoantigens, or as a secondary screening protocol to determine which neoantigens a patient may have already been exposed to, thereby facilitating the selection of candidate neoantigens for the vaccine of the present invention.

[0227] The present invention further comprises isolated peptides (e.g., neoantigenic peptides containing tumor-specific mutations identified by the described method, peptides containing known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the described method). These peptides and polypeptides are referred to herein as “neoantigenic peptides” or “neoantigenic polypeptides.” The polypeptides or peptides may be of varying lengths and will contain a small region expected to bind to a patient’s HLA molecule (“epitope”), as well as a minimum of further adjacent amino acids extending at both the N-terminus and C-terminus. The polypeptides or peptides may be in either a neutral (uncharged) form or a salt form, and may be without modifications such as glycosylation, side-chain oxidation, or phosphorylation, or may contain such modifications, subject to the condition that the modifications do not impair the biological activity of the polypeptide as described herein.

[0228] In certain embodiments, the size of at least one neoantigenic peptide molecule may include, but is not limited to, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino molecular residues, and any derivable range thereof. In specific embodiments, the neoantigenic peptide molecule has 50 or fewer amino acids. In preferred embodiments, the neoantigenic peptide molecule is equal to about 20 to about 30 amino acids.

[0229] Thus, the present invention provides nanoparticles that work in conjunction with a composition comprising a drug (e.g., DAMP / PAMP) that, when administered to a target, can stimulate an innate immune response in the target, and one or more novel antigenic peptides. In some embodiments, the nanoparticles work in conjunction with two neoantigenic peptides. In some embodiments, the nanoparticles work in conjunction with at least five or more neoantigenic peptides. In some embodiments, the nanoparticles work in conjunction with at least about six, about eight, about ten, about twelve, about fourteen, about sixteen, about eighteen, or about twenty different peptides. In some embodiments, the nanoparticles work in conjunction with at least twenty or more different peptides.

[0230] Neoantigenic peptides, polypeptides, and analogues can be further modified to contain additional chemical portions that are not normally part of a protein. These derivatized portions can improve solubility, biological half-life, protein absorption, or binding affinity. These portions can also reduce or eliminate any desired side effects, such as those associated with proteins. An overview of these portions can be found in Remington's Pharmaceutical Sciences, 2020. thThis can be found in ed., Mack Publishing Co., Easton, PA (2000). For example, neoantigenic peptides and polypeptides having desired activity may be modified as needed to provide certain desired attributes, such as improved pharmacological properties, while substantially increasing, or at least retaining, the biological activity of the unmodified peptide in which it binds to the desired MHC molecule and activates the appropriate T cells. For example, neoantigenic peptides and polypeptides may undergo various modifications, such as either conservative or non-conservative substitutions, if the modification can provide certain advantages in use, such as improved MHC binding. Such conservative substitutions may include replacing one amino acid residue with another amino acid residue that is biologically and / or chemically similar (e.g., one hydrophobic residue with another hydrophobic residue, or one polar residue with another polar residue). The effect of single amino acid substitutions may also be investigated using D-amino acids. Such modifications can be carried out using well-known peptide synthesis procedures, for example, as described in Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (NY, Academic Press), pp.1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, III., Pierce), 2d Ed. (1984).

[0231] In some embodiments, neoantigenic peptides and polypeptides may be modified with a binder to facilitate interaction with nanoparticles (e.g., sHDL nanoparticles). The present invention is not limited to a particular type or variety of binder. In some embodiments, the binder is a cysteine-serine-serine (CSS) molecule.

[0232] In some embodiments where the nanoparticles are sHDL and the neoantigenic peptide or polypeptide is modified with CSS, the sHDL is further modified with dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), and upon mixing, DOPE-PDP and CSS are involved, thereby resulting in the complexation (linking) of CSS-Ag with sHDL.

[0233] Neoantigenic peptides and polypeptides can be modified by elongating or delimited (e.g., adding or deleting amino acids) the amino acid sequence of the compound. Neoantigenic peptides, polypeptides, or analogs can also be modified by altering the order or composition of specific residues. It will be understood by those skilled in the art that certain amino acid residues essential for biological activity (e.g., residues at critical contact sites or conserved residues) generally cannot be altered without adversely affecting biological activity. Non-essential amino acids do not have to be limited to naturally occurring amino acids in proteins, such as La-amino acids or their D-isomers, but may also include unnatural amino acids such as β-γ-δ-amino acids, as well as many derivatives of La-amino acids.

[0234] Typically, neoantigen polypeptides or peptides may be optimized by using a series of peptides with single amino acid substitutions to determine the effects of static 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 may be performed along the length of the peptide to reveal different patterns of sensitivity to various MHC molecules and T cell receptors. In addition, multiple substitutions using small, relatively neutral moieties such as Ala, Gly, and Pro, or multiple substitutions using similar residues, may be used. The substitutions may be homooligomeric or heterooligomeric. The number and type of residues substituted or added depend on the required spacing between essential contact points and the specific functional attributes desired (e.g., hydrophobic vs. hydrophilic). An increase in binding affinity to MHC molecules or T cell receptors compared to the affinity of the parent peptide can be achieved by such substitutions. In any case, such substitutions may use amino acid residues or other molecular fragments selected to avoid steric hindrance and charge interference that could disrupt the binding, for example. Amino acid substitutions are usually single-residue substitutions. Substitutions, deletions, insertions, or any combination thereof may be used to arrive at the final peptide.

[0235] Those skilled in the art will understand that there are various methods for producing such tumor-specific neoantigens. Generally, such tumor-specific neoantigens can be produced either in vitro or in vivo. Tumor-specific neoantigens can be produced in vitro as peptides or polypeptides, which can then be formulated into personalized neoplasm vaccines and administered to subjects. Such in vitro production can be achieved by various methods known to those skilled in the art, such as peptide synthesis or the expression of peptides / polypeptides derived from DNA or RNA molecules in any of various bacterial, eukaryotic, or viral recombinant expression systems, followed by the purification of the expressed peptides / polypeptides.

[0236] Alternatively, tumor-specific neoantigens may be produced in vivo by introducing a molecule encoding the tumor-specific neoantigen (e.g., DNA, RNA, viral expression system), in which case the encoded tumor-specific neoantigen is expressed.

[0237] Proteins or peptides can be prepared by any technique known to those skilled in the art, including the expression of proteins, polypeptides, or peptides by standard molecular biological techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides. Nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have been disclosed and can be found in computer databases known to those skilled in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information, located on the website of the National Institutes of Health. The coding regions of known genes can be obtained by amplification and / or expression using techniques disclosed herein or known to those skilled in the art. Alternatively, various commercially available preparations of proteins, polypeptides, and peptides are known to those skilled in the art.

[0238] Peptides can be readily synthesized chemically using reagents that do not contaminate bacterial or animal matter (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-54, 1963).

[0239] Further aspects of the present invention provide nucleic acids (e.g., polynucleotides) encoding the neoantigenic peptides of the present invention, which can be used to produce the neoantigenic peptides in vitro. The polynucleotides may be, for example, single-stranded and / or double-stranded DNA, cDNA, PNA, CNA, RNA, or polynucleotides in their native or stabilized forms (e.g., polynucleotides having a phosphorothioate backbone), or combinations thereof. The polynucleotides may contain or not contain introns, as long as they encode the peptide. Further aspects of the present invention provide expression vectors capable of expressing polypeptides according to the present invention. Expression vectors for different cell types are well known in the art and can be selected without excessive experimentation. Generally, DNA is inserted into an expression vector, such as a plasmid, in the appropriate orientation and correct reading frame for expression. If necessary, the DNA may be ligated to appropriate transcriptional and translational regulatory control control nucleotide sequences recognized by the desired host (e.g., bacteria), but such control is generally obtained in the expression vector. Next, the vector is introduced into the host bacterium for cloning using standard techniques (see, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0240] The present invention further encompasses mutants and equivalents that are substantially homologous to the identified tumor-specific neoantigens described herein. These may include, for example, conservative substitution mutations (i.e., the substitution of one or more amino acids with similar amino acids). For example, a conservative substitution refers to the substitution of one amino acid with another amino acid within the same general class, such as substituting one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. The purpose of conservative amino acid substitution is well known in the art.

[0241] The present invention also includes an expression vector comprising an isolated polynucleotide, and a host cell containing the expression vector. It is also considered within the scope of the present invention that the neoantigenic peptide may be provided in the form of an RNA or cDNA molecule encoding the desired neoantigenic peptide. The present invention also specifies that one or more neoantigenic peptides of the present invention may be encoded by a single expression vector. The present invention also presents that one or more neoantigenic peptides of the present invention may be encoded and expressed in vivo using a virus-based system (e.g., an adenovirus system).

[0242] The term "polynucleotide encoding a polypeptide" encompasses polynucleotides containing only the coding sequence of a polypeptide, as well as polynucleotides containing further coding and / or non-coding sequences. The polynucleotides of the present invention may take 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 a coding strand or a non-coding (antisense) strand.

[0243] In some embodiments, the polynucleotide may include, for example, a coding sequence for a tumor-specific neoantigenic peptide fused in the same leading frame as the polynucleotide to assist in the expression and / or secretion of the polypeptide from the host cell (e.g., a leader sequence that functions as a secretion sequence to control the transport of the polypeptide out of the cell). The polypeptide having the leader sequence is a preprotein and may have the leader sequence that is cleaved by the host cell to form the mature form of the polypeptide.

[0244] In some embodiments, the polynucleotide may include, for example, a coding sequence for a tumor-specific neoantigenic peptide fused in the same reading frame as a marker sequence that enables the purification of the encoded polypeptide, which may later be incorporated into a personalized neoplasm vaccine. For example, the marker sequence may be a hexahistidine tag supplied by a pQE-9 vector to provide purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence may be a hemagglutinin (HA) tag derived from influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. Further tags include, but are not limited to, calmodulin tags, FLAG tags, Myc tags, S tags, SBP tags, Softag1, Softag3, 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 some embodiments, the polynucleotide may contain coding sequences for one or more tumor-specific neoantigenic peptides fused to the same reading frame in order to create a single concatemerized neoantigenic peptide construct capable of producing multiple neoantigenic peptides.

[0245] In some embodiments, the present invention provides an isolated nucleic acid molecule having a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 96%, 97%, 98%, or 99% identical to a polynucleotide encoding the tumor-specific neoantigenic peptide of the present invention.

[0246] 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 that of the reference sequence, except that the polynucleotide sequence may contain up to five point mutations for every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to that of the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with other nucleotides, or 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-terminus or carboxyl-terminus of the reference nucleotide sequence, or somewhere between those terminal positions, and may be scattered separately among the nucleotides in the reference sequence or in one or more adjacent groups within the reference sequence.

[0247] In practice, whether any given nucleic acid molecule is at least 80%, at least 85%, at least 90% identical to a reference sequence, and in some embodiments at least 95%, 96%, 97%, 98%, or 99% identical, can be determined conventionally using known computer programs, such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are set such that the percentage of identity is calculated over the entire full-length reference nucleotide sequence, and a homology gap of up to 5% of the total number of nucleotides in the reference sequence is permitted.

[0248] The isolated tumor-specific neoantigenic peptides described herein can be produced in vitro (e.g., in the laboratory) by any preferred method known in the art. Such methods range from direct protein synthesis to constructing DNA sequences encoding isolated polypeptide sequences and expressing those sequences in a suitable transforming host. In some embodiments, recombinant techniques are used to construct the DNA sequences by isolating or synthesizing the DNA sequences encoding the wild-type protein of interest. Optionally, these sequences can be mutagenicated by site-directed mutagenesis to provide functional analogs. See, for example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066 (1984) and U.S. Patents 4,588,585.

[0249] In embodiments, the DNA sequence encoding the target polypeptide is constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and by selecting preferred codons in the host cell from which the recombinant polypeptide of the target is to be produced. Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding the target isolated polypeptide. For example, a complete amino acid sequence can be used to construct a back-translated gene. Furthermore, it is possible to synthesize DNA oligomers containing nucleotide sequences encoding a specific isolated polypeptide. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain a 5' or 3' overhang for complementary assembly.

[0250] Once assembled (e.g., by synthesis, site-directed mutagenesis, or other means), the polynucleotide sequence encoding the specific isolated polypeptide of interest is inserted into an expression vector and optionally operably ligated to an expression regulatory sequence suitable for protein expression in the desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of the biologically active polypeptide in a suitable host. As is well known in the art, genes can be operably ligated to functional transcriptional and translational expression regulatory sequences in a selected expression host in order to obtain high expression levels of the transferred gene in the host. Recombinant expression vectors can be used to amplify and express DNA encoding tumor-specific neoantigenic peptides. A recombinant expression vector is a replicable DNA construct having a synthetic or cDNA-derived DNA fragment encoding a tumor-specific neoantigenic peptide or bioequivalent analog operably ligated to a suitable transcriptional or translational regulatory element derived from a mammalian, microorganism, virus, or insect gene. A transcription unit generally comprises an assembly of (1) a gene element or an element having a regulatory role in gene expression (e.g., a transcription promoter or enhancer), (2) a structure or coding sequence that is transcribed into mRNA and translated into a protein, and (3) appropriate transcription start and termination sequences and translation start and termination sequences, as detailed below. Such regulatory elements may include operator sequences that control transcription. Further incorporation may be made of select genes that facilitate the ability to replicate in the host, typically given by the origin of replication, and that facilitate the recognition of the transformant. DNA regions are operably ligated if they are functionally related to each other. For example, the DNA of a signal peptide (secretion leader) is operably ligated to the DNA of a polypeptide if it is expressed as a precursor involved in the secretion of the polypeptide. Promoters are operably ligated to coding sequences if they control the transcription of the sequence. Alternatively, ribosome binding sites are operably ligated to coding sequences if they are positioned to enable translation.Generally, being operable-linked means being adjacent, and in the case of a secretion leader, it means being adjacent and within the reading frame. Structural elements intended for use in yeast expression systems include a leader sequence that enables the extracellular secretion of proteins translated by the host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, the recombinant protein may contain an N-terminal methionine residue. This residue can then be optionally cleaved from the expressed recombinant protein to provide the final product.

[0251] The selection of expression regulatory sequences and expression vectors will depend on the host selection. A wide variety of expression host / vector combinations can be used. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids such as Escherichia coli plasmids containing pCR1, pBR322, pMB9, and their derivatives, as well as broad-host-range plasmids such as M13 and filamentous single-stranded DNA phages.

[0252] Suitable host cells for polypeptide expression include prokaryotes, yeasts, insects, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include Gram-negative or Gram-positive bacteria, such as Escherichia coli (E. coli) or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems can also be used. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985).

[0253] Furthermore, various mammalian or insect cell culture systems are advantageously used for the expression of recombinant proteins. Since such proteins are generally correctly folded, appropriately modified, and fully functional, recombinant protein expression can be carried out in mammalian cells. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells described by Gluzman (Cell 23:175, 1981), as well as other cell lines capable of expressing suitable vectors, such as L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Mammalian expression vectors may include non-transcription elements (e.g., origin of replication, suitable promoters and enhancers linked to the gene to be expressed, and other 5' or 3' flanking non-transcription sequences), 5' or 3' untranslated sequences (e.g., required ribosome binding sites, polyadenylation sites, splice donor sites, and acceptor sites), and transcription termination sequences. Baculovirus systems for the production of heterologous proteins in insect cells are outlined by Luckow and Summers, Bio / Technology 6:47 (1988).

[0254] Proteins produced by transformed hosts can be purified according to any preferred method. Such standard methods include chromatography (e.g., ion-exchange chromatography, affinity chromatography, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Affinity tags (e.g., hexahistidine, maltose-binding domains, influenza coat sequences, glutathione-S-transferases, etc.) can be attached to the protein and facilitate purification by passing it through a suitable affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0255] For example, the supernatant from a system secreting recombinant protein into culture medium can be initially concentrated using a commercially available protein concentration filter (e.g., an Amicon or Millipore Pellicon ultrafiltration unit). After the concentration step, the concentrate can be spread onto a suitable purification matrix. Alternatively, an anion exchange resin (e.g., a matrix or substrate having pendant diethylaminoethyl (DEAE) groups) can be used. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, to further purify the cancer stem cell protein-Fc composition, one or more reversed-phase high-performance liquid chromatography (RP-HPLC) steps using hydrophobic RP-HPLC medium (e.g., silica gel having pendant methyl or other aliphatic groups) can be used. Various combinations of some or all of the above purification steps can also be used to provide homogeneous recombinant protein. For example, recombinant proteins produced in bacterial cultures can be isolated by an 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 as the final purification step. Microbial cells used for recombinant protein expression can be destroyed by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysis agents.

[0256] Thus, in certain embodiments, the present invention relates to personalized strategies for treating disorders (e.g., neoplasms), more specifically, tumors, by administering to a subject (e.g., a mammal such as a human) a therapeutically effective amount of a composition (e.g., a vaccine composition capable of eliciting a specific T-cell response) comprising an agent (e.g., DAMP / PAMP) that, when administered to the subject, can stimulate an innate immune response in the subject (as described herein) and one or more neoplasm / tumor-specific neoantigens. In some embodiments, such compositions further work in conjunction with nanoparticles. In fact, in certain embodiments, whole-genome / exome sequencing may be used to identify all or nearly all mutant neoantigens uniquely present in an individual patient's neoplasm / tumor, and this collection of mutant neoantigens may be analyzed to identify a specific optimized subset of neoantigens to be used as a personalized cancer vaccine for the treatment of the patient's neoplasm / tumor. For example, in some embodiments, a population of neoplasm / tumor-specific neoantigens may be identified by sequencing each patient's neoplasm / tumor and normal DNA to identify tumor-specific mutations and determining the patient's HLA allotype. Next, bioinformatics analysis may be performed using validated algorithms to predict which tumor-specific mutations will generate epitopes capable of binding to a patient's HLA allotype, and in particular, which tumor-specific mutations will generate epitopes capable of binding to the patient's HLA allotype more effectively than their congeneral natural antigens, for a population of neoplasm / tumor-specific neoantigens and their related native antigens. Based on this analysis, one or more peptides corresponding to subsets of these mutations may be designed and synthesized for each patient and pooled together for use as a cancer vaccine when immunizing the patient. Neoantigen peptides may be combined with other antineoplastic agents. In some embodiments, such neoantigens are expected to bypass central thymic tolerance (thereby enabling a stronger antitumor T cell response) while reducing the likelihood of autoimmunity (e.g., by avoiding targeting of normal autoantigens).

[0257] The present invention further provides a method for treating and / or alleviating cancer symptoms in a subject by inducing a neoplasm / tumor-specific immune response in the subject, vaccinating the neoplasm / tumor, and administering the neoantigenic peptide or vaccine composition of the present invention to the subject.

[0258] According to the present invention, the cancer vaccine can be used in patients diagnosed with cancer or at risk of developing cancer. In one embodiment, the patient may have solid tumors (e.g., tumors of the breast, ovaries, prostate, lungs, kidneys, stomach, colon, testes, head and neck, pancreas, brain, melanoma, and other tumors of tissue organs) as well as hematological malignancies (e.g., lymphomas and leukemias including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma).

[0259] The peptide or composition of the present invention is administered in an amount sufficient to induce a CTL response. The neoantigenic peptide, polypeptide, or vaccine composition of the present invention can be administered alone or in combination with other therapeutic agents. The therapeutic agents are, for example, chemotherapeutic agents or biological agents, radiation, or immunotherapy. Any suitable therapeutic treatment for a particular cancer may be administered. Examples of chemotherapeutic agents and biological agents include aldesleukin, altretamine, amiphostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin α, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, and iho Examples of chemotherapy drugs that can be combined with anti-CTLA-4 for the treatment of prostate cancer include, but are not limited to, sphamide, interferon-alpha, irinotecan, lansoprazole, revamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol®), pilocarpine, prochloroperazine, rituximab, tamoxifen, Taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate. For the treatment of prostate cancer, a preferred chemotherapy agent that can be combined with anti-CTLA-4 is paclitaxel (Taxol®).

[0260] In addition, subjects may be further administered immunosuppressants or immunostimulants. For example, subjects may be further administered inhibitors of 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. Blocking CTLA-4 or PD-1 / PD-L1 with antibodies can enhance the patient's immune response to cancer cells. In particular, CTLA-4 blockade has been shown to be effective when following a vaccination protocol.

[0261] Those skilled in the art can determine the optimal amount of each peptide to be included in the vaccine composition and the optimal dosing regimen without excessive experimentation. For example, peptides or their variants can be prepared for intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), and intramuscular (im) injection. Preferred methods of peptide injection include sc, id, ip, im, and iv. Preferred methods of DNA injection include id, im, sc, ip, and iv. For example, doses of 1 to 500 mg, 50 μg to 1.5 mg, preferably 10 μg to 500 μg of peptide or DNA may be administered, depending on the peptide or DNA. The use of this dose range has been successful in previous clinical trials (Brunsvig PF, et al., Cancer Immunol Immunother. 2006; 55(12): 1553-1564; M. Staehler, et al., ASCO meeting 2007; Abstract No. 3017). Other methods of administration of the vaccine composition are known to those skilled in the art.

[0262] The vaccine of the present invention may be formulated such 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 may depend on the specific type of cancer, the disease state, the early treatment regimen, the patient's immune status, and, of course, the patient's HLA-haplotype. Furthermore, the vaccine according to the present invention may contain individualized components according to the personal needs of a particular patient. Examples include modifying the amount of peptides according to the expression of relevant neoantigens in a particular patient, personal allergies or undesirable side effects from other treatments, and adjustments for secondary treatments after the first round or scheme of treatment.

[0263] Such vaccines may be administered to individuals who already have cancer. For therapeutic use, such vaccines are administered to the patient in an amount sufficient to induce an effective CTL response to the tumor antigen and to cure or partially cessate the symptoms and / or complications. An amount sufficient to achieve this is defined as a “therapeutic dose.” The dose for this use will depend, for example, on the peptide composition, the mode of administration, the stage and severity of the disease being treated, the patient’s weight and overall health, and the judgment of the prescribing physician. Generally, for a 70 kg patient, the dose for this use is in the range of approximately 1.0 μg to approximately 50,000 μg of peptide for initial immunization (for therapeutic or prophylactic administration), followed by a range of approximately 1.0 μg to approximately 10,000 μg of peptide over several weeks to several months, according to the patient’s response and condition, and, if applicable, by measuring specific CTL activity in the patient’s blood. It should be noted that the peptides and compositions of the present invention may generally be used in severe disease conditions, i.e., life-threatening or potentially life-threatening situations, particularly when cancer has metastasized. In therapeutic use, administration should be initiated as soon as possible after tumor detection or surgical removal. Thereafter, the dose should be increased at least until the symptoms are substantially in remission, and thereafter. Pharmaceutical compositions for therapeutic treatment (e.g., vaccine compositions) are for parenteral administration, topical administration, nasal administration, oral administration, or topical administration. Pharmaceutical compositions are preferably administered parenterally, for example, intravenously, subcutaneously, intradermally, or intramuscularly. The compositions may be administered to the surgical excision site to induce a local immune response to the tumor.

[0264] Such embodiments are not limited to a particular type of adjuvant. Generally, an adjuvant is any substance that, when mixed into a vaccine composition, increases or otherwise modifies the immune response to a variant peptide. The carrier is a scaffold structure, e.g., a polypeptide or polysaccharide, to which an antigenic peptide (e.g., a neoantigenic peptide) can be coupled. Optionally, the adjuvant is covalently or noncovalently bonded to the peptide or polypeptide of the present invention.

[0265] The ability of adjuvants to enhance the immune response to antigens is usually demonstrated by a significant increase in immune-mediated responses or a reduction in disease symptoms. For example, increased humoral immunity is usually demonstrated by a significant increase in the titer of antibodies induced against the antigen, and increased T cell activity usually manifests as increased cell proliferation or cytotoxicity or cytokine secretion. Adjuvants can also alter the immune response, for example, by changing a primarily humoral or Th2 response to a primarily cellular or Th1 response.

[0266] Suitable adjuvants include 1018 ISS, aluminum salt, 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 Examples of adjuvants include, but are not limited to, ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, vector systems, PLG microparticles, reciquimod, SRL172, virosoms and other virus-like particles, YF-17D, VEGF traps, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulon derived from saponins (Aquila Biotech, Worcester, Massachusetts, USA), mycobacterial extracts and synthetic bacterial cell wall mimics, as well as other trademarked or patented adjuvants such as Ribi's Detox. Quil or Superfos. Several dendritic cell-specific immunological adjuvants (e.g., MF59) and their preparations have been previously described (Dupuis M, et al., Cell Immunol. 1998;186(1):18-27; Allison AC; Dev Biol Stand. 1998;92:3-11). Cytokines may also be used. Some cytokines are directly related to influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-α), accelerating the maturation of dendritic cells into antigen-presenting cells effective against T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (in particular U.S. Patent No. 5,849,589, which is incorporated herein by reference in whole), and acting as immunoadjuvants (e.g., IL-12) (Gabrilovich DI, et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418).Toll-like receptors (TLRs) are also sometimes used as adjuvants and are important members of the pattern recognition receptor (PRR) family, which recognize conserved motifs shared by many microorganisms, known as "pathogen-associated molecular patterns" (PAMPS).

[0267] The 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 localized to different cellular compartments, including the plasma membrane, lysosomes, endosomes, and endolysosomes. Different TLRs recognize different PAMPS. For example, TLR4 is activated by LPS contained in bacterial cell walls, TLR9 is activated by CpG DNA of unmethylated bacteria or viruses, and TLR3 is activated by double-stranded RNA. TLR ligand binding results in the activation of one or more intracellular signaling pathways, ultimately leading to the production of many important molecules related to inflammation and immunity (particularly the transcription factor NF-κB and type I interferons). TLR-mediated DC activation leads to increased expression of CCR7, which enables DC activation, phagocytosis, activation, and the upregulation of co-stimulatory markers such as CD80, CD83, and CD86, facilitates DC migration to accretionary lymph nodes, promotes antigen presentation to T cells, and increases the secretion of cytokines such as type I interferons, IL-12, and IL-6. All of these downstream events are important for inducing an adaptive immune response.

[0268] Other receptors that can be targeted include Toll-like receptors (TLRs). TLRs recognize and bind to pathogen-associated molecular patterns (PAMPs). PAMPs target TLRs on the surface of dendritic cells, signaling internally and thereby potentially increasing DC antigen uptake, maturation, and T cell stimuli. PAMPs bound to or co-encapsulated on the particle surface include unmethylated CpG DNA (bacterial), double-stranded RNA (viral), lipopolysaccharides (bacterial), peptidoglycans (bacterial), lipoarabinomannan (bacterial), zymosan (yeast), mycoplasma lipoproteins such as MALP-2 (bacterial), flagellin (bacterial), poly(inosine-cytidylic acid) (bacterial), lipoteichoic acid (bacterial), or imidazoquinoline (synthetic).

[0269] Among the most promising cancer vaccine adjuvants currently in clinical development are the TLR9 agonist CpG and the synthetic double-stranded RNA (dsRNA) TLR3 ligand polyICLC. Preclinical studies have shown that polyICLC appears to be the most potent TLR adjuvant compared to LPS and CpG, due to the absence of pro-inflammatory cytokine induction and IL-10 stimulation in DCs, as well as the maintenance of high levels of co-stimulatory molecules. Furthermore, poly-ICLC was recently directly compared to CpG from non-human primates (rhesus macaques) as an adjuvant for a protein vaccine consisting of human papillomavirus (HPV) 16 capsomeres (Stahl-Hennig C, Eisenblatter M, Jasny E, et al. Synthetic double-stranded RNAs are adjuvants for the induction of T helper 1 and humoral immune responses to human papillomavirus in rhesus macaques. PLoS pathogens. Apr 2009;5(4)).

[0270] In some embodiments, the adjuvant is a dendritic cell target molecule (DC). DCs are potent and involved in initiating antigen-specific immune responses. One biological characteristic of DCs is their ability to sense the conditions under which they encounter an antigen and initiate the process of "DC maturation." Using receptors for various microbial and inflammatory products, DCs respond to antigen exposure in different ways depending on the nature of the pathogen they encounter (virus, bacteria, protozoa). This information is transmitted to T cells by altering the cytokine release pattern upon antigen presentation in lymph nodes, thereby modifying the type of T cell response induced. Therefore, targeting DCs generally offers an opportunity not only to quantitatively enhance antigen delivery and antigen response, but also to qualitatively control the nature of the immune response in accordance with the desired vaccination outcome.

[0271] Dendritic cells express multiple cell surface receptors that can mediate the endocytosis of bound antigens. Targeting exogenous antigens that internalize surface molecules on systemically distributed antigen-presenting cells facilitates antigen uptake, thereby overcoming the major rate-limiting step in immunization and, consequently, vaccination.

[0272] Dendritic cell target molecules include monoclonal or polyclonal antibodies or fragments thereof that recognize and bind to epitopes presented on the surface of dendritic cells. Dendritic cell target molecules also include ligands that bind to cell surface receptors on dendritic cells. One such receptor, the lectin DEC-205, has been used in vitro and in mice to increase both humoral (antibody-based) and cellular (CD8 T cell) responses by two to four orders of magnitude (see, e.g., Hawiger, et al., J.Exp.Med., 194(6):769-79 (2001); Bonifaz, et al., J.Exp.Med., 196(12):1627-38 (2002); Bonifaz, et al., J.Exp.Med., 199(6):815-24 (2004)).

[0273] Various other endocytosis receptors, including mannose-specific lectins (mannose receptors) and IgGFc receptors, have also been targeted in this manner, resulting in similar enhancements to antigen presentation efficiency. Other suitable receptors that may be targeted include, but are not limited to, DC-SIGN, 33D1, SIGLEC-H, DCIR, CD11c, heat shock protein receptors, and scavenger receptors.

[0274] In some embodiments, the adjuvant is CpG. CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in a vaccine environment. While not bound by theory, CpG oligonucleotides act by activating the innate (maladaptive) immune system via Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, viable or dead viruses, dendritic cell vaccines, autologous vaccines, and polysaccharide binding in both prophylactic and therapeutic vaccines. More importantly, it enhances dendritic cell maturation and differentiation, resulting in enhanced Th1 cell activation and the generation of potent cytotoxic T lymphocytes (CTLs), even in the absence of CD4 T cells. The Th1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote Th2 bias. CpG oligonucleotides exhibit even stronger adjuvant activity when formulated or co-administered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar preparations, particularly necessary to induce a strong response when the antigen is relatively weak. They also enhance the immune response, and in several experiments, they were able to reduce the antigen dose by approximately two orders of magnitude with an equivalent antibody response to a total dose vaccine without CpG (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, Jun. 2006, 471-484). U.S. Patent No. 6,406,705B1 describes the combination of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens for inducing an antigen-specific immune response. A commercially available CpG TLR9 antagonist is dSLIM (Double Stem-Loop Immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR-binding molecules, such as RNA-binding TLR7, TLR8, and / or TLR9, may also be used.

[0275] For example, xanthenone derivatives such as Vadimezan (also known as 5,6-dimethylxanthenone-4-acetic acid (DMXAA)) or AsA404 may also be used as adjuvants according to embodiments of the present invention. Alternatively, such derivatives may be administered in parallel with the vaccine of the present invention, for example, by systemic delivery or intratumoral delivery, to stimulate immunity at the tumor site. While not bound by theory, it is thought that such xanthene derivatives act by stimulating interferon (IFN) production via stimulants of the IFN gene (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., polyi:CI2U), DNA or RNA of non-CpG bacteria, as well as immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175. These can act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives useful in connection with the present invention can be readily determined by those skilled in the art without excessive experimentation. Further adjuvants include colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sallamostim).

[0276] Poly-ICLC is a synthetically prepared double-stranded RNA consisting of polyl and polyC chains with an average length of approximately 5000 nucleotides, stabilized against thermal denaturation and hydrolysis by serum nucleases by the addition of polylysine and carboxymethylcellulose. This compound activates the RNA helicase domains of TLR3 and MDA5, both members of the PAMP family, resulting in the activation of DC and natural killer (NK) cells and the production of a "natural mixture" of type I interferons, cytokines, and chemokines. Furthermore, poly-ICLC exerts more direct, broad-spectrum host-targeted anti-infective, and potentially antitumor effects mediated by two IFN-inducible nuclear enzyme systems, 2'5'-OAS and Pl / eIF2a kinase (also known as PKR(4-6)), as well as RIG-I helicase and MDA5.

[0277] Such methods are not limited to generating sHDL nanoparticles in conjunction with a composition comprising an agent (e.g., DAMP / PAMP), an antigen, and an adjuvant (e.g., a dendritic cell target molecule) that, when administered to a target, can stimulate an innate immune response in the target. In some embodiments, the antigen and adjuvant are bound to the outer surface of the sHDL nanoparticles.

[0278] In some embodiments, sHDL nanoparticles are synthesized with thiol-reactive phospholipids that enable reduction-sensitive linkage of antigens and / or adjuvants. In some embodiments, filling of DCs within sHDL nanoparticles is facilitated by cholesterol modification of the DC molecules. In some embodiments, lyophilization is used to prepare homogeneous sHDL. In some embodiments, phospholipids and ApoA-mimicking peptides are dissolved in glacial acetic acid and lyophilized. In some embodiments, antigen peptides are incubated with sHDL in a buffer (e.g., sodium phosphate buffer (pH 7.4)) for 3 hours at room temperature (e.g., to allow conjugation of the antigen peptide). In some embodiments, unbound antigen peptides are removed using a desalting column (MWCO = 7000 Da). In some embodiments, incorporation of cholesterol-modified DCs (Cho-DCs) into sHDL involves incubation with sHDL at room temperature for about 30 minutes.

[0279] Such embodiments are not limited to specific methods for characterizing sHDL bound 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 assay.

[0280] sHDL nanoparticles configured to activate the immune response (e.g., sHDL-αGalCer) (e.g., Ag / DC-sHDL) are useful for activating T cells in a target for prophylactic and therapeutic purposes. Activation of T cells with 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. T cells activated by 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, as well as effector cells such as CTLs. T cells also include regulatory cells such as Th1, Tc1, Th2, Tc2, Th3, Treg, and Tr1 cells. T cells also include NKT cells and similar distinct classes of T cell lineages. In some embodiments, the activated T cells are CD8+ T cells.

[0281] In general, compositions comprising sHDL nanoparticles configured to activate an immune response (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL) are useful for treating subjects with any disease or disorder to which their own immune system initiates an immune response, or who are prone to having such a disease or disorder. Compositions are useful as prophylactic vaccines, which confer resistance to subsequent exposure to infectious agents to subjects. Compositions are also useful as therapeutic vaccines, which can be used to initiate or enhance an immune response in subjects to pre-existing antigens, such as tumor antigens in subjects with cancer or viral antigens in subjects infected with a virus. Compositions are also useful as desensitization vaccines, which function to "immunely tolerate" individuals to environmental antigens such as allergens.

[0282] The ability of these compositions to target professional antigen-presenting cells such as dendritic cells, and their ability to induce a T cell-mediated immune response by causing cross-presentation of antigens, makes these compositions particularly useful for inducing a cell-mediated response to disease-associated antigens in order to attack disease. Therefore, in some embodiments, the type of disease to be treated or prevented is a malignant tumor or chronic infection caused by bacteria, viruses, protozoa, helminths, or other microbial pathogens that invade cells, i.e., are attacked by cytotoxic T lymphocytes.

[0283] The desired outcomes of prophylactic, therapeutic, or desensitized immune responses can vary depending on the disease, according to principles well known in the field. For example, an immune response to an infectious agent may completely prevent colonization and replication of the infectious agent, thereby influencing "germ-free immunity" and the absence of disease symptoms. However, a vaccine against an infectious agent may be considered effective if it reduces the number, severity, or duration of symptoms; reduces the number of individuals in a symptomatic population; or reduces the transmission of the infectious agent. Similarly, immune responses to cancer, allergens, or infectious agents may be one aspect of a comprehensive therapeutic intervention for the disease, whether it is to completely cure the disease, alleviate symptoms, or otherwise address the disease. For example, stimulation of an immune response to cancer may be combined with surgical, chemotherapeutic, radiological, hormonal, and other immunological approaches to influence treatment.

[0284] Subjects possessing or exposed to infectious agents 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). Infectious agents include bacteria, viruses, and parasites. In some cases, subjects can be treated prophylactically, for example, if they may be at risk of developing a disease from an infectious agent. Individuals traveling to or living in areas with specific infectious diseases may be considered at risk and may be candidates for vaccination against specific infectious agents. Prophylactic treatment can be applied to any number of diseases for which there is a known relationship between a particular disease and specific risk factors such as geographical location or work environment.

[0285] Subjects with malignant tumors, or at risk of developing malignant tumors, 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 between cell regeneration and cell death is normally maintained in most organs and tissues. Various types of mature cells in the body have a given lifespan. When these cells die, new cells are produced by the proliferation and differentiation of various types of stem cells. Under normal circumstances, the production of new cells is regulated so that the number of any particular type of cell remains constant. However, occasionally, cells arise that no longer respond to normal growth control mechanisms. These cells grow to considerable size, giving rise to clones of cells capable of producing tumors or neoplasms. Tumors that cannot grow indefinitely and do not invade extensively into healthy surrounding tissue are benign. Tumors that continue to grow, progress, and become invasive are malignant. The term cancer specifically refers to malignant tumors. In addition to uncontrolled growth, malignant tumors exhibit metastasis. In this process, small clusters of cancer cells migrate from the tumor, infiltrate the blood or lymphatic vessels, and are carried to other tissues where they continue to proliferate. In this way, a primary tumor in one site can give rise to a secondary tumor in another site. sHDL nanoparticles configured to activate immune responses such as those disclosed herein (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL) are useful for treating subjects with malignant tumors.

[0286] Malignant tumors that can be treated are classified herein according to the embryonic origin of the tissue from which the tumor originates. Carcinomas are tumors arising from endodermal or ectoderm tissue, such as the inner lining of the epithelium of the skin or viscera and glands. Melanoma is a type of cutaneous carcinoma for which the present invention is particularly useful. Sarcomas, which occur less frequently, originate from mesodermal connective tissue, such as bone, fat, and cartilage. Leukemia and lymphoma are malignant tumors of hematopoietic cells in the bone marrow. Leukemia proliferates as single cells, while lymphoma tends to grow as a tumor mass. Malignant tumors can appear in and establish cancer in multiple organs or tissues of the body.

[0287] Types of cancer that can be treated with sHDL nanoparticles provided configured to activate an immune response (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL) include, but are not limited to, the following: bladder, brain, breast, cervix, colorectal, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, and uterus. Administration is not limited to the treatment of existing tumors or infections, 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., those with a personal or family history of certain types of cancer.

[0288] Subjects with allergens, or subjects at risk of allergen exposure, 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). Such sHDL nanoparticles may be administered to subjects for the purpose of preventing and / or mitigating allergic reactions, such as allergic reactions leading to anaphylaxis. Allergic reactions are caused by the presence of IgE antibodies against the antigen. H It can be characterized by two responses. HStimulation of an immune response and production of IgG antibodies can alleviate allergic diseases. Therefore, sHDL nanoparticles configured to activate immune responses such as those disclosed herein (e.g., sHDL-STING agonist-αGalCer) (e.g., Ag / DC-STING agonist-sHDL) are useful for producing antibodies that prevent and / or mitigate allergic reactions in subjects exposed to allergens.

[0289] Subjects with immunosuppressed conditions, or those at risk of such conditions, can be therapeutically or prophylactically treated 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). The sHDL nanoparticle vaccines disclosed herein can be used to treat conditions characterized by immunosuppression, including but not limited to AIDS or AIDS-related syndromes, idiopathic immunosuppression, drug-induced immunosuppression, other viral or environmentally induced conditions, and certain congenital immunodeficiencies. Such sHDL nanoparticle vaccine compositions can also be used to enhance immune function impaired by the use of immunosuppressants (e.g., certain chemotherapeutic agents) in radiotherapy. Therefore, the sHDL nanoparticle vaccine compositions may be particularly useful when used in conjunction with such drugs or radiotherapy.

[0290] In general, methods for administering vaccines such as those 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 those skilled in the art may be used to administer the formulation to a subject. Administration may be local (i.e., administration to a specific area, physiological system, tissue, organ, or cell type) or systemic. Vaccines may be administered by a number of routes, including, but not limited to, oral, inhalation (nasal or pulmonary), intravenous, intraperitoneal, intramuscular, percutaneous, subcutaneous, topical, sublingual, or transrectal means. Injections may be, for example, intravenous, intradermal, subcutaneous, intramuscular, or intraperitoneal injections. In some embodiments, injections may be administered at multiple locations.

[0291] The administration of the formulation may be achieved by any acceptable method that allows an effective dose of the vaccine to reach the target. The particular mode of administration chosen will depend on factors such as the specific formulation, the severity of the condition of the subject being treated, and the dose required to induce an effective immune response. As commonly used herein, “effective dose” is the amount that can induce an immune response in the subject being treated. The actual effective dose of the vaccine may vary depending on the specific antigen or combination used, the specific composition being formulated, the mode of administration, and the age, weight, condition, and route of administration and disease or disorder of the individual being vaccinated.

[0292] In certain embodiments, glycolipids encapsulated within sHDL nanoparticles are used as stimulants for natural killer T cell-mediated immune responses.

[0293] Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties with both T cells and natural killer cells. Many of these cells recognize antigen-presenting molecules that bind to non-polymorphic CD1d molecules, self- and foreign lipids and glycolipids. NKT cells constitute only about 0.1% of all peripheral blood T cells. NKT cells are a subset of T cells that co-express αβ T cell receptors, but also express various molecular markers normally associated with NK cells, such as NK1.1. The best-known NKT cells differ from conventional αβ T cells in that their T cell receptor diversity is far more limited ("invariant" or "type 1" NKT). They and other CD1d-restricted T cells ("type 2" NKT) recognize lipids and glycolipids presented by CD1d molecules, which are members of the CD1 family of antigen-presenting molecules, rather than the peptide major histocompatibility complex (MHC). + and NK1.1 - Both as well as CD4 + CD4 - CD8 + , and CD8 - Contains cells.

[0294] In certain embodiments, a composition comprising an agent (e.g., DAMP / PAMP) that, when administered to a target, can stimulate an innate immune response in that target, further interacts with one or more therapeutic agents (e.g., complexation, binding, encapsulation, absorption, adsorption, mixing). Such embodiments are not limited to a specific type or category of therapeutic agent.

[0295] In some embodiments, therapeutic agents are configured to treat and / or prevent cancer. Examples of such therapeutic agents include, but are not limited to, chemotherapeutic agents, antitumor agents, anti-angiogenic agents, tumor suppressants, and antibacterial agents.

[0296] In some embodiments, the therapeutic agent is configured to treat and / or prevent autoimmune disorders and / or inflammatory disorders. Examples of such therapeutic agents include disease-modifying antirheumatic drugs (e.g., leflunomide, methotrexate, sulfasalazine, hydroxychloroquine), biological agents (e.g., rituximab, infliximab, etanercept, adalimumab, golimumab), nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, celecoxib, ketoprofen, naproxen, piroxicam, diclofenac), analgesics (e.g., acetaminophen, tramadol), immunomodulators (e.g., anakinra, abatacept), glucocorticoids (e.g., prednisone, methylprednisone), TNF-α inhibitors (e.g., adalimumab, certolizumab pegol, etanercept, golimumab, infliximab), IL-1 inhibitors, and metalloproteinase inhibitors. In some embodiments, the therapeutic agent includes, but is not limited to, infliximab, adalimumab, etanercept, parenteral gold, or oral gold.

[0297] In some embodiments, therapeutic agents are configured to treat and / or prevent cardiovascular disorders (e.g., atherosclerosis, heart failure, arrhythmias, atrial fibrillation, hypertension, coronary artery disease, angina pectoris, etc.). Examples of therapeutic agents known to be useful in treating and / or preventing cardiovascular disorders include angiotensin-converting enzyme (ACE) inhibitors (e.g., benazepril, enalapril, lisinopril, perindopril, ramipril), adenosine, alpha-blockers (alpha-adrenergic antagonists) (e.g., clonidine, guanabenz, labetalol, phenoxybenzamine, terazosin, doxazosin, guanfacine, methyldopa, prazosin), and angiotensin II receptor blockades. Drugs (ARBs) (e.g., candesartan, irbesartan, olmesartan medoxomil, telmisartan, eprosartan, losartan, tasosartan, valsartan), anticoagulants (e.g., heparin, fondaparinux, warfarin, aldepardin, enoxaparin, reviparin, dalteparin, nadroparin, tinzaparin), antiplatelet agents (e.g., absiximab, clopidogrel, eptifivatide, ticlopidine, cilostazol, dipyridamole, sulfinpyrazone, tyrofoam) β-blockers (e.g., acebutolol, betaxolol, carteolol, metoprolol, penbutolol, propranolol, atenolol, bisoprolol, esmolol, nadolol, pindolol, timolol), calcium channel blockers (e.g., amlopidine, felodipine, isradipine, nifedipine, verapamil, diltiazem, nicardipine, nimodipine, nisoldipine), diuretics, aldosterone blockers, loop diuretics (e.g., bume Tanide, furosemide, ethacrine, torasemide), potassium-sparing diuretics, thiazide diuretics (e.g., chlorothiazide, chlorthalidone, hydrochlorothiazide, hydroflumeazide, meticlothiazide, metrazon, polythiazide, quinatazon, trichlormethiazide), cardiac stimulants, bile acid adsorbents (e.g., cholestyramine, coletipol, coleseveram), fibrates (e.g., clofibrate, gemfibrozil, fenofibrate), statins (e.g.,Examples include atorvastatin (atorvastatin, lovastatin, simvastatin, fluvastatin, pravastatin), selective cholesterol absorption inhibitors (e.g., ezetimibe), potassium channel blockers (e.g., amidarone, ibutilide, dofetilide), sodium channel blockers (e.g., disopyramide, mexiletine, procainamide, quinidine, flecainide, moriscidine, propafenone), thrombolytic agents (e.g., alteplase, leteplase, tenecteplase, anistreplase, streptokinase, urokinase), vasoconstrictors, vasodilators (e.g., hydralazine, minoxidil, mecamillamine, isorbide dintrate, isorbide mononitrate, nitroglycerin).

[0298] Generally, the nanoparticles formed in this way are spherical and have a diameter of approximately 5 nm to 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, size exclusion chromatography is performed on the sHDL nanoparticles to obtain a more homogeneous preparation.

[0299] In some embodiments, nanoparticles in conjunction with compositions such as those described herein may further interact (e.g., complex, bind, encapsulate, absorb, adsorb, or mix) with agents useful for locating the administered particles. Agents useful for this purpose include fluorescent tags, radionuclides, and contrast agents.

[0300] Suitable contrast agents include, but are not limited to, the following fluorescent molecules as listed in Molecular Probes (Handbook of fluorescent probes and research products) (e.g., rhodamine, fluorescein, Texas Red, acridine orange, Alexa Fluor (various types), allophycocyanin, 7-aminoactinomycin D, BOBO-1, BODIPY (various types), Calcium, Calcium Crimson, Calcium green, Calcium Orange, 6-carboxyrhodamine 6G, Cascade blue, Cascade yellow, DAPI, DiA, DID, Di1, DiO, DiR, ELF 97, eosin, ER Tracker Blue-White,EthD-1, Ethidium Bromide, Fluo-3, Fluo-4, FM1-43, FM4-64, Fura-2, Fura Red, Hoechst 33258, Hoechst 33342, 7-Hydroxy-4-methylcoumarin, Indo-1, JC-1, JC-9, JOE dye, Lisamin Rhodamine B, Lucifer Yellow CH, LysoSensor Blue DND-167, LysoSensor Green, LysoSensor Yellow / Blu, Lysotracker Green FM, Magnesium Green, Marina Blue, Mitotracker Green FM, Mitotracker Orange CMTMRos, MitoTracker Red CMMXRos, Monobromobimane, NBD amines, NeruoTrace 500 / 525 green, Nile Red, Oregon Green, Pacific Blue, POP-1, propidium iodide, rhodamine 110, rhodamine red, R-phycoerythrin, Resorfin, RH414, Rhod-2, rhodamine green, rhodamine 123, ROX dye, Sodium Green, SYTO blue (various), SYTO green (various), SYTO orange (various), SYTOX blue, SYTOX green, SYTOX orange, tetramethylrhodamine B, TOT-1, TOT-3, X-rhod-1, YOYO-1, YOYO-3. In some embodiments, ceramide is provided as a contrast agent. In some embodiments, an S1P agonist is provided as a contrast agent.

[0301] Furthermore, radionuclides can be used as contrast agents. Suitable radionuclides include, but are not limited to, Fe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(II), as well as indium, gallium, and technetium. Other suitable contrast agents include metal ions commonly used for chelation in paramagnetic T1-type MIR contrast agents, such as divalent and trivalent cations of 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, technetium, rhenium, platinum, thallium, and samarium. Furthermore, metal ions known to be useful in neutron capture radiotherapy include boron and other metals with large nuclear cross-sections. In addition, metal ions useful in ultrasound and X-ray contrast compositions are also suitable.

[0302] Other suitable contrast agents include radiopaque gases or gaseous compounds.

[0303] In some embodiments, nanoparticles coupled with compositions as described herein further cooperate with (e.g., complex, bind, encapsulate, absorb, adsorb, mix with) a targeting agent. In some embodiments, the targeting agent is used to help the nanoparticles coupled with compositions as described herein be delivered to a desired area of ​​the body (e.g., an area of ​​the body suffering from cardiovascular disorders). Examples of targeting agents include, but are not limited to, antibodies, receptor ligands, hormones, vitamins, and antigens, and the present invention is not limited by the properties of the targeting agent. In some embodiments, the antibody is specific to a disease-specific antigen. In some embodiments, the receptor ligand includes, but is not limited to, ligands for CFTR, EGFR, estrogen receptor, FGR2, folate receptor, IL-2 receptor, glycoprotein, and VEGFR. In some embodiments, the receptor ligand is folate.

[0304] In some embodiments, nanoparticles in conjunction with compositions such as those described herein can be delivered to a local site in a patient by a medical device. Medical devices suitable for use in the present invention include known devices for the local delivery of therapeutic drugs. Such devices include, but are not limited to, catheters such as injection catheters, balloon catheters, double balloon catheters, microporous balloon catheters, channel balloon catheters, infusion catheters, and perfusion catheters (which are, for example, coated with a therapeutic drug or through which drugs are administered); needle injection devices such as subcutaneous needles and needle injection catheters; needle-free injection devices such as jet injectors; coated stents, branched stents, artificial blood vessels, stent grafts, etc.; and coated vascular occlusion devices such as wire coils, etc.

[0305] Exemplary devices are U.S. Patent Nos. 5,935,114; 5,908,413; 5,792,105; 5,693,014; 5,674,192; 5,876,445; 5,913,894; 5,868,719; 5,851,228; 5,843,089; and 5,800,519. The following are described in Patent Nos. 5,800,508; 5,800,391; 5,354,308; 5,755,722; 5,733,303; 5,866,561; 5,857,998; 5,843,003; and 5,933,145, the entire contents of which are incorporated herein by reference. Exemplary stents that are commercially available and may be used in this application include RADIUS (SCIMED LIFE SYSTEMS, Inc.), the SYMPHONY (Boston Scientific Corporation), the Wallstent (Schneider Inc.), the PRECEDENT II (Boston Scientific Corporation), and NIR (Medinol Inc.). Such devices are delivered and / or implanted at a target site in the body by known techniques.

[0306] In some embodiments, the present invention also provides a kit comprising a composition such as those described herein. In some embodiments, the kit comprises one or more reagents and tools necessary to produce such a composition, as well as a method for using such a composition.

[0307] Nanoparticles in conjunction with compositions as described herein can be characterized for size and uniformity by any suitable analytical technique. These techniques include atomic force microscopy (AFM), electrospray ionization mass spectrometry, MALDI-TOF mass spectrometry, 13This includes, but is not limited to, 13C 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 are important for ensuring the uniformity of sHDL nanoparticle populations and for manufacturing quality control for final use in in vivo applications.

[0308] In some embodiments, gel permeation chromatography (GPC), which can separate sHDL nanoparticles from liposomes and free ApoA-I mimetic peptides, is used to analyze sHDL-TA nanoparticles. In some embodiments, the size distribution and zeta potential are measured by dynamic light scattering (DLS), for example, using a Malven Nanosizer instrument.

[0309] When clinical use is intended, in some embodiments of the present invention, sHDL nanoparticles are prepared as part of a pharmaceutical composition in a form suitable for the intended use. Generally, this requires the preparation of a composition that is essentially free of pyrogens and other impurities that may be harmful to humans or animals. However, in some embodiments of the present invention, the as-is sHDL nanoparticle formulation may be administered using one or more of the routes described herein.

[0310] In preferred embodiments, nanoparticles in conjunction with compositions such as those described herein are used with appropriate salts and buffers to ensure stable delivery of the composition, enabling uptake by target cells. Buffers are also used when sHDL nanoparticles are introduced into a patient. The aqueous composition comprises an effective amount of sHDL nanoparticles for cells, dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions are also referred to as inoculants. The term “pharmaceutically or pharmacologically acceptable” means molecular entities and compositions that, when administered to animals or humans, do not produce harmful, allergic, or other undesirable reactions. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, etc. Any conventional medium or agent is considered for use in the therapeutic composition unless it is incompatible with the vector or cells of the present invention. Co-active ingredients may also be incorporated into the composition.

[0311] In some embodiments of the present invention, the active composition includes a typical pharmaceutical formulation. Administration of these compositions according to the present invention is via any common route, as long as it can reach the target tissue via that common route. This includes oral, nasal, oral, rectal, vaginal, or topical routes. Alternatively, administration may be by orthotopic injection, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, or intravenous injection.

[0312] Active nanoparticles in conjunction with compositions as described herein may be administered parenterally, intraperitoneally, or intratumorally. Solutions of the active compound as a free base or a pharmacoagulably acceptable salt are prepared in water appropriately mixed with a surfactant such as hydroxypropyl cellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0313] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersions, and the use of surfactants. Prevention of microbial action can be achieved with various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Sustained absorption of the injection composition can be achieved by using absorption-delaying agents (e.g., aluminum monostearate and gelatin) in the composition.

[0314] Sterile injectable solutions are prepared by incorporating the required amount of active nanoparticles in conjunction with compositions such as those described herein, in a suitable solvent and, if necessary, a variety of other components listed above, and then by sterilization by filtration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques resulting from pre-sterilized filtered solutions of the active ingredients and any additional desired components.

[0315] Once formulated, nanoparticles in conjunction with compositions as described herein are administered in a manner compatible with the drug formulation and in a therapeutically effective amount. The formulations are readily administered in various dosage forms, such as injections and drug-release capsules. For parenteral administration in aqueous solutions, for example, the solution is appropriately buffered, and if necessary, the liquid diluent is first isotonicized with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. For example, a single dose may be dissolved in 1 ml of isotonic NaCl solution and then added to 1000 ml of subcutaneous injection solution, or injected into the proposed injection site (see, e.g., “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). In some embodiments of the present invention, the active particles or drug are formulated to constitute about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0, or about 10 milligrams per dose in the therapeutic mixture. Multiple doses may be administered.

[0316] Further 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 for medicinal purposes, for insertion into the rectum, vagina, or urethra. After insertion, suppositories soften, melt, or dissolve in body fluids. Generally, in the case of suppositories, conventional binders and carriers may include, for example, polyalkylene glycols or triglycerides. Such suppositories may be formed from a mixture containing 0.5% to 10%, preferably 1% to 2%, of the active ingredient. Vaginal suppositories or pessaries are usually spherical or oval in shape, each weighing about 5 g. Intravaginal administration is available in various physical forms (e.g., creams, gels, or liquids) that deviate from the typical concept of suppositories. sHDL nanoparticles may also be formulated as inhalants.

[0317] The present invention also encompasses methods comprising co-administering nanoparticles in conjunction with compositions such as those described herein, along with one or more additional activators. In fact, a further aspect of the present invention is providing a method for enhancing prior art therapeutics and / or pharmaceutical compositions by co-administering the sHDL nanoparticles of the present invention. In the co-administration procedure, the agents may be administered simultaneously or sequentially. In some embodiments, the sHDL nanoparticles in conjunction with compositions such as those described herein are administered before other activators. The agent(s) to be co-administered depend on the type of condition being treated.

[0318] This disclosure further provides compositions comprising nanoparticles in conjunction with the compositions described herein, or kits comprising components necessary for synthesizing nanoparticles as described herein. In some embodiments, the kit comprises all necessary, sufficient, or useful components for administering nanoparticles in conjunction with the compositions described herein.

[0319] [Examples] The following embodiments are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the invention, and should not be construed as limiting their scope.

[0320] (Example 1) This example describes the synthesis and characterization of CDN / Zn, CDN / Zn@liposome NPs, and CDN@CaP / PEI-PEG.

[0321] As shown in Figure 1A, CDN-Zn NPs were prepared by a simple coordination assembly. Zn, with its pyramidal coordination geometry, is assumed to be able to coordinate with both adenine and phosphate. To further enhance the stability of the resulting particles, the CDN / Zn nanoparticles were modified with liposomes. There are several different methods for MOF surface modification, including coordination modification during MOF synthesis and post-synthesis modification by ligand exchange and silica or polymer shell coating. DOPA is used during synthesis. 2+Because it is widely used for capping base MOFs, coordination modifications were applied here for the synthesis of CDN / Zn@DOPA with a lipid tail on the surface, which allowed for another lipid layer coating.

[0322] The morphology of the obtained CDN-Zn and CDN / Zn liposome NPs is shown in TEM images (Figure 2). As shown in Figure 2a, the cdAMP-Zn NPs showed a spherical shape with higher TEM contrast on the surface. Rapid nucleation of cdAMP-Zn in methanol is observed in Zn 2+ Although it caused coordination deficiency, the particle surface was Zn 2+ It is thought that the saturated coordination increased surface contrast, resulting in a "core-shell"-like structure. Since nucleation occurs more slowly in water, it was also found that a uniform spherical structure was obtained when synthesized in an aqueous medium (not shown). Consistent with TEM images, DLS and zeta potential data showed that the size of cdAMP-Zn was approximately 150 nm and the surface charge was neutral. As shown in Figure 2b, under the same synthesis conditions, cd-GMP NPs showed a uniform, irregular spherical structure of approximately 100 nm in size and an electrically neutral surface charge. In contrast to cdAMP-Zn and cdGMP-Zn, the morphology and charge of cGAMP-Zn were different (Figure 2c). The spherical nanoparticles consisted of several accumulated small clusters and had a slight positive charge on the surface. To enhance the stability of CDN-Zn NPs, CDN-Zn was modified with liposomes. As shown in Figure 2d, cdAMP-Zn@liposomes are shown as a representative CDN-Zn@liposome structure. As a result, it was found that CDN-Zn liposomes exhibited a more uniform and smaller size due to the DOPA capping effect. Furthermore, their surfaces showed a slight negative charge even after modification with liposome-PEG.

[0323] Regarding CaP / PEI-PEG preparations, experiments were initiated with clinically used adjuvant CaP hydrogels. Generally, CaP hydrogels are Ca 2+ and PO4 3-It was prepared by high-speed mixing with the other, and needle-shaped nanostructures were formed. To increase the loading of CDN onto CaP hydrogels, PEI-PEG was added to CDN to increase its charge attraction, simultaneously increasing colloidal stability (Figure 1B). Unlike conventional CaP hydrogels, which tended to aggregate into a gel, CaP / PEI-PEG dispersed well in water. As shown in Figure 2e, CDN@CaP / PEI-PEG NPs exhibited a uniform needle-like cluster structure with a size of approximately 70 nm and a surface charge of approximately +15 mV. Based on their morphology, size, and surface properties, all formulations presented here have great potential for drug delivery applications.

[0324] (Example 2) This embodiment demonstrates the release profiles and in vitro STING activation of CDN-Zn and CDN@CaP / PEI-PEG.

[0325] As two key parameters of the drug delivery system, experiments further determined the drug loading and release characteristics of CDN nanoformulations. CDN loading efficiency in nanoformulations exceeded 90% for CDN-Zn formulations and over 80% for CDN / CaP-PEI-PEG (Figure 3A). Regarding drug release, cdAMP / Zn and cdGMP / Zn showed remarkably similar release profiles (Figure 3B). During the first 18 hours, release was close to zero-order release, followed by a slightly slower release phase. The zero-order drug release from cdAMP / Zn and cdGMP / Zn is thought to be a result of stable constant dissociation of the framework. However, further research is needed in physiological states with different biomolecular interactions. For cGAMP / Zn NPs, a fast release phase was present during the first 8 hours of incubation, followed by a slower release phase (Figure 3B). The total release of cGAMP / Zn was faster than that of cdAMP / Zn and cdGMP / Zn, which may be related to its unique nanoparticle configuration. For CDN@CAP / PEI-PEG, there was a remarkably rapid drug release phase followed by another sustained release phase (Figure 3B). This profile is thought to be due to the CDN portion adhering to the CAP / PEI-PEG surface via charge interactions and being readily released under high ionic strength and high pH conditions. The release profile for CDN-Zn@liposomes is not shown here because the applicants have not developed a reliable method for quantifying the drug load after liposome coating on CDN-Zn. Liposomes on a CDN-Zn surface are expected to significantly increase particle stability and delay drug release. Sustained-release drugs are useful for increasing the degree of in situ drug exposure and immunostimulation.

[0326] We conducted experiments to determine whether a CDN delivery system can effectively activate the STING pathway in vitro and induce an immune response. This involved THP1-Blue, which contains an IFN regulator (IRF)-inducible SEAP reporter construct. TMWe used ISG (interferon-stimulated gene) cells in our experiments to monitor STING activation by CDN formulations. As shown in Figure 3C, at cdAMP concentrations of 0.25–2 μg / ml, the degree of IFN signaling pathway activation by cdAMP / Zn formulations was significantly higher than that of soluble free cdAMP. Similar improvements in stimulation were observed with CDN@CaP / PEI-PEG formulations compared to the free form (Figure 3D). These in vitro evaluation results indicate that CDN-Zn and CDN@CaP / PEI-PEG possess advantageous properties for in vivo therapeutic applications.

[0327] (Example 3) This example describes the therapeutic effects of CDN-Zn and CDN@CaP / PEI-PEG.

[0328] Finally, the therapeutic effect of CDN preparations on tumor-bearing mice was investigated. cdAMP(ps)2 was used as a representative CDN for the demonstration. Tumor size was ~60 mm. 3 When this was reached, two doses of 25 μg / dose of cdAMP(ps)2 were administered intratumorally on days 10 and 15. To evaluate the antigen-specific immune response, PBMCs were collected for tetramer staining on day 17, and ELISPOT analysis was performed using the AH1 antigen peptide on day 22. As shown in Figure 4A, mean tumor growth in mice treated with free CDN, CDN-Zn, and CDN@CaP / PEI-PEG was significantly delayed compared to the untreated group. CDN-Zn appeared to inhibit tumor growth better than CDN and CDN@CaP / PEI-PEG, but there were no statistically significant differences between them. Regarding post-treatment mouse survival, the median survival times for the untreated, CDN, CDN-Zn, and CDN@CaP / PEI-PEG groups were 23 days, 42 days, 64 days, and not reached, respectively (Figure 4B). From the individual tumor growth curves (Figure 4C), complete tumor regression was observed in 0 out of 5 mice in the untreated group; 2 out of 5 mice in the free CDN group and the CDN-Zn group; and 3 out of 5 mice in the CDN@CaP / PEI-PEG group.

[0329] No significant differences were observed between the groups in the PBMC tetramer staining assay (Figure 4D). PBMC tetramer staining may not have been sensitive enough to show antigen-specific T cell responses after nonspecific intratumoral CDN stimulation, or the timing may not have been optimal. In contrast, the ELISPOT assessment at day 22 showed a significant antigen-specific immune response (Figure 4E-F). Seven days after the second CDN administration, significant AH1 antigen-specific T cell responses were observed in the free CDN, CDN-Zn, and CDN@CaP / PEI-PEG groups. The responses to CDN-Zn and CDN@CaP / PEI-PEG were also higher than those to free CDN, and a statistical difference was observed between free CDN and CDN@CaP / PEI-PEG. Overall, these results demonstrate that the therapeutic activity of both CDN-Zn and CDN@CaP / PEI-PEG is comparable to, or even better than, that of free CDN. The therapeutic benefits of these formulations stem from the combined effect of slow release and increased cellular uptake. Based on these advantages, this CDN-Zn@liposome exhibits improved therapeutic efficacy through more sustained release and improved in vivo stability.

[0330] (Example 4) This example describes the materials and methods of Examples 1, 2, and 3.

[0331] Synthesis of CDN-Zn nanoparticles (NPs) cGAMP, cdAMP, and cdGMP were obtained from Invivogen, and cdAMP(ps)2 was obtained from MedchemExpress. CDN was dissolved in methanol before use. Meanwhile, ZnCl2 (Sigma-Aldrich) was dissolved in methanol to prepare a 100 mM storage solution. In a typical synthesis reaction, 10:1(n / n)Zn 2+ The solution was added to a 1 mg / ml CDN working solution while vigorously stirring. The solution was stirred further at room temperature for 24 hours. The resulting CDN-Zn NPs were centrifuged at 20000 × g for 15 minutes to obtain free CDN and Zn. 2+ The substance was removed, and then another wash was performed with methanol.

[0332] CDN-Zn liposome synthesis CDN-Zn liposomes were synthesized using two steps. First, CDN-Zn@DOPA NPs were synthesized by coordination-modulation. Briefly, Zn in a 10 molar ratio was used. 2+ The solution was added to CDN / DOPA (Avanti Lipids) in chloroform with vigorous stirring. After incubation for 24 hours, CDN-Zn@DOPA NPs were separated by centrifugation at 20000×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 solution of 30% (v / v) ethanol / H2O at 60°C. Finally, the THF was evaporated under reduced pressure, the final solution was allowed to cool to room temperature, and empty liposomes were removed by centrifugation at 20000×g for 20 minutes to obtain CDN-Zn@liposomes. The obtained CDN-Zn@liposomes were then resuspended in PBS for further use.

[0333] Synthesis of CDN@CaP / PEI-PEG NPs CDN@CaP / PEI-PEG NPs were prepared by a one-step precipitation method. Briefly, a solution of CaCl2 (Sigma-Aldrich) and a solution of Na2HPO4 (Sigma-Aldrich) were simultaneously added to a mixture of PEI-PEG and CDN while continuously stirring. After incubation overnight, the CDN@CaP / PEI-PEG NPs were separated by centrifugation at 18000×g for 15 minutes. The resulting NPs were washed twice with histidine buffer (pH 7.4).

[0334] In vitro release analysis Release profiles of CDN-Zn and CDN-Zn@liposomes are available in Slide-A-Lyzer. TMThe study was conducted using a MINI dialysis machine, 3.5K MWCO (Thermo Scientific). Briefly, 0.5 ml of CDN-Zn or CDN-Zn@liposomes were packed into a regenerated cellulose membrane cup, and 14 ml of release buffer (PBS) was placed in a test tube. The dialysis cup was inserted into a conical tube, capped, and the device was incubated at 37°C with continuous shaking (200 rpm). When required, 300 μl of release medium was collected and refilled with an equal volume of fresh PBS. The concentration of CDN in the release medium was analyzed by HPLC (GPC). Finally, the release rate was calculated based on the CDN concentration in the release buffer, the volume of buffer, and the total CDN load.

[0335] Evaluation of interferon-stimulated gene activation THP1-Blue purchased from Invivogen TM ISG (interferon-stimulated gene) cells were handled and cultured according to the manufacturer's instructions. In short, the cells were thawed immediately after receipt and cultured in 5 ml of growth medium in a 25 cm³ culture medium. 2 The cells were transferred to a flask. After one passage, the cells were maintained in growth medium, and a selective antibiotic was added every other passage, resulting in 7 × 10 5 Cells were passaged every three days at a starting cell concentration of cells / ml. To evaluate the biological activity of the CDN formulation, 20 μl of a pre-warmed formulation solution was added to a 96-well flat-bottom plate. Then, 180 μl of cell suspension (~100,000 cells per well) was mixed with the CDN sample. After incubation at 37°C and 5% CO2 for 18 hours, 20 μl of supernatant was collected and incubated with 180 μl of QUANTI-Blue solution (Invivogen) for colorimetric analysis. THP1 activation was quantified by measuring absorbance at 620–655 nm.

[0336] Animal experiments All animals were managed in accordance with federal, state, and local guidelines. All research conducted on animals was in compliance with and approved by the University Committee on Use and Care of Animals (UCUCA). 6-8 week old female Balb / c mice (Jackson Laboratories) were given 1 × 10⁶ doses. 5 Individual CT26 colorectal cancer cells were inoculated. The tumor size was approximately 100 mm. 3 When this was reached, 25 μg of cdAMP(ps)2 in two doses from various formulations was 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 formula: Volume = Length × Width 2 ×0.5. Animals were euthanized when the tumor reached a diameter of 1.5 cm or when the animal became mortally ill due to severe weight loss or ulceration. On day 17, the percentage of tumor antigen-specific CD8α+ T cells in PBMCs was analyzed using the tetramer staining assay described above with peptide-MHC tetramer (H-2Kb restricted AH1) (NIH Tetramer Core Facility, Atlanta, GA). On day 22, the ELISPOT assay was performed using mouse-derived PBMCs treated as described above.

[0337] (Example 5) This example describes the materials and methods of Examples 6 to 11.

[0338] In vitro screening of metal ions that modulate innate immune stimulants Mouse bone marrow-derived dendritic cells (BMDCs) were isolated and cultured. Briefly, bone marrow stem cells were harvested and seeded in bacteriological petri dishes containing GM-CSF medium. The cell culture medium was refreshed on days 3, 6, and 8. Ten days after differentiation, immature DCs were harvested for use. To screen for metal ions that can modulate the cytokine profile of innate immune stimulants, first, 100,000 BMDCs / 100 μl were seeded into each well of a 96-well plate. Then, various metal ions at different concentrations were added along with various innate immune stimulants at different concentrations. Simultaneously, free metal ions alone or free innate immune stimulants alone at the same concentrations were used as controls. After incubation at 37°C and 5% CO2 for 24 hours, the supernatant was collected for ELISA assays of various cytokines.

[0339] Cyclic innate immune-stimulating factor-metal ion coordination polymer combination formulations: cGAMP, cdAMP, and cdGMP were obtained from Invivogen, and cdAMP(ps)2 was obtained from MedchemExpress. CDN was dissolved in methanol or endotoxin-free water before use. Meanwhile, metal ions were dissolved in methanol or water to prepare a 100 mM stock solution. In a typical synthesis reaction, a 10:1 (n / n) metal ion solution was added to a 1 mg / ml CDN working solution with vigorous stirring. The solution was stirred further at room temperature for 24 hours. The resulting CDN-metal mixture was centrifuged at 20000 × g for 15 minutes to remove free CDN and metal ions, and then washed again with methanol.

[0340] CDN-metal ion liposomes: CDN-metal liposomes were synthesized using two steps. Here, CDN-Zn liposomes are given as an example. First, Zn-CDN / H11-DOPE NPs were synthesized by coordination-modulation. Briefly, a 10 molar ratio of Zn was used. 2+The solution was added to CDN / H11-DOPE (Avanti Lipids) in chloroform with vigorous stirring. After 24 hours of incubation, Zn-CDN / H11-DOPE NPs were separated by centrifugation at 20000×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, the THF was evaporated under reduced pressure, the final solution was allowed to cool to room temperature, and empty liposomes were removed by centrifugation at 20000×g for 20 minutes to obtain CDN-Zn@liposomes. The obtained CDN-Zn@liposomes were then resuspended in PBS for further use.

[0341] Metal ion-CDN / polyhistidine-PEG nanocoordinate polymer (NCP): Metal ion-CDN / polyhistidine-PEG NCP was prepared by a one-step precipitation method. Here, Co 2+ Let's take CDN / polyhistidine-PEG as an example. Briefly, a fixed-ratio solution of CoCl2 (Sigma-Aldrich), CDN, polyhistidine-PEG, and HEPES buffer was added dropwise to a mixed solution while continuously stirring. After 24 hours of incubation, Co 2+ -CDN / polyhistidine-PEG nanoparticles (NPs) were separated using a 10kD centrifugal ultrafilter to remove free metal ions and CDN.

[0342] CDN@CaP / PEI-PEG NP was prepared by a one-step precipitation method. Briefly, a solution of CaCl2 (Sigma-Aldrich) and a solution of Na2HPO4 (Sigma-Aldrich) were simultaneously added to a mixture of PEI-PEG and CDN with continuous stirring. After incubation overnight, the CDN@CaP / PEI-PEG NP was separated by centrifugation at 18000×g for 15 minutes. The obtained NP was washed twice with histidine buffer (pH 7.4).

[0343] Innate immune-stimulating factor-metal mineral@anionic polypeptide-PEG: Innate immune-stimulating factor-metal mineral@anionic polypeptide-PEG was prepared by a one-step precipitation method. Taking MnP@PGA-PEG NP as an example: MnCl2 (Sigma-Aldrich) solution and Na2HPO4 (Sigma-Aldrich) solution were simultaneously added to a mixed solution of PGA-PEG and innate immune-stimulating factor with continuous stirring. After overnight incubation, the innate immune MnP@PGA-PEG NP was separated by centrifugation at 18000×g for 15 minutes. The obtained NP was washed twice with histidine buffer (pH 7.4).

[0344] In vitro release analysis The release profile of the formulation, Slide-A-Lyzer TM The study was conducted using a MINI dialysis machine, 3.5K MWCO (Thermo Scientific). Briefly, 0.5 ml of the formulation solution was filled into a regenerated cellulose membrane cup, and 14 ml of release buffer (PBS) was placed in a test tube. The dialysis cup was inserted into a conical tube, capped, and the device was incubated at 37°C with continuous shaking (200 rpm). 300 μl of release medium was collected when needed and refilled with an equal volume of fresh PBS. The concentration of CDN in the release medium was analyzed by HPLC (GPC). Finally, the release rate was calculated based on the CDN concentration in the release buffer, the volume of buffer, and the total CDN load.

[0345] Animal experiments All animals were managed in accordance with federal, state, and local guidelines. All research conducted on animals was in compliance with and approved by the University Committee on Use and Care of Animals (UCUCA). 6-8 week old female Balb / c mice (Jackson Laboratories) were given 1 × 10⁶ doses. 5 Individual CT26 colorectal cancer cells were inoculated. The tumor size was approximately 50 mm. 3If the patient reached a certain level, the prescribed medication or preparation was administered via the prescribed route. Tumor size and survival were monitored every 2 or 3 days. Tumor size was calculated based on the following formula: Volume = Length × Width 2 ×0.5. Animals were euthanized when the tumor reached a diameter of 1.5 cm or when the animal became mortally ill due to severe weight loss or ulceration. On day 17, the percentage of tumor antigen-specific CD8α+ T cells in PBMCs was analyzed using the tetramer staining assay described above with peptide-MHC tetramer (H-2Kb restricted AH1) (NIH Tetramer Core Facility, Atlanta, GA). On day 22, the ELISPOT assay was performed using mouse-derived PBMCs treated as described above.

[0346] (Example 6) This embodiment describes the identification of metal ions that can enhance the STING activation of STING agonists.

[0347] As shown in Figures 5A and 5B, mouse bone marrow-derived dendritic cells (BMDCs) were treated with different metal ions or co-treated with different metal ions and STING agonists. The inventors selected metal ions from essential and trace mineral elements of biological systems. Mn 2+ On its own, it was possible to activate BMDC at a highly toxic dose. However, Mn 2+ When combined with a STING agonist, it significantly enhanced STING activation at much lower concentrations. Similarly, Co 2+ It did not show STING activation itself. However, with 125 μM or 250 μM Co 2+ When combined with 5 μM cGAMP, it significantly enhanced the activation of the STING pathway. Both concentrations were well tolerable. To further confirm whether this phenomenon functions in human cells, the inventors repeated similar experiments using the human monocyte cell line THP1 (Figure 5C). A similar trend was observed in human THP1 cells, demonstrating that this phenomenon is independent of the type of STING agonist.

[0348] (Example 7) This example is Co 2+ and Mn 2+ This study demonstrates the enhanced activation of STING and its anti-cancer therapeutic effect. vinegar.

[0349] We investigated whether an in vitro enhanced type I IFN response would benefit cancer treatment in vivo. In a mouse tumor model, we evaluated combinations of metal ions and STING agonists. As shown in Figures 6a and 6c, Co 2+ -CDA and Mn 2+ -CDA slowed tumor growth. In particular, the metal-CDA group had a significantly higher number of tumor-free mice than the free-CDA group, as evidenced by the survival rate of 80% in the metal-CDA group compared to 20% in the free-CDA group (Figure 6d). Furthermore, Co 2+ -CDA treatment resulted in significantly higher serum IFN beta levels 8 hours after infusion compared to free CDA treatment (Figure 6b). However, Mn 2+ -The same phenomenon was not observed with the CDA combination.

[0350] (Example 8) This embodiment demonstrates an in vivo immune response to a combination of a STING agonist and a metal.

[0351] To study the mechanism of action of the improved cancer treatment efficacy, antigen-specific T cell responses were evaluated in treated animals, and a tumor reloading test was performed 81 days after the initial treatment. CDA-Mn 2+ While the ELISPOT results on day 22 of the experiment showed a better T cell-specific response, the T cell ELISPOT results showed CDA-Co 2+ Similarities were observed between the group and the free CDA group (Figure 7b). In the re-challenging study of tumors, CDA-Co 2+ and CDA-Mn 2+ Survivors in the treatment group completely inhibited the growth of the second CT26 tumor. CDA-Co 2+The treatment group showed a significant increase in antigen-specific T cell response.

[0352] (Example 9) This embodiment demonstrates the identification of metal ions that may modulate other innate immune-stimulating factors.

[0353] Based on our results regarding the STING pathway, we also evaluated whether metal ions can modulate other innate immune stimulants. Mouse BMDCs were treated with different metal ions, or with different metal ions in combination with innate immune stimulants. Similar metal ion-innate immune stimulant synergies were observed. However, different metal ions showed synergistic effects with different DAMPs or PAMPs, including TLR3 / 4 / 7 / 8 / 9 ligands, NOD1 / 2 ligands, TLR7 / 8 ligands, RIG-I & CDS agonists, and inflammasome-induced factors. For example, Co 3+ While poly-IC dramatically increased IFNb, TNFa, IL6, and IL2 production, Mn 2+ This increased only IFNb production by poly-IC (Figures 8a-d). Mn 2+ This increased IFNb and TNFa production in MPLA, but Ni 2+ This increased TNFa production in MPLA (Figures 8e-f). Mn 2+ This increased the production of IFNb and TNFa from R848, but Ni 2+ This increased TNFa production of R848 (Figure 7g~h). Ni 2+ and Mn 2+ CpG increased the production of IFN beta and TNFα (Figures 8i-j). Cytokine profiles of NOD1 / 2 ligands, TLR 7 / 8 ligands, RIG-I & CDS agonists, and pro-inflammatory agents were observed. 2+ Co 2+ , Al 3+ Cu 2+ Fe 3+ Ni 2+It can also be regulated by (Figures 9-12). These results demonstrate that our metal ion-based approach is a simple yet effective method for regulating the cytokine profiles of a wide range of immunostimulators. Based on this finding, we anticipate that pharmaceutically acceptable formulations can be developed to produce better, more potent vaccine adjuvants or cancer immunotherapies. For example, certain metal salts of DAMP / PAMP may function better than the original form. Coordination polymers composed of selected metal ions and DAMP / PAMP with or without pharmaceutically acceptable coordination molecules may result in optimized metal ion-DAMP / PAMP combinations. Other pharmaceutically acceptable formulations are also possible for the simultaneous delivery of metal ions and DAMP / PAMP, including but not limited to metal hydroxide / carbonate / phosphate minerals, liposomes, lipid nanoparticles, PLGA particles, hydrogels, emulsions, etc.

[0354] (Example 10) This example describes representative metal ion-innate immune stimulating factor formulations.

[0355] To co-deliver metal ions and innate immune-stimulating factors to target tissues with an ideal release profile, appropriate formulations can be designed based on the physical and chemical properties of specific metal salts of DAMP / PAMP, coordination, and other pharmaceutically acceptable formulations (hydroxide / carbonate / phosphate minerals, liposomes, lipid nanoparticles, PLGA, hydrogels, emulsions, etc.). Here are some representative examples of coordination formulations: manganese-CDA-H11-DOPE@liposome nanoparticles (Mn-CDA / H11@liposome, Figure 13), Co-CDA / H33-PEG coordination nanoparticles (Co-CDA / H33-PEG, Figure 14), and CDA@Co 2+ We provide a 4-arm-PEG-His11 hydrogel (CDA@4aH11-Co hydrogel, Figure 15). CDA itself is Co via the nitrogen of the purine ring. 2+ and Mn 2+It may cooperate with and may be further stabilized by polyhistidine. Nanoparticle structures (Figures 13-14) or hydrogels (Figure 15) are generated by various building module designs, Co 2+ / Mn 2+ The reaction could be adjusted by optimizing the ratio and concentration of :CDA:polyhistidine-PEG, reaction time, and pH. The loading effect was Co 2+ / Mn 2+ The success rate was approximately 30% with CDA and over 70% with CDA. The inventors further tested these combination formulations in a mouse CT26 colorectal cancer model. As shown in Figures 13-15, these nanoparticle configurations or hydrogel formulations were able to significantly enhance STING activation in vivo compared to free CDA or free CDA + metal ions. In particular, liposome-coated nanoparticles, CDA-Mn-His11-DOPE@liposome (Mn-CDA / H11@lip), could be used for systemic delivery of the STING agonist and eradicated 60% of established CT26 colorectal cancers (Figure 13); Co-CDA / His33-PEG significantly extended IFNb production and was detectable even 4 days after injection (Figure 14); and the injectable CDA@4aH11-Co hydrogel produced a very strong local resection immune response and 1 st Significant ulcers formed after administration were induced (Figure 15f). These improved therapeutic effects were also characterized by increased antigen-specific T cell responses, type I IFN responses, and pro-inflammatory cytokine releases.

[0356] In addition to the above formulations, many other formulations exist that can be synthesized to deliver metal-innate immune-stimulating factors. Here, we provide several examples having the morphology shown in the TEM images (Figure 16). As shown in Figure 16(a), CDA-Zn NPs exhibited a spherical shape with higher TEM contrast on their surface, resulting in a "core-shell" structure. We also found that a uniform spherical structure was obtained when synthesized in an aqueous medium, as slower nucleation occurred in water. Consistent with the TEM images, DLS and zeta potential data showed that the size of cdAMP-Zn was approximately 150 nm and the surface charge was neutral. Under the same synthesis conditions, CDA-Co 2+ NP stands for cross-linked nanoparticle cluster. This showed; CDG-Zn 2+ It exhibited a uniform, irregular spherical structure of approximately 100 nm in size and a neutral surface charge; cGAMP-Zn 2+ The sample showed spherical nanoparticles composed of accumulated small clusters, with a slightly positively charged surface. CDN-Zn 2+ To enhance the stability of the NPs, other polyvalent coordinators such as liposomes (Figure 16b), polyhistidine (Figure 16c), and polyhistidine-PEG (Figure 16d) were also added. Furthermore, innate immune stimulants loaded into nanoscale metal minerals can also be prepared for the delivery of metal ion-innate immune stimulant combinations (Figures 16d-e). To increase the stability of the nanoparticles, surface modifications with PEI-PEG, PGA-PEG, and other anionic polypeptide-PEGs can be applied.

[0357] The inventors also evaluated a subset of the above formulations in tumor-bearing mice. The tumor size was 60 mm. 3 If the target was reached, two doses of the indicated formulation containing 25 μg / dose of adAMP(ps)2 were administered intratumorally on days 10 and 15. As shown in Figure 17, free CDN and CDN-Zn were administered. 2+ Tumor growth in mice treated with CDN@CaP / PEI-PEG was significantly delayed compared to the untreated group. 2+Compared to CDN and CDN@CaP / PEI-PEG, there was no statistically significant difference between them, but it inhibited tumor growth more efficiently. Regarding post-treatment mouse survival, the comparison was between untreated, CDN, and CDN-Zn. 2+ The median survival times for the CDN@CaP / PEI-PEG groups were 23 days, 42 days, 64 days, and not reached, respectively (Figure 17d). From the individual tumor growth curves (Figure 17e), we determined that 0 out of 5 mice in the untreated group; the free CDN group and the CDN-Zn group were different. 2+ Complete tumor regression was observed in 2 out of 5 mice in the group and in 3 out of 5 mice in the CDN@CaP / PEI-PEG group. No significant differences were observed between the groups in the PBMC tetramer staining assay (Figure 17f). PBMC tetramer staining may not have been sensitive enough to show an antigen-specific T cell response after nonspecific intratumoral CDN stimulation, or the timing may not have been optimal. In contrast, the ELISPOT evaluation on day 22 showed a significant antigen-specific immune response (Figure 17f-g). Seven days after the second CDN administration, free CDN and CDN-Zn were evaluated. 2+ A significant AH1 antigen-specific T cell response was observed in the CDN@CaP / PEI-PEG group. CDN-Zn 2+ Furthermore, the response of CDN@CaP / PEI-PEG was higher than that of free CDN, and a statistical difference was observed between free CDN and CDN@CaP / PEI-PEG.

[0358] (Example 11) This example describes the chelation of metal ions to inhibit the innate immune response.

[0359] Considering the intriguing role of metal ions in modulating innate immune responses in our findings, we further evaluated whether chelated metal ions could inhibit metal ions according to innate immune pathways that could be used to treat autoimmune diseases such as systemic lupus erythematosus, Eicardi-Gautier syndrome, acute pancreatitis, age-related macular degeneration, alcoholic liver disease, hepatic fibrosis, metastasis, myocardial infarction, non-alcoholic steatohepatitis (NASH), Parkinson's disease, polyarthritis / fetal and neonatal anemia, sepsis, inflammatory bowel disease, and multiple sclerosis. Through unbiased selection, we identified several chelating agents that exhibited significant inhibitory activity against innate immune responses (Figures 18-19). As shown in Figures 18a-b, chelating agents exhibited higher inhibitory activity as structural complexity increased. This is consistent with our hypothesis that higher structural complexity of chelating agents leads to higher chelating ability. THP1 dual KI-hSTING WT(R232)Using reporter cell lines, DNA / lipofectamine complex challenges, which are thought to have very high activity in activating the cGAS-STING-Type I IFN pathway, were co-incubated with these chelating agents. The degree of inhibition could be determined by ISRE-induced luminescence. The inventors found that the IC50 of DNA-induced Type I IFN responses to punicalagin (PC) and tannic acid (TA) was as low as the nanomolar level, and that these were well acceptable in in vitro assays (Figure 18b-d). Inhibitory effects were also confirmed with another human STING allele, HAQ, and similar results were obtained (Figure 18e). To investigate which stage of the cGAS-STING-Type I IFN pathway the chelating agents affect, we examined whether they could inhibit Type I IFN induced by cGAMP (Figure 18f). The inventors found that the inhibitory effect was eliminated, indicating that these chelating agents can primarily act on cGAS inhibition. Note that the chelating agents presented here are mostly natural polyphenols. Polyphenols have been widely reported to deprive cells of ROS and anti-inflammatory properties. However, few have recognized their potent inhibitory effects against DNA-induced inflammation. Similarly, we have also found that these chelating agents can be used to inhibit poly-IC-induced inflammatory responses in STING knockout THP1 reporter cell lines (Figure 19). We anticipate that many other chelating agents, particularly chelating agents of polyphenol structures (shown in Figure 20), can be used as innate immune inhibitors for DNA and RNA-induced inflammation.

[0360] Built-in by reference The entirety of each patent and scientific paper disclosure referenced herein is incorporated by reference for all purposes.

[0361] Equal parts The present invention may be embodied in other specific forms without departing from its spirit or essential features. Therefore, the embodiments described above should be considered in all respects illustrative rather than limiting the invention as described herein. Accordingly, the scope of the invention is indicated more by the appended claims than by the above specification, and all modifications falling within the meaning and scope of the equivalents of the claims are included therein.

Claims

1. One or more circular dinucleotide interferon gene stimulator (STING) agonists, Zn ２＋ 、Mn ２＋ 、Fe ２＋ 、Fe ３＋ 、Cu ２＋ 、Ni ２＋ 、Co ２＋ 、Pb ２＋ 、Sn ２＋ 、Ru ２＋ 、Au ２＋ 、Mg ２＋ 、VO ２＋ 、Al ３＋ 、Co ３＋ 、Cr ３＋ 、Ga ３＋ 、Tl ３＋ 、Ln ３＋ 、MoO ３＋ 、Cu ＋ 、Au ＋ 、Tl ＋ 、Ag ＋ 、Hg ２＋ 、Pt ２＋ 、Pb ２＋ 、Hg ２＋ 、Cd ２＋ 、Pd ２＋ 、and Pt ４＋ and one or more cations selected from the group consisting of A composition containing nanoparticles is available, The aforementioned nanoparticles are selected from the group consisting of metal-polyhistidine-DOPE liposomes, metal-polyhistidine-PEG, 4arm-PEG-polyhistidine-metal hydrogels, and liposomes. The nanoparticles further comprise poly(histidine) polyethylene glycol (PH-PEG), lipid poly-histidine, or poly(lysine) polyethylene glycol (PK-PEG). composition.

2. The STING agonist of one or more cyclic dinucleotides is cGAMP, cdiAMP, cdiGMP, cAIMP, 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP difluor, cAIM(PS)2, difluor(Rp / Sp), 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP fluoride, c-di-AMP fluoride, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp, Rp), c-di-GMP fluoride Fluoride, 2'3'-c-di-GMP, c-di-IMP, cGAM(PS)2, 2'2'-cGAM(PS)2, 2'3'-cGAM(PS)2, cGAMP fluoride, 2'3'-cGAMP fluoride, 2'2'-cGAMP fluoride, 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, 2'3'-cdAMP fluoride, 2'2'-cdAMP fluoride, 3'3'-cdAMP fluoride, cd GMP, 2'3'-cdGMP, 2'2'-cdGMP, 3'3'-cdGMP, c-di-GM (PS)2, 2'3'-c-di- GM(PS)2, 2'2'-c-di-GM(PS)2, 3'3'-c-di-GM(PS)2, cdGMP fluoride, 2'3'-c dGMP fluoride, 2'2'-cdGMP fluoride, 3'3'-cdGMP fluoride, 2'3'-cAIMP, 2'2'-cA IMP, 3'3'-cAIMP, cAIMP difluoro, 3'3'-cAIMP fluoride, 2'3'-cAIMP fluoride, 2'2 '-cAIMP fluoride, cAIMP(PS)2 difluoro, 3'-3'-cAIMP(PS)2 difluoro(Rp / Sp), 2'3'-cAIMP(PS)2 difluoro, 2'2'-cAIMP(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, c-di-IMMP fluoride, 2'3'-cdIMP fluoride, 2'2'-cdIMP fluoride,The composition according to claim 1, selected from the group consisting of and 3'3'-cdIMP fluoride.

3. The aforementioned nanoparticles further interact with the antigen, and this interaction is selected from complexation, binding, encapsulation, absorption, adsorption, and mixing. The antigen is either derived from an autoantigen or from α-actinin-4, Bcr-Abl fusion protein, Casp-8, β-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, Mum-2, and Mum-3, Neo-PAP, myosin class I, OS-9, pml- RARα fusion protein, PTPRK, K-ras, N-ras, triose phosphate isomerase, Bage-1, GAGE3, GAGE4, ​​GAGE5, GAGE6, GAGE7, GnTV, Herv-K-mel, Lage-1, Mage-A1, Mage-2, Mage-3, Mage-4, Mage-6, Mage-10, Mage-12, MAGE-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, 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, CA19-9, CA-72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T 4,791Tgp72, α-fetoprotein, 13hCG, BCA225, BTAA, CA125, CA15-3 (CA27.29 / BCAA), CA195, CA242, CA-50, CAM43, CD68 / KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), human EGFR protein or fragment thereof, human EGFR residues 306-325 (SCVRACGADSYEMEEDGVRK (sequence number) WT1 (and WT1-derived peptide sequences: WT1 126-134 (RMFP NAPYL (SEQ ID NO: 375)), WT1 122-140 (SGQARMFPNAPYLPSCLES (SEQ ID NO: 377)), WT1122-144 (SGQARMFPNAPYLPSCLESQPTI (SEQ ID NO: 378)), MUC1 (and MUC1-derived peptides and glycopeptides, RPAPGS (SEQ ID NO: 379), PPAHGVT (SEQ ID NO: 380), and PDTRP (SEQ ID NO: 381)), LMP2, EGFRvIII, idiotype, GD2, Ras variant, p53 variant, proteinase 3 (PR1), survivin, hTERT, sarcoma translocation cleavage, EphA2, EphA4, LMW-PTP, PAP, ML-IAP, AFP, ERG (TMPRSS2) ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GM1, mesothelin, sLe (animal), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK The composition according to claim 1, selected from the group consisting of HMWMAA, AKAP-4, XAGE1, B7H3, Regmine, Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-α, PDGFR-β, MAD-CT-2, Fos-related antigen 1, ERBB2, folate receptor 1 (FOLR1 or FBP), IDH1, IDO, LY6K, fms-related tyrosine kinase 1 (FLT1), KDR, PADRE, TA-CIN (recombinant HPV16 L2E7E6), SOX2, novel antigen, and aldehyde dehydrogenase.

4. The composition according to claim 3, wherein the antigen is bound to the outer surface of the nanoparticle.

5. The composition interacts with the adjuvant, and this interaction is selected from complexation, binding, encapsulation, absorption, adsorption, and mixing. The adjuvants mentioned above are CPG, polyIC, polyICLC, 1018 ISS, aluminum salt, BCG, CP-870, CP-893, CpG7909, CyaA, dSLIM, cytokine, IC30, IC31, and ImuFact. IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, Monophosphoryl Lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTelRTM, Vector System, PLGA Microparticles, Imiquimod, Regiquimod, Gardikimod, 3M-052, SRL172, Virosom and Other Virus-like Particles, YF-17D, VEGF Trap, β-Glucan, Pam3Cys, Aquila's QS21 Stimulon, Basimezan, AsA404 (DMXAA), 3M The composition according to claim 1, selected from the group consisting of MEDI9197, glucopyranosyllipid adjuvant (GLA), GLA-SE, CD1d ligand, STING agonist, CL401, CL413, CL429, flagellin, RC529, E6020, imidazoquinoline small molecule TLR-7 / 8a, AS01, AS02, AS03, AS04, AS15, IC31, CAF01, ISCOM, cytokines, and bacterial toxins.

6. The composition according to claim 1, wherein the average particle size of the nanoparticles is between 6 and 500 nanometers.

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