Compositions and methods for delivering biopolymer drugs

Abstract

The present invention provides nanoparticles conjugated with biopolymer drugs configured to treat, prevent, or ameliorate various types of disorders, and methods for their synthesis. [Solution] Provided are compositions comprising nanoparticles (e.g., synthetic high-density lipoprotein (sHDL)) carrying biopolymer drugs (e.g., nucleic acids, peptides, glycolipids, etc.), methods for synthesizing such nanoparticles, and systems and methods for utilizing such nanoparticles (e.g., in diagnostic and / or therapeutic settings).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 138,186, filed March 25, 2015, and U.S. Provisional Patent Application No. 62 / 248,908, filed October 30, 2015, which are incorporated by reference in their entireties. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under AI097291 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] The present invention relates to nanoparticles conjugated with biopolymer drugs and methods for synthesizing the same, which are configured to treat, prevent, or ameliorate various types of disorders. In particular, the present invention relates to compositions comprising nanoparticles (e.g., synthetic high-density lipoprotein (sHDL)) carrying biopolymer drugs (e.g., nucleic acids, peptides, glycolipids, etc.), methods for synthesizing such nanoparticles, and systems and methods for utilizing such nanoparticles (e.g., in diagnostic and / or therapeutic settings). [Background technology]

[0003] Peptide- and nucleic acid-based drugs hold great potential as next-generation therapeutics. Despite this potential, their clinical translation has been difficult, in part, due to the lack of a drug delivery platform that can efficiently deliver drugs to the site of action while protecting the cargo material against enzymatic degradation in vivo. One prime example is the area of ​​cancer vaccines. Although numerous clinical trials have been conducted using defined tumor-associated antigen peptides, they have failed to demonstrate clinical efficacy because soluble peptides do not sufficiently reach the site of action (e.g., lymphoid tissues) and do not generate a strong immune response.

[0004] Improved compositions and techniques for stable and targeted delivery (eg, in vitro or in vivo) of biopolymers (eg, peptides, nucleic acids, glycolipids) are needed. Summary of the Invention

[0005] Despite the great potential of peptide-based cancer vaccines, their efficacy has been limited in humans. Recent advances in tumor exome sequencing have signaled a new era of personalized immunotherapy using patient-specific neoantigens (see, e.g., Yadav, M. et al. Nature 515, 572-576 (2014); Kreiter, S. et al. Nature 520, 692-696 (2015); Schumacher, T. N. & Schreiber, RD Science 348, 69-74 (2015)). However, a general methodology for stimulating potent CD8α+ cytotoxic T lymphocyte (CTL) responses remains lacking. Experiments conducted in the course of developing embodiments of the present invention demonstrated that preformed high-density lipoprotein-mimetic nanodiscs can be readily conjugated with antigen (Ag) peptides and adjuvants to yield stable, ultrasmall nanoparticles that significantly improve Ag / adjuvant co-delivery to lymphoid organs and achieve sustained Ag presentation on dendritic cells. Surprisingly, these nanodiscs were shown to elicit CTL frequencies up to 41-fold higher than soluble vaccines and 9-fold higher than perhaps the most potent adjuvant (i.e., CpG in Montanide) in clinical trials (see, e.g., Speiser, DEet (See, e.g., Fourcade, J. et al. J. Clin. Invest. 115, 739-746 (2005); Fourcade, J. et al. J. Immunother. 31, 781-791 (2008)). Furthermore, the nanodisc platform has been shown to be easily adaptable to neoantigens and to generate potent antitumor immunity. These results suggest a new and powerful approach for cancer immunotherapy and, more generally, a general strategy for personalized nanomedicine.

[0006] These results have significant clinical significance because these nanodiscs, with their established manufacturing procedures and excellent safety profile in humans, can significantly improve the simultaneous delivery of antigens and adjuvants to LNs, maintain antigen presentation on DCs, and trigger T cell responses with potent antitumor effects. Because most tumor mutations are unique to each patient, cancer vaccines will require a personalized approach (see, e.g., Yadav, M. et al. Nature 515, 572-576 (2014); Kreiter, S. et al. Nature 520, 692-696 (2015); Schumacher, T. N. & Schreiber, R. D. Science 348, 69-74 (2015)). Coupled with recent innovations in neoantigen screening, this approach offers a powerful and easier strategy for producing cancer vaccines designed for each patient. Furthermore, this platform technology is generally applicable to personalized therapeutics using a wide range of bioactive molecules and imaging agents.

[0007] Thus, in certain embodiments, the present invention provides methods for producing a personalized neoplasm vaccine for a subject diagnosed with a neoplasm. The present invention is not limited to a particular method for producing a personalized neoplasm vaccine for a subject diagnosed with a neoplasm. In some embodiments, such methods include obtaining a biological sample of the neoplasm from the subject; identifying a plurality of mutations in the neoplasm; analyzing the plurality of mutations to identify one or more neoantigenic mutations predicted to encode neoantigenic peptides, where the neoantigenic mutations are selected from the group consisting of missense mutations, neoORF mutations, and any combination thereof; and producing a personalized neoplasm vaccine, where the personalized neoplasm vaccine comprises microparticles or nanoparticles complexed with one or more neoantigenic peptides specific for the analyzed and identified neoantigenic mutations predicted to encode neoantigenic peptides. In some embodiments, the nanoparticles are further complexed or mixed with an adjuvant. In some embodiments, the identifying further comprises sequencing the genome, transcriptome, or proteome of the neoplasm.

[0008] In some embodiments, the microparticles are between 0.5 microns and 100 microns in size.

[0009] In some embodiments, one or more neoantigenic peptides range from about 5 to about 50 amino acids in length. In some embodiments, one or more neoantigenic variant peptides range from about 15 to about 35 amino acids in length. In some embodiments, one or more neoantigenic peptides range from about 18 to about 30 amino acids in length. In some embodiments, one or more neoantigenic peptides range from about 6 to about 15 amino acids in length.

[0010] In some embodiments, the adjuvant is CPG, polyIC, poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide The adjuvant is selected from the group consisting of ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, vector systems, PLGA microparticles, imiquimod, resiquimod, gargicomod, 3M-052, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, β-glucan, Pam3Cys, Aquila's QS21 Stimulon, vadimesan, and AsA404 (DMXAA). In some embodiments, the adjuvant is any derivative of an adjuvant (e.g., cholesterol-modified CpG).

[0011] The method is not limited to a particular nanoparticle. In some embodiments, the average size of the nanoparticles is between 6 and 500 nm. In some embodiments, the nanoparticles are sHDL nanoparticles. In some embodiments, the sHDL nanoparticles comprise a mixture of at least one phospholipid and at least one HDL apolipoprotein or apolipoprotein mimetic. In some embodiments, the average size of the nanoparticles is between 6 and 500 nm. In some embodiments, the average particle size of the sHDL nanoparticles is between 6 and 70 nm.

[0012] In some embodiments, the phospholipid is dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[ 4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexanecarboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexanecarboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.

[0013] In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein AI (ApoA-I), apolipoprotein A-II (ApoA-II), apolipoprotein A4 (ApoA4), apolipoprotein Cs (ApoCs), and apolipoprotein E (ApoE). In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is set forth by any of SEQ ID NOs: 1-336.

[0014] In certain embodiments, the present invention provides methods for treating a subject diagnosed with a neoplasm with a personalized neoplasm vaccine. The present invention is not limited to a specific method for treating a subject diagnosed with a neoplasm with a personalized neoplasm vaccine. In some embodiments, such methods include obtaining a biological sample of the neoplasm from a subject; identifying one or more mutations in the neoplasm; analyzing the plurality of mutations to identify one or more neoantigenic mutations predicted to encode expressed neoantigenic peptides, where the neoantigenic mutations are selected from the group consisting of missense mutations, neoORF mutations, and any combination thereof; producing a personalized neoplasm vaccine, the personalized neoplasm vaccine comprising microparticles or nanoparticles conjugated to one or more neoantigenic peptides specific for the analyzed and identified neoantigenic mutations predicted to encode neoantigenic peptides; and administering the personalized neoplasm vaccine to the subject, thereby treating the neoplasm. In some embodiments, the personalized neoplasm vaccine is co-administered with an adjuvant. In some embodiments, the nanoparticles are further complexed or mixed with an adjuvant. In some embodiments, identifying further comprises sequencing the genome, transcriptome, or proteome of the neoplasm.

[0015] In some embodiments, one or more neoantigenic peptides range from about 5 to about 50 amino acids in length. In some embodiments, one or more neoantigenic variant peptides range from about 15 to about 35 amino acids in length. In some embodiments, one or more neoantigenic peptides range from about 18 to about 30 amino acids in length. In some embodiments, one or more neoantigenic peptides range from about 6 to about 15 amino acids in length.

[0016] In some embodiments, the adjuvant is CPG, polyIC, poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide The adjuvant is selected from the group consisting of ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, vector systems, PLGA microparticles, imiquimod, resiquimod, gargicomod, 3M-052, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, β-glucan, Pam3Cys, Aquila's QS21 Stimulon, vadimesan, and AsA404 (DMXAA). In some embodiments, the adjuvant is any derivative of an adjuvant (e.g., cholesterol-modified CpG).

[0017] The method is not limited to a particular nanoparticle. In some embodiments, the nanoparticles have an average size of 6 to 500 nm. In some embodiments, the nanoparticles are sHDL nanoparticles. In some embodiments, the sHDL nanoparticles comprise a mixture of at least one phospholipid and at least one HDL apolipoprotein or apolipoprotein mimetic. In some embodiments, the sHDL nanoparticles have an average particle size of 6 to 70 nm.

[0018] In some embodiments, the phospholipid is dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[ 4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexanecarboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexanecarboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.

[0019] In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein AI (ApoA-I), apolipoprotein A-II (ApoA-II), apolipoprotein A4 (ApoA4), apolipoprotein Cs (ApoCs), and apolipoprotein E (ApoE). In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is set forth by any of SEQ ID NOs: 1-336.

[0020] In some embodiments, the personalized neoplasm vaccine is co-administered with an anti-immunosuppressant or immunostimulatory agent, hi some embodiments, the anti-immunosuppressant or immunostimulatory agent is selected from the group consisting of an anti-CTLA antibody, anti-PD-1, anti-PD-L1, anti-TIM-3, anti-BTLA, anti-VISTA, anti-LAG3, anti-CD25, anti-CD27, anti-CD28, anti-CD137, anti-OX40, anti-GITR, anti-ICOS, anti-TIGIT, and an inhibitor of IDO.

[0021] In certain embodiments, the present invention provides compositions comprising microparticles or nanoparticles conjugated with one or more neoantigenic peptides, each of which is specific for a neoantigenic mutation identified in a neoplastic biological sample obtained from a subject. In some embodiments, the subject is a human.

[0022] In some embodiments, the microparticles have a size between 0.5 microns and 100 microns, and in some embodiments, the nanoparticles have an average size between 6 and 500 nm.

[0023] In some embodiments, the one or more neoantigenic peptides range from about 5 to about 50 amino acids in length. In some embodiments, the one or more neoantigenic peptides range from about 15 to about 35 amino acids in length. In some embodiments, the one or more neoantigenic peptides range from about 18 to about 30 amino acids in length. In some embodiments, the one or more neoantigenic peptides range from about 6 to about 15 amino acids in length.

[0024] In some embodiments, the nanoparticles are further complexed or mixed with an adjuvant, such as CPG, polyIC, poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide The adjuvant is selected from the group consisting of ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, vector systems, PLGA microparticles, imiquimod, resiquimod, gargicomod, 3M-052, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, β-glucan, Pam3Cys, Aquila's QS21 Stimulon, vadimesan, and AsA404 (DMXAA). In some embodiments, the adjuvant is any derivative of an adjuvant (e.g., cholesterol-modified CpG).

[0025] In some embodiments, the nanoparticles are sHDL nanoparticles. In some embodiments, the sHDL nanoparticles comprise a mixture of at least one phospholipid and at least one HDL apolipoprotein or apolipoprotein mimetic. In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein AI (ApoA-I), apolipoprotein A-II (ApoA-II), apolipoprotein A4 (ApoA4), apolipoprotein Cs (ApoCs), and apolipoprotein E (ApoE). In some embodiments, the phospholipid is dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[ 4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexanecarboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexanecarboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof. In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is described by any of SEQ ID NOs: 1-336. In some embodiments, the average particle size of the sHDL nanoparticles is 6-70 nm.

[0026] Additionally, the present invention relates to nanoparticles conjugated with biopolymer drugs configured to treat, prevent, or ameliorate various types of disorders, and methods for synthesizing the same. In particular, the present invention relates to compositions comprising synthetic high-density lipoprotein (sHDL) nanoparticles loaded with biopolymer drugs (e.g., nucleic acids, peptides, glycolipids, etc.), methods for synthesizing such sHDL nanoparticles, and systems and methods for utilizing such sHDL nanoparticles (e.g., in diagnostic and / or therapeutic settings).

[0027] Thus, in certain embodiments, the present invention provides a method for inhibiting a target gene in a cell, the method comprising introducing into the cell a composition comprising an siRNA encapsulated in an sHDL nanoparticle, wherein the siRNA can inhibit the target gene by RNA interference, and the siRNA comprises two RNA strands that are complementary to each other. In some embodiments, the siRNA is modified with cholesterol at the 3' sense strand. In some embodiments, the cell is in vivo, in vitro, or ex vivo. In some embodiments, the cell is in a human. In some embodiments, an imaging agent is encapsulated in the sHDL nanoparticle.

[0028] In certain embodiments, the present invention provides a method for reducing serum LDL-C levels in a patient, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a PCSK9 siRNA encapsulated in nanoparticles, wherein the PCSK9 siRNA can inhibit the PCSK9 gene by RNA interference, the PCSK9 siRNA comprising two RNA strands complementary to each other, and the inhibition of the PCSK9 gene results in a reduction in the patient's serum LDL-C levels. In some embodiments, the patient is a human patient. In some embodiments, the PCSK9 siRNA is modified with cholesterol at the 3' sense strand. In some embodiments, an imaging agent is encapsulated in the nanoparticles. In some embodiments, the nanoparticles are selected from the group consisting of sHDL nanoparticles, fullerenes, endohedral metallofullerenes, buckyballs, trimetallic nitride-templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitride nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold nanoparticles, silver nanoparticles, carbon nanoparticles, iron nanoparticles, and modified micelles. In some embodiments, the nanoparticles are sHDL nanoparticles.

[0029] In certain embodiments, the present invention provides a method for treating coronary heart disease in a patient by reducing the patient's serum LDL-C level, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a PCSK9 siRNA encapsulated in nanoparticles, wherein the PCSK9 siRNA can inhibit the PCSK9 gene by RNA interference, the PCSK9 siRNA comprising two RNA strands complementary to each other, and the inhibition of the PCSK9 gene results in a reduction in serum LDL-C level. In some embodiments, the patient is a human patient. In some embodiments, the PCSK9 siRNA is modified with cholesterol at the 3' sense strand. In some embodiments, an imaging agent is encapsulated in the nanoparticles. In some embodiments, the nanoparticles are selected from the group consisting of sHDL nanoparticles, fullerenes, endohedral metallofullerenes, buckyballs, trimetallic nitride-templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitride nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold nanoparticles, silver nanoparticles, carbon nanoparticles, iron nanoparticles, and modified micelles. 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.

[0030] In certain embodiments, the present invention provides methods for inducing a natural killer T cell-mediated immune response in cells, the methods comprising exposing the cells to a composition comprising an αGalCer glycolipid encapsulated in a nanoparticle, wherein such exposure results in the induction of a natural killer T cell-mediated immune response. In some embodiments, the cells are in vivo cells, ex vivo cells, or in vitro cells. In some embodiments, the nanoparticles are selected from the group consisting of sHDL nanoparticles, fullerenes, endohedral metallofullerenes, buckyballs, trimetallic nitride-templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitride nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold nanoparticles, silver nanoparticles, carbon nanoparticles, iron nanoparticles, and modified micelles. In some embodiments, the nanoparticles are sHDL nanoparticles.

[0031] In certain embodiments, the present invention provides a method for inducing an immune response to an antigen, the method comprising administering to a subject in need thereof an effective amount of a composition comprising nanoparticles, wherein the antigen is complexed with the nanoparticles and an adjuvant is complexed or mixed with the nanoparticles.

[0032] In some embodiments, the antigen is directed against PCSK9. In some embodiments, the antigen is directed against gp100 melanoma. 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, inactivated organism-based antigens, attenuated organism-based antigens, viral antigens, bacterial antigens, parasitic antigens, antigens derived from allergens, and tumor antigens. In some embodiments, the antigen is selected from the group consisting of α-actinin-4, Bcr-Abl fusion protein, Casp-8, β-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, Mum-2, and M um-3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Bage-1, Gage3, Gage4, Gage5, Gage6, Gage7, GnTV, Herv-K-mel, Lage-1, Mage-A1, Mage-A2, Mage-A3, Mage-A4, Mage-A6, Mage-A10, Mage-A12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TR P-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO(LAGS), SCP-1, Hom / Mel-40, PRAME, p53, H-R as, 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, and 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 The tumor antigen is selected from the group consisting of: IFN-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS.

[0033] In some embodiments, the adjuvant is a dendritic cell targeting molecule. In some embodiments, the adjuvant is CpG. In certain embodiments, the present invention provides a method for inducing an immune response to an antigen, the method comprising administering to a subject in need thereof an effective amount of a composition comprising nanoparticles, wherein the antigen is complexed with the nanoparticles. In some embodiments, the antigen is directed against PCSK9. In some embodiments, the nanoparticles are further complexed or mixed with an adjuvant. In some embodiments, the nanoparticles are co-administered with an adjuvant.

[0034] In some embodiments, the nanoparticles are selected from the group consisting of sHDL nanoparticles, fullerenes, endohedral metallofullerenes, buckyballs, trimetallic nitride-templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitride nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold nanoparticles, silver nanoparticles, carbon nanoparticles, iron nanoparticles, and modified micelles. In some embodiments, the nanoparticles are sHDL nanoparticles.

[0035] In some embodiments, the adjuvant is a dendritic cell targeting molecule. In some embodiments, the adjuvant is CpG. In some embodiments, the adjuvant is CPG, polyIC, poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide The adjuvant is selected from the group consisting of ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, vector systems, PLGA microparticles, imiquimod, resiquimod, gargicomod, 3M-052, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, β-glucan, Pam3Cys, Aquila's QS21 Stimulon, vadimesan, and AsA404 (DMXAA). In some embodiments, the adjuvant is any derivative of an adjuvant (e.g., cholesterol-modified CpG).

[0036] In some embodiments, the antigen is conjugated to the exterior surface of the nanoparticle. In some embodiments, the adjuvant is conjugated to the exterior surface of the nanoparticle. In some embodiments, the adjuvant is encapsulated within the nanoparticle.

[0037] In some embodiments, the composition is co-administered with a chemotherapeutic agent, such as aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alfa, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, and one or more of interferon alpha, irinotecan, lansoprazole, levamisole, 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.

[0038] In certain embodiments, the present invention provides compositions comprising nanoparticles, wherein the antigen is complexed to the nanoparticles. In some embodiments, the nanoparticles are further complexed or mixed with an adjuvant.

[0039] In some embodiments, the antigen is derived from an autoantigen.

[0040] In some embodiments, the antigen is directed against PCSK9. In some embodiments, the antigen is directed against gp100 melanoma. 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, inactivated organism-based antigens, attenuated organism-based antigens, viral antigens, bacterial antigens, parasitic antigens, antigens derived from allergens, and tumor antigens. In some embodiments, the antigen is selected from the group consisting of α-actinin-4, Bcr-Abl fusion protein, Casp-8, β-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, Mum-2, and M um-3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Bage-1, Gage3, Gage4, Gage5, Gage6, Gage7, GnTV, Herv-K-mel, Lage-1, Mage-A1, Mage-A2, Mage-A3, Mage-A4, Mage-A6, Mage-A10, Mage-A12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TR P-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO(LAGS), SCP-1, Hom / Mel-40, PRAME, p53, H-R as, 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, and 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 The tumor antigen is selected from the group consisting of: IFN-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS.

[0041] In some embodiments, the adjuvant is a dendritic cell targeting molecule. In some embodiments, the adjuvant is an immunostimulatory agent that activates dendritic cells. In some embodiments, the adjuvant is CpG. In some embodiments, the adjuvant is CPG, polyIC, poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide The adjuvant is selected from the group consisting of ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, vector systems, PLGA microparticles, imiquimod, resiquimod, gargicomod, 3M-052, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, β-glucan, Pam3Cys, Aquila's QS21 Stimulon, vadimesan, and AsA404 (DMXAA). In some embodiments, the adjuvant is any derivative of an adjuvant (e.g., cholesterol-modified CpG).

[0042] In some embodiments, the antigen is conjugated to the exterior surface of the nanoparticle. In some embodiments, the adjuvant is conjugated to the exterior surface of the nanoparticle. In some embodiments, the adjuvant is encapsulated within the nanoparticle.

[0043] In some embodiments, the nanoparticles are selected from the group consisting of sHDL nanoparticles, fullerenes, endohedral metallofullerenes, buckyballs, trimetallic nitride-templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitride nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold nanoparticles, silver nanoparticles, carbon nanoparticles, iron nanoparticles, and modified micelles. In some embodiments, the nanoparticles are sHDL nanoparticles.

[0044] In certain embodiments, the present invention provides a composition comprising siRNA encapsulated in nanoparticles, wherein the siRNA can inhibit a target gene by RNA interference, and the siRNA comprises two RNA strands that are complementary to each other. In some embodiments, the siRNA is modified with cholesterol at the 3' sense strand. In some embodiments, an imaging agent is encapsulated in the nanoparticles.

[0045] In some embodiments, the nanoparticles are selected from the group consisting of sHDL nanoparticles, fullerenes, endohedral metallofullerenes, buckyballs, trimetallic nitride-templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitride nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold nanoparticles, silver nanoparticles, carbon nanoparticles, iron nanoparticles, and modified micelles. In some embodiments, the nanoparticles are sHDL nanoparticles.

[0046] In certain embodiments, the present invention provides a pharmaceutical composition comprising a PCSK9 siRNA encapsulated in nanoparticles, wherein the PCSK9 siRNA can inhibit the PCSK9 gene by RNA interference, and the PCSK9 siRNA comprises two complementary RNA strands. In some embodiments, the PCSK9 siRNA is modified with cholesterol at the 3' sense strand. In some embodiments, an imaging agent is encapsulated in the nanoparticles.

[0047] In some embodiments, the average size of the nanoparticles is between 6 and 500 nm.

[0048] In some embodiments, the nanoparticles are selected from the group consisting of sHDL nanoparticles, fullerenes, endohedral metallofullerenes, buckyballs, trimetallic nitride-templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitride nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold nanoparticles, silver nanoparticles, carbon nanoparticles, iron nanoparticles, and modified micelles. In some embodiments, the nanoparticles are sHDL nanoparticles.

[0049] In certain embodiments, the present invention provides compositions comprising αGalCer glycolipids encapsulated within nanoparticles.

[0050] Such methods and compositions are not limited to a particular size, type, or variety of nanoparticles. In some embodiments, the nanoparticles are selected from the group consisting of sHDL nanoparticles, fullerenes, endohedral metallofullerenes, buckyballs, trimetallic nitride-templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitride nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold nanoparticles, silver nanoparticles, carbon nanoparticles, iron nanoparticles, and modified micelles.

[0051] 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.

[0052] In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein AI (ApoA-I), apolipoprotein A-II (ApoA-II), apolipoprotein A4 (ApoA4), apolipoprotein Cs (ApoCs), and apolipoprotein E (ApoE). In some embodiments, the HDL apolipoprotein is selected from preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, preproApoA-IV, proApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-lMilano, proApoA-IMilano, ApoA-lMilano, preproApoA-IParis, proApoA-IParis, and ApoA-IParis, and peptidomimetics of these proteins, and mixtures thereof.

[0053] In some embodiments, the phospholipid is dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[ 4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexanecarboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexanecarboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.

[0054] In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is set forth by any of SEQ ID NOs: 1-336. In some embodiments, the average particle size of the sHDL nanoparticles is between 6-70 nm.

[0055] In certain embodiments, the present invention provides methods for inducing an immune response to one or more antigens, the method comprising administering to a subject in need thereof an effective amount of a composition comprising nanoparticles, wherein the one or more antigens are complexed with the nanoparticles and an adjuvant is complexed with the nanoparticles. In certain embodiments, the present invention provides compositions comprising nanoparticles, wherein the one or more antigens are complexed with the nanoparticles and an adjuvant is complexed with the nanoparticles. In some embodiments, the average size of the nanoparticles is between 6 and 500 nm.

[0056] In some embodiments, the one or more antigens are to PCSK9, M30, M27, Adpgk, and ASMTNMELM, hi some embodiments, the one or more antigens are conjugated to the outer surface of the nanoparticle.

[0057] In some embodiments, the adjuvant is CPG, polyIC, poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide The adjuvant is selected from the group consisting of ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, vector systems, PLGA microparticles, imiquimod, resiquimod, gargicomod, 3M-052, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, β-glucan, Pam3Cys, Aquila's QS21 Stimulon, vadimesan, and AsA404 (DMXAA). In some embodiments, the adjuvant is any derivative of an adjuvant (e.g., cholesterol-modified CpG). In some embodiments, the adjuvant is conjugated to the outer surface of the nanoparticle. In some embodiments, the adjuvant is encapsulated within the nanoparticle.

[0058] In some embodiments, the nanoparticles are selected from the group consisting of sHDL nanoparticles, fullerenes, endohedral metallofullerenes, buckyballs, trimetal nitride templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitride nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold nanoparticles, silver nanoparticles, carbon nanoparticles, iron nanoparticles, modified micelles.

[0059] In some embodiments, the nanoparticles are sHDL nanoparticles. In some embodiments, the nanoparticles are sHDL, and the sHDL nanoparticles comprise a mixture of at least one phospholipid and at least one HDL apolipoprotein or apolipoprotein mimetic.

[0060] In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein AI (ApoA-I), apolipoprotein A-II (ApoA-II), apolipoprotein A4 (ApoA4), apolipoprotein Cs (ApoCs), and apolipoprotein E (ApoE). The phospholipids were dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p- maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexanecarboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexanecarboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.

[0061] In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic and the thiol-reactive phospholipid is dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP). In some embodiments, the ApoA-I mimetic is described by any of SEQ ID NOs: 1-336.

[0062] Further embodiments based on the teachings contained herein will be apparent to those skilled in the art. [Brief explanation of the drawings]

[0063] [Figure 1A]TEM images and size distribution of sHDL are shown. [Figure 1B] 1 shows the biodistribution of DiR-labeled sHDL in mice. [Figure 1C] Cellular uptake of DiO-sHDL without or with excess blank sHDL by SR-BI negative or positive cells is shown. [Figure 1D] FIG. 1 is a schematic diagram of HDL-siRNA. [Figure 1E] 1 shows a GPC assay of sHDL loaded with different concentrations of PCSK9 siRNA. [Figure 1F] Western blot analysis showed that PCSK9 siRNA-sHDL could knockdown PCSK9 better than free PCSK9 Cho-siRNA in HepG2 cells. [Figure 2] (Figure 2A) Schematic diagram of antigen- and adjuvant-loaded sHDL. (Figure 2B) Addition of antigen to functional lipid-containing sHDL resulted in the formation of lipid-antigen conjugates as measured by HPLC. (Figure 2C) Cho-CpG could be quantitatively incorporated into sHDL as measured by GPC. (Figure 2D) Colocalized delivery of antigen (Ag) and adjuvant (CpG) by sHDL resulted in a stronger cellular response than a mixture of antigen and adjuvant in Montanide. [Figure 3] Schematic diagram of the synthesis of sHDL-CSSIINFEK(FITC)L / CpG. [Figure 4] The homogeneous particle size of sHDL-Ag / CpG is shown as analyzed by cryoEM and dynamic light scattering. [Figure 5A] Compared to free antigen forms, antigen delivery by sHDL has been shown to significantly prolong antigen presentation by dendritic cells. [Figure 5B] Compared to free antigen forms, antigen delivery by sHDL has been shown to significantly prolong antigen presentation by dendritic cells. [Figure 6]We show that sHDL-Ag / CpG significantly enhances the induction of antigen-specific CD8+ T cells compared to vaccination with free antigen mixed with conventional adjuvants. [Figure 7] We show that sHDL-Ag / CpG vaccination induces strong CD8+ T cell responses and reduces tumor growth in tumor-bearing mice. [Figure 8] We show that compared with the free soluble form, α-GalCer delivered via sHDL significantly enhanced CD1d presentation on antigen-presenting cells. [Figure 9] We show that lyophilization provides a convenient method for the large-scale synthesis of α-GalCer-loaded sHDL. [Figure 10] FIG. 1 shows a schematic diagram of the lyophilization method for the rapid preparation of sHDL containing encapsulated siRNA. [Figure 11] 1 shows a schematic diagram of using sHDL to regulate PCSK9 for LDL-C management. As shown, (A) LDL is cleared by LDLR via endocytosis; (B) PCSK9 binding to LDLR leads to degradation of LDLR in lysosomes, preventing LDLR recycling; (C) knockdown of PCSK9 can upregulate LDLR and reduce LDL-C; and (D) PCSK9 antibodies induced by PCSK9 vaccine can block the interaction between PCSK9 and LDLR, thereby upregulating LDLR and reducing LDL-C. [Figure 12]Design of an sHDL nanodisc platform for "personalized" cancer vaccines is shown. (a) sHDL nanodiscs composed of phospholipids and an apolipoprotein-1 mimetic peptide (22A) were engineered for co-delivery of antigen (Ag) peptides and adjuvants. Preformed sHDL nanodiscs displaying 4 mol% DOPE-PDP (insert) were mixed with cysteine-modified Ag peptides containing tumor-associated antigens (TAAs) and tumor-specific mutant neoantigens identified by tumor exome DNA sequencing, followed by incubation with cholesterol-modified immunostimulatory molecules (Cho-CpG), resulting in the formation of sHDL nanodiscs co-loaded with Ag and CpG (sHDL-Ag / CpG). (b) Upon administration, sHDL nanodiscs efficiently co-deliver Ag and CpG to draining lymph nodes, promoting strong and sustained Ag presentation by dendritic cells (DCs) (signal 1), inducing DC maturation (signal 2), and leading to the induction of potent Ag-specific CD8α+ cytotoxic T lymphocyte (CTL) responses. Activated CTLs recognize and kill target cancer cells in peripheral tissues, exerting potent antitumor effects. [Figure 13] The effects of 22A variants and lipids on sHDL nanodisc formation are shown. (a) sHDL was prepared using DMPC (containing 4 mol% DOPE-PDP) and different 22A variants. In addition to the 22A we use throughout this study, several other 22A variants, including 22A composed of D-amino acids, formed homogeneous sHDL nanodiscs that remained stable (as analyzed by dynamic light scattering) at 4°C for up to one month. ND indicates no evidence of aggregation. (b) sHDL synthesis requires a high transition temperature (Tm) and phospholipids bearing ApoA-mimetic peptides. DPPC and DMPC (Tm = 41°C and 24°C, respectively), but not POPC or DOPC (Tm = -2°C and -17°C, respectively), formed homogeneous sHDL in the presence of 22A and 4 mol% DOPE. [Figure 14]Synthesis of the functional lipid DOPE-PDP is shown. (a) DOPE, SPDP (succinimidyl 3-(2-pyridyldithio)propionate), and triethylamine (1:1:1.5 molar ratio) were dissolved in chloroform and reacted in the dark for 5 hours with stirring. (b) The reaction progress was monitored by thin-layer chromatography (TLC) using the following mixture of chloroform / methanol / water (volume ratio: 65 / 25 / 4) as the developing solvent. (c-d) The reaction mixture was purified using a silica gel column, and the purity was evaluated by (c) TLC and (d) HPLC using the conditions described in Example VI. [Figure 15]

[0023] Figure 1 shows the preparation and characterization of sHDL-CSSSIINFEKL / CpG, sHDL-gp100 / CpG, and sHDL-Adpgk / CpG. CSSSIinFEKL, CSS-gp100, or CSS-Adpgk was incubated with sHDL-PDP, followed by insertion of Cho-CpG into sHDL-CSSSIINFEKL, sHDL-gp100, or sHDL-Adpgk. HPLC chromatograms confirming (a) conjugation of CSSSIINFEKL to sHDL-PDP, (c) conjugation of gp100 to sHDL-PDP, or (e) conjugation of Adpgk to sHDL-PDP are shown. (b) GPC of sHDL-CSSSIINFEKL / CpG, (d) GPC of sHDL-gp100 / CpG, and (f) GPC of sHDL-Adpgk / CpG demonstrated homogeneity of all formulations and efficient loading of Cho-CpG into sHDL nanodiscs. [Figure 16]Figure 1 shows robust and sustained Ag presentation mediated by sHDL nanodiscs. (a) Dynamic light scattering analysis and (b) transmission electron microscopy imaging demonstrated uniform sHDL-Ag / CpG (10.5 nm ± 0.5 mean diameter) with nanodisc-like morphology. (c) Nanodisc homogeneity was maintained after sterile filtration (0.22 μm), long-term storage (8 weeks) at -20°C, and subsequent thawing at 37°C. BMDCs were incubated with the vaccine formulation for (d) 24 h and (e) the indicated times. Ag presentation was quantified by flow cytometry analysis of DCs stained with 25D1.16 mAb, which recognizes the SIINFEKL-H-2Kb complex. (f-g) Confocal microscopy images of JAWSII cells (immature DCs) are shown. (f) JAWSII cells were incubated with free Ag+CpG or sHDL-Ag / CpG for 24 h and stained with 25-D1.16 mAb. Scale bar = 20 μm. g. JAWS II cells were incubated with free CSSSIINFEK(FITC)L + CpG or sHDL-CSSSIINFEK(FITC)L / CpG for 6, 24, or 48 hours, followed by staining with Hochest and Lysotracker. Scale bar = 10 μm. h. BMDCs were incubated with different concentrations of the indicated formulations: low dose = 20 nM SIINFEKL and 3 nM CpG; medium dose = 100 nM SIINFEKL and 15 nM CpG; and high dose = 500 nM SIINFEKL and 75 nM CpG. After 24 or 48 hours of incubation, BMDCs were cocultured with SIINFEKL-specific B3Z T cell hybridoma for an additional 24 hours, followed by assessment of T cell activation. Data show mean ± standard deviation from a representative experiment (n = 3) out of 2–4 independent experiments. ****p<0.0001, analyzed by two-way ANOVA with Tukey's HSD post-hoc test. [Figure 17]Figure 1 shows robust and sustained Ag presentation mediated by sHDL-Ag / CpG. BMDCs were incubated with the vaccine formulation for (a and b) 24 hours or (c) the indicated time points, and Ag presentation was quantified by flow cytometry analysis of DCs stained with 25D1.16 mAb, which recognizes the SIINFEKL-H-2Kb complex. (a) Percentage of antigen-presenting BMDCs at 24 hours; (b) Representative histogram; (c) Percentage of antigen-presenting BMDCs over 48 hours. Data are shown as mean ± standard deviation from a representative experiment (n = 3) out of 2-4 independent experiments. ****p < 0.0001, analyzed by two-way ANOVA with Tukey's HSD post-hoc test. [Figure 18] Ag delivery and presentation mediated by sHDL-Ag / CpG (wide field). JAWS II cells were incubated with free CSSSIINFEK(FITC)L + CpG or sHDL-CSSSIINFEK(FITC)L / CpG for 6, 24, or 48 hours and stained with Hochest and Lysotracker. Scale bar = 50 μm. [Figure 19] Intracellular delivery of sHDL (wide field). JAWS II cells were incubated with sHDL containing either rhodamine-labeled DOPE (DOPE-Rhod) or Texas Red-labeled 22A for 24 hours and stained with Hochest and Lysotracker. Scale bar = 50 μm. [Figure 20] Stimulation of bone marrow-derived dendritic cells (BMDCs) with CpG-containing formulations is shown. BMDCs were incubated with blank sHDL or 75 nM CpG formulations for 24 hours. After staining with corresponding fluorophore-conjugated antibodies, the expression levels of CD40, CD80, and CD86 were measured by flow cytometry. Data show the mean ± standard deviation from a representative experiment (n = 3) from three independent experiments. [Figure 21]Vaccine nanodiscs for LN targeting of Ag and adjuvant and elicitation of CTL responses are shown. C57BL / 6 mice were subcutaneously administered (a) 31 nmol of FITC-tagged Ag (CSSSIINFEK(FITC)L) or (b) 2.3 nmol of Cho-CpG (20% Cy5-labeled) in free soluble or sHDL form at the base of the tail. Fluorescence signals in the draining inguinal LN were quantified 24 hours later using IVIS. (c-f) C57BL / 6 mice were immunized with the indicated formulations (15.5 nmol of Ag peptide and 2.3 nmol of CpG) on days 0, 21, and 42. (c) The frequency of SIINFEKL-specific CD8α+ T cells in peripheral blood was measured 7 days after each immunization by flow cytometry analysis of tetramer+CD8α+ T cells. (d) Representative scatter plots at day 49 are shown. (e-f) On day 50, prevaccinated animals received 2 × 10 B16OVA cells via subcutaneous flank injection. (e) Tumor growth and (f) overall survival are shown. (g-h) C57BL / 6 mice were immunized every other week with the indicated formulations. (g) Percentage of SIINFEKL-specific CD8α T cells among PBMCs is shown. (h) ELISPOT analysis of IFN-γ spot-forming cells among splenocytes after ex vivo restimulation with SIINFEKL on day 42 is shown. Data are shown as mean ± standard deviation from a representative experiment (n = 4-5) from two to three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, analyzed by (a-b) unpaired two-tailed Student's t test, (c, d, e) two-way ANOVA with Tukey's HSD post-hoc test, or (f) log-rank (Mantel-Cox) test. The asterisk in panel e indicates a statistically significant difference between sHDL-Ag / CpG and SIINFEKL+CpG+Montanide. [Figure 22]Colocalization of antigenic peptide and sHDL in the dLN after subcutaneous administration. Cy5-labeled 22A-incorporated sHDL-CSSIINFEK(FITC)L nanodiscs were injected subcutaneously into the base of the tail of C57BL / 6 mice (31 nmol of antigenic peptide / mouse). 24 hours later, the draining inguinal lymph nodes were harvested and cryosections were prepared for confocal microscopy. Confocal images show that antigenic peptide and 22A colocalized in the lymph node (indicated by white arrows). Scale bar = 50 μm. [Figure 23] Induction of CTL responses by sHDL-Ag / CpG vaccination. C57BL / 6 mice were immunized every other week with the indicated formulations. Representative scatter plots of SIINFEKL-specific CD8+ T cells in PBMCs on day 35 and their effector CD8+ T cell phenotypes analyzed by CD44 and CD62L staining are shown. [Figure 24] Therapeutic vaccination against melanoma using sHDL-Ag / CpG is shown. C57BL / 6 mice (n = 5) were subcutaneously inoculated with 2 × 10 B16OVA cells and vaccinated with the indicated formulations (equivalent to 15.5 nmol of Ag peptide and 2.3 nmol of CpG) on days 4 and 11. (a) The frequency of SIINFEKL-specific CD8α T cells in PBMCs measured by tetramer staining, (b) representative scatter plots of these on day 17, (c) B16OVA tumor growth, and (d) animal survival. Data are shown as means ± standard deviations from a representative experiment (n = 5) out of two to three independent experiments. *p < 0.05, and ****p < 0.0001, analyzed by (a, c) two-way ANOVA with Tukey's HSD post-hoc test, or (d) log-rank (Mantel-Cox) test. Asterisks in panel c indicate statistically significant differences between sHDL-Ag / CpG and all other groups. [Figure 25]Nanodisc vaccination with tumor-associated antigens and tumor-specific neoantigens for the treatment of melanoma and colon adenocarcinoma. (a-c) C57BL / 6 mice were subcutaneously inoculated with 2 × 10 nonimmunogenic B16F10 melanoma cells and vaccinated on days 4 and 11 with the indicated formulations (equivalent to 15.5 nmol of Ag peptide and 2.3 nmol of CpG). (a) Frequency of gp100-specific CD8α T cells in PBMCs, (b) B16F10 tumor growth, and (c) animal survival are shown. (d) Adpgk mutations were confirmed in MC-38 murine colon adenocarcinoma cells by sequencing the Adpgk cDNA. (e-h) C57BL / 6 mice were subcutaneously inoculated with 10 MC-38 tumor cells and vaccinated with the indicated formulations (equivalent to 15.5 nmol of mutant Adpgk peptide and 2.3 nmol of CpG) on days 10, 17, and 24. (e) Representative scatter plots of the frequency of Adpgk-specific CD8α T cells and Adpgk-tetramer CD8α T cells in PBMCs on day 23, (f) representative scatter plots of the percentages of intracellular IFN-γ, TNF-α, and IFN-γ+TNF-α+CD8α T cells in PBMCs on day 30 after ex vivo restimulation with mutant Adpgk Ag, (g) MC-38 tumor mass growth, and (h) animal survival. Data are shown as means ± standard deviations from a representative experiment (n = 5-8) from two to three independent experiments. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, (a, b, e, g) analyzed by two-way ANOVA with Tukey's HSD post-hoc test, (c, h) analyzed by log-rank (Mantel-Cox) test. (b, g) Asterisks indicate statistically significant differences between sHDL-Ag / CpG and all other groups. [Figure 26](a) Therapeutic vaccination against melanoma using sHDLAg / CpG. C57BL / 6 mice were inoculated subcutaneously with 2×10 B16F10 cells and vaccinated with the indicated formulations (equivalent to 15.5 nmol Ag peptide and 2.3 nmol CpG) on days 4 and 11. (b) Representative scatter plots of gp100-specific CD8α T cells in PBMCs from B16F10 tumor-bearing mice on day 17 are shown. [Figure 27] cDNA sequencing of MC-38 cells for mutant Adpgk neoantigens. Two different sets of primers were used to prepare cDNAs of two different lengths (485 bp and 250 bp) for the neoantigen Adpgk mRNA (amino acid sequence ASMTNMELM). The sequences of the cDNAs were analyzed by DNA sequencing. (A) Two different lengths of cDNA bands on an agarose gel, (B) the results of Sanger DNA sequencing for the 485 bp cDNA, and (C) the results of Sanger DNA sequencing for the 250 bp cDNA. The arrow indicates the G→T mutation. [Figure 28] Nanodisc-based vaccination with multivalent neoantigen peptides induced strong CD4+ and CD8+ T cell responses. (a) PBMCs from mice vaccinated with sHDL-M30 / M27 / CpG showed strong IFNγ secretion from CD4+ T cells upon restimulation with M30 peptide. (b) PBMCs from mice vaccinated with sHDL-M30 / M27 / CpG showed strong IFNγ secretion from CD8+ T cells upon restimulation with M27 peptide. Data represent mean ± standard deviation (n = 3-4). [Figure 29]Nanoparticle formulations improve CD8+ T cell responses and the therapeutic efficacy of neoantigen peptide vaccination. C57BL / 6 mice were inoculated with tumor cells (1 x 105 MC38 cells per mouse) by subcutaneous injection into the right flank on day 0. Mice were vaccinated with 15.5 nmol of ASMTNMELM and 2.3 nmol of CpG in either soluble or liposomal form on days 10 and 17. AuNP (gold nanoparticle) groups were immunized on day 10, exposed to laser on day 11 or not, followed by tetramer staining on day 17. (a) Percentage of antigen-specific CD8+ T cells in PBMCs induced by different formulations 7 days after the last vaccination. (b) Tumor growth curves for the indicated formulations. Data represent mean ± standard deviation (n = 3-5). Definitions: To facilitate understanding of the present invention, several terms and phrases are defined below.

[0064] As used herein, the term "lipid" refers to a water-insoluble fatty substance, including fats, oils, waxes, and related compounds. They can be either produced in the blood (endogenous) or ingested through the diet (exogenous). Lipids are essential for normal body function, and whether produced from exogenous or endogenous sources, they must be transported and then released for use by cells. The production, transport, and release of lipids for use by cells is referred to as lipid metabolism. There are several classes of lipids, but the two major classes are cholesterol and triglycerides. Cholesterol can be ingested through the diet and manufactured by cells in most organs and tissues in the body, primarily the liver. Cholesterol can be found in its free form or combined with fatty acids, often referred to as cholesterol esters.

[0065] As used herein, the term "lipoprotein" refers to a spherical compound structured so that water-insoluble lipids are partially contained within a water-soluble outer shell. Depending on the type of lipoprotein, the content includes various amounts of free and esterified cholesterol, triglycerides, and apoproteins or apolipoproteins. There are five major types of lipoproteins, which differ in function and lipid and apoprotein content and are classified according to increasing 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 associated with lipoproteins.

[0066] As used herein, the term "HDL" or "high-density lipoprotein" refers to high-density lipoprotein. HDL contains approximately equal amounts of lipid and protein complexes and functions as a transporter of cholesterol in the blood. HDL is primarily synthesized in and secreted from epithelial cells in the liver and small intestine. Immediately after secretion, HDL is in the form of discoidal particles containing apolipoprotein AI (also known as ApoA-I) and phospholipids as major components, and is also called nascent HDL. This nascent HDL receives free cholesterol from the cell membranes of peripheral cells in the blood or is produced during the hydrolysis of other lipoproteins. It forms mature spherical HDL while retaining cholesterol esters converted from the cholesterol by the action of LCAT (lecithin cholesterol acyltransferase) in its hydrophobic center. HDL plays a crucial role in the lipid metabolic process known as "reverse cholesterol transport," taking up cholesterol from peripheral tissues into the blood and transporting it to the liver. Reverse cholesterol transport is thought to be one of the major mechanisms of HDL's protective effect against atherosclerosis, and high levels of HDL are therefore associated with a reduced risk of atherosclerosis and coronary heart disease (CHD).

[0067] As used herein, the terms "synthetic HDL," "sHDL," "reconstituted HDL," or "rHDL" refer to particles structurally similar to native HDL, composed of lipid(s) associated with at least one protein of HDL, preferably ApoA-I or a mimetic thereof, which exhibit all of the known physiological functions of HDL. Typically, the components of sHDL are derived from blood or may be produced by recombinant technology.

[0068] As used herein, the term "conjugated" is used in the broadest sense. Examples of conjugation include, but are not limited to, chemical conjugation, surface adsorption, internal loading, and physical mixture of antigen and adjuvant molecules.

[0069] As used herein, the term "biological biopolymer" or "biopolymer" refers to a molecule of molecular weight greater than 1 kDa that can be isolated from an organism or cell culture, such as a eukaryotic (e.g., mammalian) cell culture or a prokaryotic (e.g., bacterial) cell culture. In some embodiments, use of the term refers to polymers, e.g., biopolymers, such as nucleic acids (e.g., DNA, RNA), polypeptides (e.g., proteins), carbohydrates, and lipids. In some embodiments, the term "biopolymer" refers to a protein. In some embodiments, the term "biopolymer" refers to a recombinant protein or a fusion protein. In some embodiments, the protein is soluble. In some embodiments, the biopolymer is an antibody, e.g., a monoclonal antibody.

[0070] As used herein, the term "antigen" is defined herein as a molecule containing one or more epitopes that will stimulate the host immune system to elicit a cellular antigen-specific immune response and / or a humoral antibody response. Antigens can be peptides, proteins, polysaccharides, sugars, lipids, nucleic acids, and combinations thereof. Antigens can be derived from viruses, bacteria, parasites, plants, protozoa, fungi, tissues, or transformed cells such as cancer or leukemia cells, and can be whole cells or immune components thereof, such as cell wall components. Antigens can be oligonucleotides or polynucleotides that express the antigen. Antigens can be natural or synthetic, e.g., haptens, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens (see, e.g., Bergmann, et al., Eur. J. Immunol., 23:2777-2781 (1993); Bergmann, et al., J. Immunol., 157:3242-3249 (1996); Suhrbier, Immunol. and Cell Biol., 75:402-408 (1997)).

[0071] As used herein, the term "neoantigen" or "neoantigenicity" refers to a class of tumor antigens that arise from tumor-specific mutation(s) that alter the amino acid sequence of a genome-encoded protein.

[0072] As used herein, the term "tumor-specific antigen" is defined herein as an antigen that is unique to tumor cells and does not occur in or on other cells in the body.

[0073] As used herein, the term "tumor-associated antigen" is defined herein as an antigen that is not unique to tumor cells and is also expressed in or on normal cells under conditions that are incapable of inducing an immune response against the antigen.

[0074] As used herein, the term "adjuvant" is defined herein as a substance that, when administered together with other antigens, increases the immune response to the other antigens. Adjuvants are also referred to herein as "immune enhancing agents" and "immunomodulating agents."

[0075] As used herein, the term "antigen-presenting cell" is defined herein as a highly specialized cell that can process antigens and present 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 cells for B cells are follicular dendritic cells.

[0076] As used herein, the term "cross-presentation" is defined herein as the ability of antigen-presenting cells to take up extracellular antigens, process them, and present them to CD8 T cells (cytotoxic T cells) via 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 the induction of cytotoxic immunity in vaccination with protein antigens, for example, tumor vaccination.

[0077] As used herein, the terms "immune," "immunological," or "immune" response is the development of a humoral and / or cellular response to an antigen.

[0078] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of sHDL nanoparticles (e.g., compositions comprising sHDL nanoparticles encapsulating siRNA) as described herein (e.g., compositions comprising sHDL nanoparticles configured to activate an immune response), such delivery systems include systems that allow for the storage, transportation, or delivery of such compositions and / or supplemental materials (e.g., written instructions for using the materials, etc.) from one location to another. For example, a kit includes one or more enclosures (e.g., boxes) containing the necessary agents and / or supplemental materials. As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers, each containing a portion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain a composition comprising sHDL nanoparticles or components necessary to synthesize such sHDL nanoparticles, while a second container contains a second agent (e.g., siRNA, antigen, adjuvant) (e.g., antibiotic or spray applicator). Indeed, any delivery system containing two or more separate containers, each containing a portion of the overall kit components, is encompassed by the term "fragmentation kit." In contrast, a "combination kit" refers to a delivery system containing all of the components necessary to synthesize and utilize any of the sHDL nanoparticles as described (e.g., in a single box housing each of the desired components). The term "kit" includes both fragmentation kits and combination kits.

[0079] As used herein, the term "subject" refers to any animal (e.g., mammal) that is to be the recipient of a particular treatment, including, but not limited to, humans, non-human primates, rodents, etc. Generally, the terms "subject" and "patient" are used interchangeably herein with respect to a human subject.

[0080] As used herein, the term "sample" is used in the broadest sense. In one sense, it is meant to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, e.g., plasma, serum, etc. Environmental samples include environmental materials such as surface material, soil, water, quartz, and industrial samples. However, such examples should not be construed as limiting the types of samples applicable to the present invention.

[0081] As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell cultures. The term "in vivo" refers to a natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

[0082] As used herein, the term "drug" or "therapeutic agent" is meant to include any molecule, molecular complex, or substance that is administered to a living organism for diagnostic or therapeutic purposes, including medical imaging, monitoring, contraceptive, cosmetic, nutraceutical, pharmaceutical, and prophylactic uses. The term "drug" is further meant to include any such molecule, molecular complex, or substance that is chemically modified and / or operably linked to a biological or biocompatible structure.

[0083] As used herein, the term "solvent" refers to the medium in which a reaction is carried out. Solvents may be liquids, but are not limited to liquid form. Solvent categories include, but are not limited to, non-polar, polar, protic, and aprotic. DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention relates to nanoparticles conjugated with biopolymer drugs and methods for synthesizing the same, which are configured to treat, prevent, or ameliorate various types of disorders. In particular, the present invention relates to compositions comprising nanoparticles (e.g., synthetic high-density lipoprotein (sHDL)) carrying biopolymer drugs (e.g., nucleic acids, peptides, glycolipids, etc.), methods for synthesizing such nanoparticles, and systems and methods for utilizing such nanoparticles (e.g., in diagnostic and / or therapeutic settings). nanoparticles The present invention is not limited to any particular type or variety of nanoparticles for conjugation with biopolymer drugs configured to treat, prevent, or ameliorate various types of disorders.

[0085] Examples of nanoparticles include, but are not limited to, fullerenes (also known as C60, C70, C76, C80, and C84), endohedral metallofullerenes containing additional atoms, ions, or clusters within the fullerene cage, buckyballs (EMIs), trimetallic nitride-templated endohedral metallofullerenes (TNT EMEs, encapsulating highly symmetrical four-atom molecular clusters formed in a trimetallic nitride template within a carbon cage), single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitride nanotubes, carbon nanotube peapods (carbon nanotubes with internal metallofullerene and / or other internal chemical structures), carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, quantum dots, superparamagnetic nanoparticles, nanorods, and cellulose nanoparticles. Particle embodiments can also include microparticles capable of enhancing efficacy or selectivity. Other non-limiting exemplary nanoparticles include glass and polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold, silver, carbon, and iron nanoparticles.

[0086] 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 in itself. As used herein, the term "micelle" refers to an aggregate of molecules dispersed in a liquid. Typical micelles in aqueous solution form aggregates with hydrophilic "head" regions in contact with the surrounding solvent, sequestering a single hydrophobic tail region in the center of the micelle. In some embodiments, the head region may be, for example, a surface region of the polyol polymer, while the tail region may be, for example, a hydrophobic polymer block region of the polyol polymer.

[0087] The present invention further encompasses the use of particles on the micrometer scale, in addition to the nanometer scale. When microparticles are used, they are preferably relatively small, about 1 to 50 micrometers. For ease of discussion, the use of "nanoparticle" herein encompasses true nanoparticles (about 1 nm to about 1000 nm in size), microparticles (e.g., about 1 micrometer to about 50 micrometers), or both.

[0088] Examples of nanoparticles include, by way of example and not limitation, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers with covalently bound metal chelates, nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In some embodiments, the nanoparticles are metal nanoparticles (e.g., nanoparticles of gold, palladium, platinum, silver, copper, nickel, cobalt, iridium, or alloys of two or more thereof). The nanoparticles can comprise a core or a core and a shell, such as core-shell nanoparticles.

[0089] In some embodiments, the nanoparticles are sHDL nanoparticles. Generally, sHDL nanoparticles consist of a mixture of HDL apolipoproteins and amphipathic lipids.

[0090] The present invention is not limited to the use of any particular type or variety of HDL apolipoproteins, including, for example, apolipoprotein AI (ApoA-I), apolipoprotein A-II (ApoA-II), apolipoprotein A4 (ApoA4), apolipoprotein Cs (ApoCs), and apolipoprotein E (ApoE). In some embodiments, the HDL apolipoprotein is selected from preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, preproApoA-IV, proApoA-IV, ApoA-IV, preproApoE, proApoE, ApoE, preproApoA-I Milano, proApoA-I Milano, ApoA-I Milano, preproApoA-IParis, proApoA-IParis, and ApoA-IParis, as well as peptidomimetics of these proteins and mixtures thereof. Preferably, the carrier particles are composed of ApoA-I or ApoA-II, although other lipoproteins, including apolipoprotein A4, apolipoprotein Cs, or apolipoprotein E, may be used alone or in combination to formulate carrier particle mixtures for delivery of therapeutic agents. In some embodiments, mimetics of such HDL apolipoproteins are used.

[0091] ApoA-I is synthesized by the liver and small intestine as a preproapolipoprotein, secreted as a proprotein that is rapidly cleaved to generate a mature polypeptide of 243 amino acid residues. ApoA-I consists of six to eight distinct 22-amino acid repeats separated by linker moieties, primarily proline, and in some cases, stretches of several residues. ApoA-I forms three types of stable complexes with lipids: small, lipid-poor complexes called pre-β-1 HDL; flattened, discoidal particles containing polar lipids (phospholipids and cholesterol) called pre-β-2 HDL; and spherical or mature HDL (HDL3 and HDL2), spherical particles containing both polar and nonpolar lipids. Most HDL in the circulating population contains both ApoA-I and ApoA-II (the second major HDL protein).

[0092] In some embodiments, ApoA-I agonists or mimetics are provided. In some embodiments, such ApoA-I mimetics can form an amphipathic α-helix that mimics the activity of ApoA-I and have specific activity that approaches or exceeds that of the native molecule. In some embodiments, the ApoA-I mimetics are peptides or peptide analogs that form amphipathic 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 HDL fractions, and promote cholesterol efflux.

[0093] The present invention is not limited to the use of a particular ApoA-I mimetic. In some embodiments, any of the ApoA-I mimetics described in Srinivasa, et al., 2014 Curr. Opinion Lipidology Vol. 25(4):304-308 are utilized. In some embodiments, any of the ApoA-I mimetics described in U.S. Patent Application Publication Nos. 20110046056 and 20130231459 are utilized.

[0094] In some embodiments, the "22A" ApoA-I mimetic is used (PVLDLFRELLNELLEALKQKLK) (SEQ ID NO: 4) (see Examples I-IV) (see, e.g., U.S. Patent No. 7,566,695). In some embodiments, any of the following ApoA-I mimetics shown in Table 1, as described in U.S. Patent No. 7,566,695, are utilized:

[0095] [Table 1] TIFF0007812573000002.tif252169 TIFF0007812573000003.tif252169 TIFF0007812573000004.tif253169 TIFF0007812573000005.tif252169 TIFF0007812573000006.tif253169 TIFF0007812573000007.tif47169

[0096] * indicates an N-terminally acetylated and C-terminally amidated peptide; indicates an N-terminally dansylated peptide; sp indicates a peptide that showed solubility problems under the experimental conditions; X is Aib; Z is NaI; O is Orn; He (%) indicates percent helicity; mics indicates micelles; ~ indicates a missing amino acid.

[0097] In some embodiments, an ApoA-I mimetic having the following sequence as described in U.S. Patent No. 6,743,778 is utilized: Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (SEQ ID NO: 255).

[0098] In some embodiments, any of the following ApoA-I mimetics shown in Table 2 as described in U.S. Patent Application Publication No. 2003 / 0171277 are utilized:

[0099] [Table 2] TIFF0007812573000009.tif255169 TIFF0007812573000010.tif100169

[0100] In some embodiments, an ApoA-I mimetic is utilized having the following sequence as described in US Patent Application Publication No. 2006 / 0069030: FAEKFKEAVKDYFAKFWD (SEQ ID NO: 333).

[0101] In some embodiments, ApoA-I mimetics having the following sequences as described in U.S. Patent Application Publication No. 2009 / 0081293 are utilized: DWFKAFYDKVAEKFKEAF (SEQ ID NO: 334); DWLKAFYDKVAEKLKEAF (SEQ ID NO: 335); PALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ (SEQ ID NO: 336).

[0102] Amphipathic lipids include, for example, any lipid molecule having both a hydrophobic portion and a hydrophilic portion. Examples include phospholipids or glycolipids. Examples of phospholipids that can be used in sHDL-TA nanoparticles include dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], and 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine. Examples of suitable phospholipids include, but are not limited to, 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexanecarboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(P-maleimidomethyl)cyclohexanecarboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof. In some embodiments, the phospholipid is conjugated with an imaging agent (e.g., rhodamine (Rhod)-labeled DOPE (DOPE-Rhod)). In some embodiments, the phospholipid is a thiol-reactive phospholipid such as, for example, dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dihexadecanoyl-sn-glycero-3-phosphothioethanol, or N-4-(p-maleimidophenyl)butyryl)dipalmitoylphosphatidylethanolamine (MPB-DPPE)).

[0103] In some embodiments, exemplary phospholipids include small alkyl chain phospholipids, egg phosphatidylcholine, soy phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidylcholine, 1-stearoyl-2-palmito ... Dioleoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, dilauroylphosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, dimyristoylphosphatidylglycerol, diphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol , dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, egg sphingomyelin, milk sphingomyelin, palmitoyl sphingomyelin, phytosphingomyelin, dipa These include, but are not limited to, lumitoyl sphingomyelin, distearoyl sphingomyelin, dipalmitoyl phosphatidylglycerol salts, phosphatidic acid, galactocerebrosides, gangliosides, cerebrosides, dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3) diglyceride, aminophenylglycosides, 3-cholesteryl-6'-(glycosylthio)hexyl ether glycolipids, and cholesterol and its derivatives.The phospholipid fraction containing SM and palmitoylsphingomyelin can optionally contain small amounts of any type of lipid, including, but not limited to, lysophospholipids, sphingomyelins other than palmitoylsphingomyelin, galactocerebrosides, gangliosides, cerebrosides, glycerides, triglycerides, and cholesterol and its derivatives.

[0104] In some embodiments, the sHDL nanoparticles have a phospholipid / HDL apolipoprotein molar ratio of 2 to 250 (eg, 10 to 200, 20 to 100, 20 to 50, 30 to 40).

[0105] Generally, the sHDL nanoparticles so formed are spherical and have a diameter of about 5 nm to about 20 nm (e.g., 4-75 nm, 4-60 nm, 4-50 nm, 4-22 nm, 6-18 nm, 8-15 nm, 8-10 nm, etc.). In some embodiments, the sHDL nanoparticles are subjected to size exclusion chromatography to obtain a more homogeneous preparation.

[0106] The present invention addresses the need for improved, stable, and targeted delivery (e.g., in vitro or in vivo) of biopolymers (e.g., peptides, nucleic acids, glycolipids). Indeed, the present invention addresses such a need by providing synthetic high-density lipoprotein (sHDL) nanoparticles for the stable, targeted delivery of biopolymers, including peptides, nucleic acids, and glycolipids.

[0107] Compared to other approaches involving traditional nanoparticle vehicles, sHDL nanoparticles possess attractive biocompatibility and cargo-loading capabilities. For example, their ultrasmall yet tunable size (e.g., 10–20 nm) allows sHDL nanoparticles to efficiently drain into lymph nodes and deliver cargo peptide antigens and nucleic acid-based adjuvants to lymph node-resident dendritic cells, thereby positioning them as an efficient platform for the co-delivery of antigens and adjuvants for tumor immunotherapy. In addition, experiments conducted during the development of embodiments of the present invention demonstrated a broad range of sHDL-based approaches by (1) targeting siRNA to hepatocytes, the natural target cells for endogenous HDL, and (2) targeting the immunostimulatory agent glycolide (α-galactosylceramide) to antigen-presenting cells.

[0108] These experiments further demonstrated the engineering of sHDL nanoparticles prepared with phospholipids and apolipoprotein AI-mimetic peptides and loaded with biopolymer drugs. To load peptide drugs into HDL nanodiscs, synthetic thiol-reactive phospholipids were utilized, enabling reduction-sensitive linkage of peptides to the surface of HDL nanodiscs. To load nucleic acids (including CpG motifs and siRNA), the nucleic acids were modified with cholesteryl moieties, which were found to enable facile insertion of nucleic acids into sHDL nanoparticles. To load glycolipids into HDL, hydrophobic interactions between glycolipids and HDL were utilized. These experiments further demonstrated the stable delivery of such cargoes to target tissues in vitro and in vivo. RNA interference In certain embodiments, sHDL nanoparticles are used within methods and systems for RNA interference.

[0109] RNA interference is a highly conserved mechanism induced by double-stranded RNA (dsRNA) that can downregulate the transcription of genes homologous to the dsRNA. dsRNA is initially processed by Dicer into short, 21-23 nt double-stranded RNAs called short interfering RNAs (siRNAs). Once incorporated into the RNA-induced silencing complex (RISC), they can mediate gene silencing by cleaving target mRNAs. "siRNA" or "small interfering ribonucleic acid" refers to two strands of ribonucleotides that hybridize along complementary regions under physiological conditions. An siRNA molecule contains a double-stranded region that is substantially identical to a region of the target gene's mRNA. A region with 100% identity to the corresponding sequence of the target gene is preferred. This condition is referred to as "fully complementary." However, depending on the length of the targeted mRNA region, the region may contain one, two, or three mismatches compared to the corresponding region of the target gene and therefore may not be fully complementary. The method of analyzing and identifying siRNA that has sufficient sequence identity to effectively inhibit the expression of specific target sequence is known in the art.Preferred mRNA target region is coding region.In addition, untranslated region such as 5'-UTR, 3'-UTR and splice site are suitable as long as this region is unique to mRNA target and not related to the poly A tail of mRNA.

[0110] In some embodiments, siRNA encapsulated within sHDL nanoparticles is utilized to practice methods and systems involving RNA interference.

[0111] Such embodiment is not limited to a particular size or type of siRNA molecule.For example, the length of the region of siRNA that is complementary to target can be 15-100 nucleotides, 18-25 nucleotides, 20-23 nucleotides, or more than 15, 16, 17, or 18 nucleotides.If there is mismatch with the corresponding target region, the length of complementary region generally needs to be somewhat longer.

[0112] In certain embodiments, it is believed that the siRNA delivery approach using sHDL nanoparticles disclosed herein (e.g., by encapsulating siRNA within sHDL nanoparticles) can inhibit any gene of interest.

[0113] The present invention is not limited to a specific method for producing sHDL nanoparticles containing encapsulated siRNA molecules. For example, in some embodiments, a lyophilization method is used to prepare homogeneous sHDL. In some embodiments, phospholipids and ApoA mimetic peptides are dissolved in glacial acetic acid and lyophilized. In some embodiments, the loading of siRNA molecules into sHDL nanoparticles is facilitated by cholesterol modification of the siRNA molecules. For example, siRNA is modified with cholesterol at the 3' sense strand (e.g., Kuwahara, H.; et al., Molecular Therapy 2011, 19(12), 2213-2221), and intermediate levels of chemical modification are used to stabilize siRNA in serum without significantly impairing silencing efficacy (see, e.g., Behlke, MA, Oligonucleotides 2008, 18(4), 305-319). In some embodiments, lyophilized phospholipids and ApoA-mimetic peptides are hydrated (e.g., in PBS (pH 7.4)) and thermocycled above and below the transition temperature (Tm) of the phospholipids to form blank sHDL, which are then incubated with cholesterol-modified siRNA at room temperature for an optimal time (e.g., 5, 10, 20, 25, 30, 35, 50, 80, 120, or 360 minutes) to form sHDL containing encapsulated siRNA. Figure 10 shows a schematic diagram of the lyophilization method for the rapid preparation of sHDL containing encapsulated siRNA.

[0114] Such embodiments are not limited to a particular technique for characterizing sHDL containing encapsulated siRNA. 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 Malvern Nanosizer instrument and a GPC assay.

[0115] Such embodiments are not limited to specific methods for evaluating the delivery profile of siRNA in vitro and in vivo. In some embodiments, labeling siRNA molecules with an imaging agent (e.g., the fluorescent dye Cy3) allows visualization of the organ-level biodistribution and intracellular delivery profile of siRNA molecules. In some embodiments, RT-PCR and Western blot are used to analyze target proteins at the mRNA and protein levels, respectively.

[0116] Therefore, in certain embodiments, the present invention provides a method for inhibiting a target gene in a cell, the method comprising introducing an siRNA capable of inhibiting the target gene by RNA interference into a cell, the siRNA comprising two complementary RNA strands, and the siRNA being encapsulated in an sHDL nanoparticle. In some embodiments, the 3' sense strand of the siRNA is modified with cholesterol. In some embodiments, the cell is in a human.

[0117] In certain embodiments, provided is sHDL nanoparticle, wherein the siRNA specific for proprotein convertase subtilisin / kexin 9 (PCSK9) is encapsulated in the sHDL nanoparticle.Compelling evidence shows that elevated plasma levels of low-density lipoprotein cholesterol (LDL-C) are the main risk factor for coronary heart disease (CHD) (see for example, Law, MR; et al., British Medical Journal 2003,326(7404),1423-1427; Boekholdt, SM; et al., Jama-Journal of the American Medical Association 2012,307(12),1302-1309; Sniderman, AD; et al., Circulation-Cardiovascular Quality and Outcomes 2011,4(3),337-U144). PCSK9 synthesized in the liver plays an important role in the regulation of LDL-C. PCSK9 can bind to the LDL receptor (LDLR) on hepatocytes and prevent the recycling of LDLR from lysosomes to the cell surface, which in turn leads to downregulation of LDLR and increased levels of LDL-C (e.g., Maxwell, KN; et al., Proceedings of the National Academy of Sciences of the United States, pp. 111-114, 2014). States of America 2004, 101(18), 7100-7105; Dadu, RT; et al., Nature Reviews Cardiology 2014, 11(10), 563-575; Horton, JD; et al., Trends in Biochemical Sciences 2007, 32(2), 71-77). Therefore, PCSK9 inhibition could potentially reduce LDL-C (see, e.g., Shen, L.; et al., Pharmacological Research 2013, 73, 27-34). Therapeutic approaches under development for in vivo PCSK9 inhibition include siRNA-mediated knockdown of PCSK9 and vaccination against PCSK9 (see, e.g., Fitzgerald, K.; et al., Lancet 2014, 383(9911), 60-68; Galabova, G.; et al., Circulation 2013, 128(22)). However, both strategies face a major challenge: how to effectively deliver therapeutic agents to target cells, i.e., hepatocytes and immune cells, respectively, to maximize the in vivo efficacy of each strategy.

[0118] Previously, PCSK9 siRNA has been delivered to hepatocytes by lipid nanoparticles (see, e.g., Frank-Kamenetsky, M.; et al., Proceedings of the National Academy of Sciences of the United States of America 2008, 105(33), 11915-11920) or by conjugating siRNA to N-acetylgalactosamine (GalNAc) ligands (see, e.g., Akinc, A.; et al., Molecular Therapy 2010, 18(7), 1357-1364), which allow siRNA to be passively targeted to hepatocytes or by recognition of the asialoglycoprotein receptor (ASGPR) on hepatocytes. However, these conventional delivery methods can expose siRNA molecules to the intracellular endosomal / lysosomal pathway, where the siRNA cargo may be degraded, resulting in suboptimal knockdown of PCSK9. Therefore, there is an urgent need to develop strategies that can both target hepatocytes and bypass the endosomal / lysosomal pathway.

[0119] The use of sHDL nanoparticles containing encapsulated PCSK9 siRNA molecules overcomes these limitations. Indeed, sHDL nanoparticles have properties similar to endogenous HDL, and they can inherently target hepatocytes after intravenous injection, allowing direct delivery of siRNA cargo to the cytosol of hepatocytes and knockdown of PCSK9 without passing through the intracellular endosomal / lysosomal pathway.

[0120] In certain embodiments, sHDL containing encapsulated PCSK9 siRNA molecules are delivered to the cytosol, where they can bind to the RNA-induced silencing complex (RISC) to knock down PCSK9 protein (see, e.g., Chendrimada, TP; et al., Nature 2005, 436(7051), 740-744; Matranga, C.; et al., Cell 2005, 123(4), 607-620) in SR-BI-expressing hepatocytes (see, e.g., Goldstein, JL; Brown, MS, Arteriosclerosis Thrombosis and Vascular Biology 2009, 29(4), 431-438; Wolfrum, C.; et al., Nature Biotechnology 2007, 25(10), 1149-1157).

[0121] FIG. 11 shows a schematic diagram of using sHDL to regulate PCSK9 for LDL-C management.

[0122] The present invention is not limited to the use of a specific PCSK9 siRNA sequence. In some embodiments, the PCSK9 siRNA sequence is cross-reactive with mouse, rat, non-human primate, and human PCSK9 mRNA (see, e.g., Frank-Kamenetsky, et al., Proceedings of the National Academy of Sciences of the United States of America 2008, 105(33), 11915-11920).

[0123] In certain embodiments, the present invention provides a method for inhibiting the PCSK9 gene in a cell, the method comprising: The method comprises introducing siRNA into cells, wherein the PCSK9 siRNA comprises two complementary RNA strands, and the PCSK9 siRNA is encapsulated in sHDL nanoparticles. In some embodiments, the PCSK9 siRNA is modified with cholesterol at the 3' sense strand. In some embodiments, the cells are in humans.

[0124] In certain embodiments, the present invention provides a method for reducing serum LDL-C levels in a patient (e.g., a human patient), the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising PCSK9 siRNA encapsulated in sHDL nanoparticles, wherein the PCSK9 siRNA is capable of inhibiting the PCSK9 gene by RNA interference, the PCSK9 siRNA comprising two RNA strands that are complementary to each other, and wherein inhibition of the PCSK9 gene results in a reduction of serum LDL-C levels.

[0125] In certain embodiments, the present invention provides a method for treating coronary heart disease in a patient by reducing the patient's serum LDL-C level, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising PCSK9 siRNA encapsulated in sHDL nanoparticles, wherein the PCSK9 siRNA is capable of inhibiting the PCSK9 gene by RNA interference, the PCSK9 siRNA comprising two RNA strands complementary to each other, and wherein inhibition of the PCSK9 gene results in a reduction of serum LDL-C level.

[0126] In certain embodiments, sHDL nanoparticles are used to stimulate an immune response. Such embodiments are not limited to a particular manner of stimulating an immune response. Immune response stimulation The immune system can be divided into two functional subsystems: the innate immune system and the adaptive immune system. The innate immune system is the first line of defense against infection; most potential pathogens are rapidly neutralized by this system before they can cause significant infection. The adaptive immune system responds to molecular structures called antigens on invading organisms. There are two types of adaptive immune responses: humoral and cell-mediated. In humoral immune responses, antibodies secreted by B cells into bodily fluids bind to antigens from pathogens, leading to their elimination through various mechanisms, such as complement-mediated lysis. In cell-mediated immune responses, T cells are activated, which can destroy other cells. For example, disease-related proteins, when present within cells, are proteolytically fragmented into peptides within the cell. Specific cellular proteins then attach themselves to the antigens or peptides thus formed, transport them to the cell surface, and present them to the body's molecular defense mechanisms, particularly T cells. Cytotoxic T cells recognize these antigens and kill the antigen-bearing cells.

[0127] Molecules that transport and present peptides on the cell surface are called major histocompatibility complex (MHC) proteins. MHC proteins are classified into two types, MHC class I and MHC class II. The structures of the two MHC class proteins are very similar. However, they have very different functions. MHC class I proteins are present on the surface of almost all cells in the body, including most tumor cells. MHC class I proteins are usually loaded with antigens derived from endogenous proteins or from intracellular pathogens and then presented to naive T lymphocytes or cytotoxic T lymphocytes (CTLs). MHC class II proteins are present on dendritic cells, B lymphocytes, macrophages, and other antigen-presenting cells. They primarily present external antigen sources, i.e., extracellularly processed peptides, to T helper (Th) cells. Most peptides bound by MHC class I proteins are derived from cytoplasmic proteins produced by the body's own healthy host cells and do not usually stimulate an immune response. Thus, cytotoxic T lymphocytes that recognize such self-peptide-presenting MHC class I molecules are either defective in the thymus (central tolerance) or become defective or inactivated, i.e., tolerized, after release from the thymus (peripheral tolerance). When MHC molecules present peptides to non-tolerized T lymphocytes, they can stimulate an immune response. Cytotoxic T lymphocytes have both T cell receptors (TCRs) and CD8 molecules on their surface. T cell receptors can recognize and bind peptides complexed with MHC class I molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor that can bind to a specific MHC / peptide complex.

[0128] Before being presented on the cell surface, peptide antigens attach themselves to MHC class I molecules in the endoplasmic reticulum through competitive affinity binding. Here, the affinity of an individual peptide antigen is directly related to its amino acid sequence and the presence of specific binding motifs at defined positions within the amino acid sequence. If the sequences of such peptides are known, it is possible to manipulate the immune system against diseased cells using, for example, peptide vaccines.

[0129] Peptide-based cancer vaccines have been widely investigated due to their favorable safety profile and ease of production and quality control. However, their antitumor effects in clinical trials have been disappointing, a phenomenon attributed to the inefficient co-delivery of Ag peptides and adjuvants to draining lymph nodes (dLNs) and subsequent immune tolerance and CTL fratricidality (see, for example, Toes, RE, et al., Proc. Natl. Acad. Sci. USA 93, 7855-7860 (1996); Su, MW, et al., J. Immunol. 151, 658-667 (1993); Melief, CJ & van der Burg, SH Nat. Rev. Cancer 8, 351-360 (2008)). Depot-forming water-in-oil adjuvant systems can improve immunogenicity (see, e.g., Speiser, DE et al. J. Clin. Invest. 115, 739-746 (2005); Fourcade, J. et al. J. Immunother. 31, 781-791 (2008)), and booster immunizations can cause sequestration of T cells at the vaccine site, leading to T cell exhaustion and deficiency (see, e.g., Rezvani, K. et al. Haematologica 96, 432-440 (2011); Hailemichael, Y. et al. Nat. Med. 19, 465-472 (2013)). To address these issues, various nanoparticle-based vaccine systems have been evaluated in animal models (see, e.g., Reddy, ST et al. Nat. Biotechnol. 25, 1159-1164 (2007); Li, AV et al. Sci. Transl. Med. 5, 204ra130 (2013); Jeanbart, L. et al. Cancer. Immunol. Res. 2, 436-447 (2014); Xu, Z., et al., ACS Nano 8, 3636-3645 (2014); Liu, H. et al. Nature 507, 519-522 (2014); Fan, Y. & Moon, JJ Vaccines (Basel) 3, 662-685 (2015)).However, potential safety concerns and scale-up manufacturing of nanoparticles remain major challenges, especially in approaches suitable for personalized therapy using patient-specific neoantigens.

[0130] Experiments conducted in the course of developing embodiments of the present invention developed an alternative, simple approach in which preformed nanoparticles, with established clinical manufacturing procedures and an excellent safety profile in humans, were mixed with Ag peptides and adjuvants to produce a "personalized" cancer vaccine (Figure 12). The approach was based on synthetic high-density lipoprotein (sHDL) nanodiscs composed of phospholipids and apolipoprotein A1 (ApoA1)-mimetic peptides. Other HDLs containing the 243 amino acid ApoA-I purified from human plasma or recombinantly produced (e.g., Wolfrum, C. et al. Nat. Biotechnol. 25, 1149-1157 (2007); Diditchenko, S. et al. al. Arterioscler. Thromb. Vasc. Biol. 33, 2202-2211 (2013); Fischer, N.O. et al. J. Am. Chem. Soc. 135, 2044-2047 (2013); Tardy, C. et al. Atherosclerosis 232, 110-118 (2014); Duivenvoorden, R. et al. Nat. Commun. 5, 3065 (2014)). In comparison with endogenous ApoA-I, we synthesized sHDL nanodiscs bearing a 22-mer peptide (22A) derived from the repeat α-helical domain of ApoA1, which has no sequence homology with endogenous ApoA-I (see, e.g., U.S. Patent No. 6,734,169; U.S. Patent No. 8,378,068; Li, D., Gordon, S., Schwendeman, A. & Remaley, A. Apolipoprotein A. mimetic peptides for stimulating cholesterol efflux.in Apolipoprotein Mimetics in the Management of Human Disease (eds. Anantharamaiah, GM & Goldberg, D.) 29-42 (Springer, Switzerland, 2015)). This avoids the potential induction of autoimmunity. Importantly, sHDL has previously been produced for clinical trials and has been shown to be approximately 2.2 g / m in humans. 2 It has been proven safe at the maximum tolerated dose (e.g., Khan, M., et al., Circulation 108(Suppl IV), 563-564(2003); Miles, J., et al., Proceedings of Arteriosclerosis Thrombosis and Vascular Biology 24, E19-E19 (2004)), values ​​one to two orders of magnitude greater than most polymeric or inorganic nanoparticles in clinical trials (see, e.g., Alexis, F., et al., Mol. Pharm. 5, 505-515 (2008); Anselmo, AC & Mitragotri, SA, AAPS J 17, 1041-1054 (2015)).

[0131] Experiments conducted during the development of an embodiment of the present invention developed a nanodisc-based platform for neoantigen vaccination (Figure 12). Taking advantage of HDL's inherent role as a nanoparticle for cholesterol, the immunostimulant CpG, a potent Toll-like receptor-9 agonist, was modified with cholesterol (Cho-CpG) to enhance in vivo delivery. Preformed sHDL nanodiscs were shown to be capable of simply mixing with cholesteryl-CpG containing neoantigens identified by tumor DNA sequencing and tumor Ag peptides to produce homogeneous and stable ultrasmall nanodiscs in less than 2 hours at room temperature (RT). The nanodiscs efficiently promoted Ag / CpG co-delivery to dLNs, prolonged Ag presentation on antigen-presenting cells (APCs), and elicited significant levels of CTL responses with antitumor efficacy. Because of the previously demonstrated facile manufacturing process, potent therapeutic efficacy, and clinical safety (see, e.g., Khan, M., et al., Circulation 108(Suppl IV), 563-564 (2003); Miles, J., et al. Proceedings of Arteriosclerosis Thrombosis and Vascular Biology 24, E19-E19 (2004)), this approach provides an attractive platform technology for patient-tailored cancer vaccines and other bioactive therapeutics.

[0132] Thus, in certain embodiments, nanoparticles conjugated with an antigen (e.g., sHDL nanoparticles) are used to induce an immune response. In some embodiments, the nanoparticles are further complexed or mixed with an adjuvant (e.g., a dendritic cell targeting molecule (DC)). In some embodiments, the nanoparticles are co-administered with an adjuvant.

[0133] Such embodiments are not limited to a particular antigen. Indeed, the antigen can be a peptide, protein, polysaccharide, sugar, lipid, glycolipid, nucleic acid, or a combination thereof. The antigen can be derived from any source, including, but not limited to, a virus, a bacterium, a parasite, a plant, a protozoan, a fungus, a tissue, or a transformed cell, such as a cancer or leukemia cell, and can be a whole cell or an immune component thereof, such as a cell wall component or a molecular component thereof.

[0134] In some embodiments, antigens are known in the art and available from commercial, government, and scientific sources. In some embodiments, antigens are fully inactivated or attenuated organisms. These organisms may be infectious organisms such as viruses, parasites, and bacteria. These organisms may also be tumor cells. Antigens may be purified or partially purified polypeptides derived from tumor or viral or bacterial sources. Criteria for identifying and selecting effective antigenic peptides (e.g., minimal peptide sequences capable of eliciting an immune response) can be found in the art. Antigens may be recombinant polypeptides produced by expressing DNA encoding the polypeptide antigen in a heterologous expression system. Antigens may be DNA encoding all or part of the antigen protein. The DNA may be in the form of vector DNA, such as plasmid DNA.

[0135] Antigens may be provided as single antigens or in combination. Antigens may also be provided as complex mixtures of polypeptides or nucleic acids.

[0136] In some embodiments, the antigen is an autoantigen. As used herein, the term "autoantigen" refers to an immunogenic antigen or epitope that is unique to a mammal and that may be involved in the pathogenesis of an autoimmune disease.

[0137] In some embodiments, the antigen is a viral antigen, selected from the following virus families: Arenaviridae, Artervirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., coronaviruses such as Severe Acute Respiratory Syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltaviridae, and the like. viruses, Dianthoviruses, Enamoviruses, 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 1, Human herpesvirus 3, Human herpesvirus 4, Human herpesvirus 5, and Human herpesvirus 6) , and cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Liposthrixviridae, Microviridae, Orthomyxoviridae (e.g., influenza virus A, influenza virus B, and influenza virus C), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentiviruses such as human immunodeficiency virus (HIV)-1 and HIV-2), Rhabdoviridae (e.g., rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (e.g., rubella virus, dengue virus, etc.), and Totiviridae.Suitable viral antigens also include all or part of dengue protein M, dengue protein E, dengue D1NS1, dengue D1NS2 and dengue D1NS3.

[0138] Viral antigens may be derived from specific strains of papillomavirus, herpesvirus, i.e., herpes simplex 1 and herpes simplex 2; hepatitis virus, 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 virus; parainfluenza, varicella-zoster, cytomegalovirus, Epstein-Barr, rotavirus, rhinovirus, adenovirus, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choriomyelitis.

[0139] In some embodiments, the antigen is a bacterial antigen, including Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Haemophilus influenzae type B (HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, Meningococcus B, and Meningococcus C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochlorous, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, as well as Treponema, Vibrio, and Yersinia.

[0140] In some embodiments, the antigen is a parasitic antigen. Parasitic antigens include, but are not limited to, parasites such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial The antigens can be derived from antigens derived from Plasmodium trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni. These include sporozoan antigens, plasmodial antigens, such as all or part of Plasmodium sporozoite surface proteins, sporozoite surface proteins, liver stage antigens, apical membrane-associated proteins, or merozoite surface proteins.

[0141] In some embodiments, the antigens are allergens and environmental antigens, including, but not limited to, antigens derived from natural allergens, such as pollen allergens (tree pollen allergens, herb pollen allergens, weed pollen allergens, and grass pollen allergens), insect allergens (inhalant allergens, saliva allergens, and venom allergens), animal hair and dander allergens, and food allergens. Important pollen allergens from trees, grasses and herbs arise from the taxonomic orders Fagales, Oleaceales, Pinales and Platanales (including, inter alia, birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedars (Cryptomeria and Juniperus), sycamore (Platanus)), Poales (i.e., including grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale and Sorghum), Asterales and Urticales (including, inter alia, herbs of the genera Ambrosia, Artemisia and Parietaria). Other allergen antigens that may be used include allergens derived from venom allergens, including those derived from stinging insects such as house dust mites of the genera Dermatophagoides and Euroglyphus, dust mites (e.g., Lepidoglyphys, Glycyphagus, and Tyrophagus), cockroaches, horseflies, and fleas (e.g., Blatella, Periplaneta, Chironomus, and Ctenocepphalides), mammals such as cats, dogs, horses, and birds, and those from the taxonomic order Hymenoptera, which includes bees (superfamily Apidae), hornets (superfamily Vespidae), and ants (superfamily Formicidae). Still other allergen antigens that may be used include inhalant allergens derived from fungi such as the genera Alternaria and Cladosporium.

[0142] In some embodiments, the antigen is a tumor antigen, such as α-actinin-4, Bcr-Abl fusion protein, Casp-8, β-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, Mum-2, and Mum-3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, or the like. Protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Bage-1, Gage3, Gage4, Gage5, Gage6, Gage7, GnTV, Herv-K-mel, Lage-1, Mage-A1, Mage-A2, Mage-A3, Mage-A4, Mage-A6, Mage-A10, Mage-A12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGS), SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, 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).The antigen may be a tumor antigen, including tumor-associated or tumor-specific antigens, such as, but not limited to, 29 BCAAs), CA195, CA242, CA-50, CAM43, CD68 KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein, cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS.

[0143] One of the major obstacles to the development of curative and tumor-specific immunotherapy is the identification and selection of highly specific and restricted tumor antigens to circumvent autoimmunity. Tumor neoantigens, which arise as a result of genetic alterations in malignant cells (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.), represent the most tumor-specific antigen class.

[0144] In some embodiments, the antigen is a neoantigen. The term neoantigen is used herein to define any newly expressed antigenic determinant. Neoantigens can arise as newly expressed determinants (especially on the surface of transformed or infected cells) upon protein conformational changes, as a result of complex formation of one or more molecules, or as a result of molecular cleavage resulting in the presentation of new antigenic determinants. Thus, as used herein, the term neoantigen encompasses antigens expressed during infection (e.g., viral, protozoan, or bacterial infection), prion-mediated diseases, and cell transformation (cancer); in the latter case, neoantigens are sometimes referred to as tumor-associated antigens.

[0145] The present invention is not limited to a particular technique for identifying neoantigens. In some embodiments, identifying neoantigens involves identifying all or nearly all mutations in a neoplasm / tumor at the DNA level using whole-genome sequencing of the tumor, whole-exome (e.g., captured exons only) sequencing of the tumor, or RNA sequencing of the tumor on matched germline samples from each patient. In some embodiments, identifying neoantigens involves analyzing the identified mutations with one or more peptide-MHC binding prediction algorithms to generate a plurality of candidate neoantigen T-cell epitopes expressed in the neoplasm / tumor and capable of binding to patient HLA alleles. In some embodiments, identifying neoantigens involves synthesizing a plurality of candidate neoantigen peptides selected from a set of all neo open reading frame peptides and predicted binding peptides used in cancer vaccines.

[0146] Thus, the present invention is based, at least in part, on the ability to identify all or nearly all mutations within a neoplasm / tumor (e.g., translocations, inversions, large and small deletions and insertions, missense mutations, splice site mutations, etc.). In particular, these mutations are present in the genome of a subject's neoplasm / tumor cells but are absent in normal tissues derived from the subject. Such mutations are of particular interest if they result in changes that result in proteins with altered amino acid sequences unique to the patient's neoplasm / tumor (e.g., neoantigens). For example, useful mutations may include: (1) nonsynonymous mutations resulting in different amino acids in the protein; (2) read-through mutations in which a stop codon is altered or deleted, resulting in translation of a longer protein with a novel tumor-specific sequence at the C-terminus; (3) splice site mutations that contain introns in the mature mRNA, thereby resulting in a unique tumor-specific protein sequence; (4) chromosomal rearrangements (i.e., gene fusions) that result in chimeric proteins with tumor-specific sequences at the junction of two proteins; (5) frameshift mutations or deletions that result in new open reading frames with novel tumor-specific protein sequences; and the like. For example, peptides with mutations or mutant polypeptides resulting from splice site, frameshift, readthrough, or gene fusion mutations in tumor cells may be identified by sequencing DNA, RNA, or protein of tumor versus normal cells.

[0147] Individualized neoantigen peptides derived from common tumor driver genes are also within the scope of the present invention and may further include previously identified tumor-specific mutations.

[0148] Preferably, any suitable sequencing-by-synthesis platform can be used to identify mutations. Four major sequencing-by-synthesis platforms are currently available: the Genome Sequencers from Roche / 454 Life Sciences, the SOLiD system from Applied BioSystems, and the Heliscope system from Helicos Biosciences. Sequencing-by-synthesis platforms have also been described by Pacific Biosciences and VisiGen Biotechnologies. Each of these platforms can be used in the methods of the present invention. In some embodiments, multiple nucleic acid molecules to be sequenced are attached to a support (e.g., a solid support). To immobilize the nucleic acids on the support, a capture sequence / universal priming site can be added to the 3' and / or 5' end of the template. The nucleic acid may be attached to the support by hybridizing the capture sequence to a complementary sequence covalently attached to the support. The capture sequence (also called a universal capture sequence) is a nucleic acid sequence complementary to the support-attached sequence that can double as a universal primer.

[0149] Instead of capture sequences, members of a coupling pair (e.g., antibody / antigen, receptor / ligand, or avidin-biotin pair, as described in U.S. Patent Application Publication No. 2006 / 0252077) may be linked to each fragment to be captured on a surface coated with the second member of that binding pair. Following capture, the sequence may be analyzed, for example, by single-molecule detection / sequencing, including sequencing by template-dependent synthesis, as described in the Examples and U.S. Patent No. 7,283,337. In decoding-by-synthesis methods, surface-bound molecules are exposed to multiple labeled nucleotide triphosphates in the presence of a polymerase. The sequence of the template is determined by the order of labeled nucleotides incorporated at the 3' end of the growing strand. This can be done in real time or step-and-repeat mode. For real-time analysis, different optical labels for each nucleotide may be incorporated, and multiple lasers may be utilized for stimulation of the incorporated nucleotides.

[0150] Any cell type or tissue can 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 body fluids, such as blood, by known techniques (e.g., venipuncture) or saliva.Alternatively, nucleic acid testing can be performed on dry samples (e.g., hair or skin).

[0151] Various methods are available for detecting the presence of specific mutations or alleles in an individual's DNA or RNA. Advances in this field have provided accurate, easy, and inexpensive large-scale SNP genotyping. More recently, several new techniques have been described, including dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMan system, and various DNA "chip" technologies such as the Affymetrix SNP chip. These methods require amplification of the target gene region, usually by PCR. Yet other newly developed methods based on invasive cleavage followed by mass spectrometry or the generation of small signal molecules by immobilized padlock probes and rolling circle amplification may ultimately eliminate the need for PCR. Some of the methods known in the art for detecting specific single nucleotide polymorphisms are summarized below. It is understood that the method of the present invention encompasses all available methods.

[0152] PCR-based detection means may involve multiplex amplification of multiple markers simultaneously, for example, it is well known in the art to select PCR primers to generate PCR products that do not overlap in size and can be analyzed simultaneously.

[0153] Alternatively, different markers can be amplified using primers that are differently labeled and therefore can be differentially detected. Naturally, hybridization-based detection means allow for differential detection of multiple PCR products in a sample. Other techniques that allow for multiplex analysis of multiple markers are known in the art.

[0154] Several methods have been developed to facilitate the analysis of single nucleotide polymorphisms in genomic DNA or cellular RNA. In one embodiment, single nucleotide polymorphisms can be detected by using specialized exonuclease-resistant nucleotides, as disclosed in U.S. Pat. No. 4,656,127, for example. According to this method, a primer complementary to the allelic sequence immediately 3' from the polymorphic site is allowed to hybridize to a target molecule 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 present, that derivative will be incorporated into the end of the hybridized primer. This incorporation confers exonuclease resistance to the primer, thereby enabling its detection. Since the identity of the exonuclease-resistant derivative in the sample is known, the finding that the primer is exonuclease-resistant indicates that the nucleotide present at 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 large amounts of unrelated sequence data.

[0155] In another embodiment of the present invention, a solution-based method is used to determine the identity of the nucleotide at a polymorphic site (see, e.g., French Patent No. 2,650,840; PCT Application No. WO 1991 / 02087). As in the method of U.S. Patent No. 4,656,127, a primer complementary to the allelic sequence immediately 3' to the polymorphic site may be used. This method determines the identity of the nucleotide at that site using a labeled dideoxynucleotide derivative that, if complementary to the nucleotide at the polymorphic site, becomes incorporated onto the end of the primer.

[0156] An alternative method known as Genetic Bit Analysis (GBA®) is described in International Patent Publication No. WO 1992 / 15712. GBA® uses a mixture of labeled terminators and primers complementary to the sequence 3' to the polymorphic site. The labeled terminators incorporated are therefore determined by and complementary to the nucleotides present at the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent No. 2,650,840; International Patent Application No. WO 1991 / 02087), the GBA® method is preferably a heterogeneous assay in which either the primers or the target molecule are immobilized on a solid phase. Recently, several primer-directed nucleotide incorporation procedures for assaying polymorphic sites in DNA have been described (e.g., Komher, J. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990); Kuppuswamy, M. et al., Proc. Natl. Acad. Sci. (USA) 88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. et al. (See, e.g., Syvanen, A.-C, et al., Anal. Biochem. 208:171-175 (1993)). These methods differ from GBA® in that they all rely on the incorporation of labeled deoxynucleotides to discriminate between bases at polymorphic sites. In such formats, signal is proportional to the number of deoxynucleotides incorporated, so that polymorphisms occurring in runs of the same nucleotide can result in a signal proportional to the length of the run (see, e.g., Syvanen, A.-C, et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

[0157] An alternative method for identifying tumor-specific neoantigens is direct protein sequencing. Protein sequencing of enzymatic digests using multidimensional MS techniques (MSn), including tandem mass spectrometry (MS / MS), can also be used to identify neoantigens of the present invention. Such proteomic approaches allow for rapid and highly automated analysis (see, e.g., K. Gevaert and J. Vandekerckhove, Electrophoresis 21:1145-1154 (2000)). It is further contemplated within the scope of the present invention that high-throughput methods for de novo sequencing of unknown proteins can be used to analyze the proteome of a patient's tumor to identify expressed neoantigens. For example, meta-shotgun protein sequencing may be used to identify expressed neoantigens (see, e.g., Guthals et al. (2012) Shotgun Protein Sequencing with Meta-contig Assembly, Molecular and Cellular Proteomics 11(10):1084-96).

[0158] Tumor-specific neoantigens may also be identified using MHC multimers to identify neoantigen-specific T cell responses. For example, high-throughput analysis of neoantigen-specific T cell responses in patient samples may be performed using MHC tetramer-based screening technologies (see, e.g., Hombrink et al. (2011) High-Throughput Identification of Potential Minor Histocompatibility Antigens by MHC Tetramer-Based Screening: Feasibility and Limitations 6(8):1-11; Hadrup et al. (2009) Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers, Nature Methods, 6(7):520-26; van Rooij et al. (2013) Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an ipilimumab-responsive melanoma, Journal of Clinical Oncology, 31:1-4; and Heemskerk et al. (2013) The cancer antigenome, EMBO Journal, 32(2):194-203). It is considered within the scope of the present invention that such tetramer-based screening techniques may be used for the initial identification of tumor-specific neoantigens or as a secondary screening protocol to evaluate neoantigens to which patients may have already been exposed, thereby facilitating the selection of candidate neoantigens for the vaccines of the present invention.

[0159] The present invention further includes isolated peptides (e.g., neoantigenic peptides containing tumor-specific mutations identified by the described methods, peptides containing known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the described methods). These peptides and polypeptides are referred to herein as "neoantigenic peptides" or "neoantigenic polypeptides." The polypeptides or peptides can be of various lengths and will minimally contain a small region predicted to bind to a patient's HLA molecule (the "epitope") and additional flanking amino acids extending at both the N- and C-termini. The polypeptides or peptides can be in either neutral (uncharged) or salt form and can be either free of or contain modifications such as glycosylation, side chain oxidation, or phosphorylation, provided that the modifications do not destroy the biological activity of the polypeptide as described herein.

[0160] In certain embodiments, the size of at least one neoantigenic peptide molecule can include, but is not limited to, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino acid residues, and any range derivable therein. In specific embodiments, the neoantigenic peptide molecule is 50 amino acids or less. In preferred embodiments, the neoantigenic peptide molecule is equal to about 20 to about 30 amino acids.

[0161] Thus, the present invention provides nanoparticles (e.g., sHDL nanoparticles) conjugated to one or more neoantigenic peptides. In some embodiments, the nanoparticles (e.g., sHDL nanoparticles) are conjugated to one neoantigenic peptide. In some embodiments, the nanoparticles (e.g., sHDL nanoparticles) are conjugated to two neoantigenic peptides. In some embodiments, the nanoparticles (e.g., sHDL nanoparticles) are conjugated to at least five or more neoantigenic peptides. In some embodiments, the nanoparticles (e.g., sHDL nanoparticles) are conjugated to at least about 6, about 8, about 10, about 12, about 14, about 16, about 18, or about 20 different peptides. In some embodiments, the nanoparticles (e.g., sHDL nanoparticles) are conjugated to at least 20 or more different peptides.

[0162] Neoantigenic peptides, polypeptides, and analogs can be further modified to contain additional chemical moieties not normally part of proteins. These derivatized moieties can improve solubility, biological half-life, protein absorption, or binding affinity. The moieties can also reduce or eliminate any desired side effects of the protein. A summary of these moieties is provided in Remington's Pharmaceutical Sciences, 2009. thed., Mack Publishing Co., Easton, PA (2000). For example, neoantigenic peptides and polypeptides with desired activity may be modified as needed to provide certain desired attributes, e.g., improved pharmacological properties, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide in binding to the desired MHC molecule and activating appropriate T cells. For example, neoantigenic peptides and polypeptides may undergo various changes, such as conservative or non-conservative substitutions, which may provide certain advantages in use, such as improved MHC binding. Such conservative substitutions may involve exchanging one amino acid residue for another that is biologically and / or chemically similar, e.g., one hydrophobic residue for another hydrophobic residue, or one polar residue for another polar residue. The effects of single amino acid substitutions may also be investigated using D-amino acids. Such modifications may be carried out using well-known peptide synthesis procedures, as described, for example, in Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (NY, Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, III., Pierce), 2nd Ed. (1984).

[0163] In some embodiments, neoantigenic peptides and polypeptides may be modified with a linking agent intended to facilitate complexation with nanoparticles (e.g., sHDL nanoparticles). The present invention is not limited to a particular type or kind of linking agent. In some embodiments, the linking agent is a cysteine-serine-serine (CSS) molecule.

[0164] 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 when mixed, the DOPE-PDP and CSS engage, thereby resulting in the complexation (linkage) of the CSS-Ag with the sHDL.

[0165] Neoantigenic peptides and polypeptides may also be modified by extending or reducing the amino acid sequence of the compound, for example, by adding or deleting amino acids. Neoantigenic peptides, polypeptides, or analogs may also be modified by changing the order or composition of specific residues. Those skilled in the art will understand that certain amino acid residues essential for biological activity, such as residues at critical contact sites or conserved residues, generally will not be altered without adversely affecting the biological activity. Non-essential amino acids need not be limited to amino acids naturally occurring in proteins, such as La-amino acids or their D-isomers, but may also include unnatural amino acids, such as β-γ-δ-amino acids, as well as many derivatives of La-amino acids.

[0166] Typically, neoantigen polypeptides or peptides may be optimized by using a series of peptides with single amino acid substitutions to determine the effect of electrostatic charge, hydrophobicity, and other factors 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 made 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, or Pro or similar residues may be used. Substitutions may be homo- or hetero-oligomeric. The number and type of residues substituted or added depend on the spacing required between essential contact points and the specific functional attributes desired (e.g., hydrophobicity vs. hydrophilicity). Increased binding affinity for MHC molecules or T cell receptors may also be achieved by such substitutions compared to the affinity of the parent peptide. In any case, such substitutions should use amino acid residues or other molecular fragments selected to prevent, for example, steric hindrance and charge interference that could disrupt binding. Amino acid substitutions are usually of single residues. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at a final peptide.

[0167] Those skilled in the art will appreciate that there are various methods for producing such tumor-specific neoantigens. Generally, such tumor-specific neoantigens may be produced either in vitro or in vivo. Tumor-specific neoantigens may be produced in vitro as peptides or polypeptides, which may then be formulated into personalized neoplasia vaccines and administered to subjects. Such in vitro production may occur by various methods known to those skilled in the art, such as, for example, peptide synthesis or expression of peptides / polypeptides from DNA or RNA molecules in any of a variety of bacterial, eukaryotic, or viral recombinant expression systems, followed by purification of the expressed peptides / polypeptides.

[0168] Alternatively, tumor-specific neoantigens may be produced in vivo by introducing a molecule (e.g., DNA, RNA, viral expression system, etc.) encoding the tumor-specific neoantigen into a subject, whereupon the encoded tumor-specific neoantigen is expressed.

[0169] Proteins or peptides may be produced by any technique known to those of skill in the art, including expression of the protein, polypeptide, or peptide by standard molecular biology techniques, isolation of the protein or peptide from a natural source, or chemical synthesis of the protein or peptide. Nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have been previously disclosed and may be found in computer databases known to those of skill in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information at the National Institutes of Health website. The coding regions of known genes may be amplified and / or expressed using techniques disclosed herein or known to those of skill in the art. Alternatively, various commercially available preparations of proteins, polypeptides, and peptides are known to those of skill in the art.

[0170] Peptides can be easily synthesized chemically using reagents that do not contain contaminating bacterial or animal materials (Merrifield RB: Solid phase peptide synthesis). synthesis.I.The synthesis of a tetrapeptide.J.Am.Chem.Soc.85:2149-54,1963).

[0171] A further aspect of the present invention provides nucleic acids (e.g., polynucleotides) encoding the neoantigenic peptides of the present invention, which may 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 natural or stabilized forms, such as polynucleotides with phosphorothioate backbones, or combinations thereof, which may or may not contain introns, so long as they encode the peptide. A further aspect of the present invention provides expression vectors capable of expressing the polypeptides of the present invention. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, DNA is inserted into an expression vector, such as a plasmid, in the appropriate orientation and correct reading frame for expression. If necessary, the DNA may be linked to appropriate transcriptional and translational control nucleotide sequences recognized by the desired host (e.g., bacteria); such controls are generally available on expression vectors. The vector is then introduced into a host bacterium for cloning using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0172] The present invention further encompasses variants and equivalents that are substantially homologous to the identified tumor-specific neoantigens described herein. These can contain, for example, conservative substitution mutations, i.e., the replacement of one or more amino acids with similar amino acids. For example, a conservative substitution refers to the substitution of an amino acid for another amino acid within the same general class, such as the substitution of one acidic amino acid for another acidic amino acid, one basic amino acid for another basic amino acid, or one neutral amino acid for another neutral amino acid. What is intended by conservative amino acid substitution is well known in the art.

[0173] The present invention also includes expression vectors containing the isolated polynucleotides and host cells containing the expression vectors. It is also contemplated within the scope of the present invention that neoantigenic peptides may be provided in the form of RNA or cDNA molecules encoding the desired neoantigenic peptides. The present invention also provides that one or more neoantigenic peptides of the present invention may be encoded by a single expression vector. The present invention also provides that one or more neoantigenic peptides of the present invention may be encoded and expressed in vivo using a viral-based system (e.g., an adenoviral system).

[0174] The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that contain only the coding sequence for a polypeptide, as well as polynucleotides that contain additional coding and / or non-coding sequences. Polynucleotides of the invention can be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or non-coding (antisense) strand.

[0175] In embodiments, the polynucleotide may include a coding sequence for a tumor-specific neoantigenic peptide fused in the same reading frame as the polynucleotide, which aids in the expression and / or secretion of the polypeptide from a host cell (e.g., a leader sequence that functions as a secretory sequence to control transport of the polypeptide out of the cell). Polypeptides with leader sequences can be preproteins, with the leader sequence being cleaved by the host cell to form the mature form of the polypeptide.

[0176] In some embodiments, the polynucleotide can include a coding sequence for a tumor-specific neoantigenic peptide fused in the same reading frame as a marker sequence, which, for example, allows for purification of the encoded polypeptide, which may then be incorporated into a personalized neoplasia vaccine. For example, the marker sequence can be a hexa-histidine tag provided by the pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. Additional tags include, but are not limited to, calmodulin tag, FLAG tag, Myc tag, S tag, SBP tag, Softag1, Softag3, V5 tag, Xpress tag, Isopeptag, SpyTag, biotin carboxyl carrier protein (BCCP) tag, GST tag, fluorescent protein tag (e.g., green fluorescent protein tag), maltose binding protein tag, Nus tag, Strep tag, thioredoxin tag, TC tag, Ty tag, etc. In some embodiments, the polynucleotide may comprise coding sequences for one or more of the tumor-specific neoantigenic peptides fused in the same reading frame to create a single concatemerized neoantigenic peptide construct capable of producing multiple neoantigenic peptides.

[0177] In some embodiments, the present invention provides isolated nucleic acid molecules having a nucleotide sequence that is at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 96%, 97%, 98%, or 99% identical to a polynucleotide encoding a tumor-specific neoantigenic peptide of the present invention.

[0178] A polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence can contain up to 5 point mutations for every 100 nucleotides of the reference nucleotide sequence.In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with other nucleotides, or up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence.These mutations of the reference sequence can occur at the amino-terminal or carboxy-terminal position of the reference nucleotide sequence, or between these terminal positions, and can be distributed separately between the nucleotides in the reference sequence or in one or more adjacent groups within the reference sequence.

[0179] In practice, whether any particular nucleic acid molecule is at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments at least 95%, 96%, 97%, 98%, or 99% identical to a reference sequence can be conventionally determined using known computer programs, such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the invention, parameters are set such that the percentage of identity is calculated across the full-length reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0180] The isolated tumor-specific neoantigenic peptides described herein can be produced in vitro (e.g., in a laboratory) by any suitable method known in the art. Such methods range from direct protein synthesis to constructing DNA sequences encoding the isolated polypeptide sequences and expressing those sequences in a suitable transformed host. In some embodiments, recombinant techniques are used to construct the DNA sequences by isolating or synthesizing a DNA sequence encoding the wild-type protein of interest. Optionally, the sequence can be mutagenized by site-directed mutagenesis to provide a functional analog thereof. See, for example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066 (1984) and U.S. Patent No. 4,588,585.

[0181] In embodiments, DNA sequences encoding polypeptides of interest are constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide, selecting codons preferred in the host cell in which the recombinant polypeptide of interest is to be produced. Standard methods can be applied to synthesize isolated polynucleotide sequences encoding isolated polypeptides of interest. For example, the complete amino acid sequence can be used to construct a reverse-translated gene. Furthermore, DNA oligomers containing nucleotide sequences encoding specific isolated polypeptides can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides usually contain 5' or 3' overhangs for complementary assembly.

[0182] Once assembled (e.g., by synthesis, site-directed mutagenesis, or another method), a polynucleotide sequence encoding a particular isolated polypeptide of interest is inserted into an expression vector and, optionally, operably linked to expression control sequences suitable for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to obtain high expression levels of a gene transfected into a host, the gene can be operably linked to transcriptional and translational expression control sequences functional in the selected expression host. Recombinant expression vectors may be used to amplify and express DNA encoding the tumor-specific neoantigenic peptide. Recombinant expression vectors are replicable DNA constructs having synthetic or cDNA-derived DNA fragments encoding the tumor-specific neoantigenic peptide or a biologically equivalent analog operably linked to suitable transcriptional or translational control elements derived from mammalian, microbial, viral, or insect genes. A transcription unit generally contains an assembly of (1) genetic elements or elements that play a regulatory role in gene expression, such as a transcriptional promoter or enhancer, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences, as described in detail below. Such regulatory elements may include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may also be incorporated. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor involved in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation.Generally, operably linked means contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Structural elements intended for use in yeast expression systems will include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue which can optionally be subsequently cleaved from the expressed recombinant protein to provide the final product.

[0183] The choice of expression control sequence and expression vector will depend on the host chosen. A wide variety of expression host / vector combinations can be used. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences derived from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids such as Escherichia coli-derived plasmids, including pCR1, pBR322, pMB9, and their derivatives, and broad-host-range plasmids such as M13 and filamentous single-stranded DNA phages.

[0184] Suitable host cells for expressing polypeptides include prokaryotes, yeast, insect, or higher eukaryotic cells under the control of an appropriate promoter. Prokaryotes include gram-negative or gram-positive bacteria, such as E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems can also be used. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985).

[0185] Various mammalian or insect cell culture systems can also be advantageously used to express recombinant proteins. Expression of recombinant proteins in mammalian cells is feasible because such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells described by Gluzman (Cell 23:175, 1981), as well as other cell lines capable of expressing suitable vectors, including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Mammalian expression vectors can include non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, as well as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0186] Proteins produced by transformed hosts can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange chromatography, affinity chromatography, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Affinity tags, such as hexahistidine, maltose-binding domain, influenza coat sequence, glutathione-S-transferase, and the like, can be attached to the protein, allowing for easy purification by passage through an appropriate affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0187] For example, supernatants from systems secreting recombinant proteins into culture media can be first concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration units. Following the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin, such as a matrix or substrate bearing pendant diethylaminoethyl (DEAE) groups, can be used. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, one or more reverse-phase high-performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium, such as silica gel bearing pendant methyl or other aliphatic groups, can be used to further purify the cancer stem cell protein-Fc composition. Various combinations of some or all of the above purification steps can also be used to provide a homogeneous recombinant protein. Recombinant proteins produced in bacterial culture can be isolated, for example, by initial extraction from a cell pellet, followed by one or more concentration, salting out, aqueous ion exchange, or size exclusion chromatography steps. High-performance liquid chromatography (HPLC) can be used for final purification steps. Microbial cells used to express recombinant proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents.

[0188] Thus, in certain embodiments, the present invention relates to a personalized strategy for the treatment of disorders (e.g., neoplasms), more particularly tumors, by administering to a subject (e.g., a mammal, such as a human) a therapeutically effective amount of sHDL molecules (e.g., a vaccine composition capable of generating a specific T cell response) complexed with one or more neoplasm / tumor-specific neoantigens. Indeed, 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. The population of neoplasm / tumor-specific neoantigens and their cognate natural antigens may then be subjected to bioinformatics analysis using validated algorithms that predict which tumor-specific mutations generate epitopes capable of binding to the patient's HLA allotype, and in particular, which tumor-specific mutations generate epitopes that can bind to the patient's HLA allotype more effectively than the cognate natural antigen. Based on this analysis, one or more peptides corresponding to a subset of these mutations may be designed and synthesized for each patient and pooled together for use as a cancer vaccine when immunizing the patient. Neoantigen peptides may also be combined with other anti-neoplastic agents. In some embodiments, such neoantigens are predicted to bypass central thymic immune tolerance (thereby enabling a strong anti-tumor T cell response) while reducing the potential for autoimmunity (e.g., by avoiding targeting normal self-antigens).

[0189] The present invention further provides methods for treating and / or alleviating symptoms of cancer in a subject by inducing a neoplasm / tumor-specific immune response in the subject, vaccinating against a neoplasm / tumor, and administering to the subject a neoantigenic peptide or vaccine composition of the present invention.

[0190] According to the present invention, the cancer vaccine may be used in patients diagnosed with cancer or at risk of developing cancer. In one embodiment, the patient may have solid tumors, such as breast, ovarian, prostate, lung, kidney, stomach, colon, testicular, head and neck, pancreas, brain, melanoma, and other tumors of tissues and organs, and blood tumors, such as lymphomas and leukemias, including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma.

[0191] The peptides or compositions of the present invention are 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, such as chemotherapeutic or biological agents, radiation, or immunotherapy. Any suitable therapeutic treatment for the particular cancer may be administered. Examples of chemotherapeutic and biological agents include aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alfa, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifophylline, iodine, iodine-3-phosphate dehydrogenase (IOD), iodine-4-phosphate dehydrogenase (IOD), iodine-5-phosphate dehydrogenase (IOD), iodine-6-phosphate dehydrogenase (IOD), iodine-7-phosphate dehydrogenase (IOD), iodine-8-phosphate dehydrogenase (IOD), iodine-9-phosphate dehydrogenase (IOD), iodine-10-phosphate dehydrogenase (IOD), iodine-11-phosphate dehydrogenase (IOD), iodine-12-phosphate dehydrogenase (IOD), iodine-13-phosphate dehydrogenase (IOD), iodine-14-phosphate dehydrogenase (IOD), iodine-15-phosphate dehydrogenase (IOD), iodine-16-phosphate dehydrogenase (IOD), iodine-17-phosphate dehydrogenase (IOD), iodine-18- These include, but are not limited to, sufamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol®), pilocarpine, prochlorperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate. For the treatment of prostate cancer, a preferred chemotherapeutic agent that can be combined with anti-CTLA-4 is paclitaxel (Taxol®).

[0192] In addition, the subject may be further administered an anti-immunosuppressant or immunostimulatory drug. For example, the subject may be further administered an inhibitor of anti-CTLA antibody, anti-PD-1, anti-PD-L1, anti-TIM-3, anti-BTLA, anti-VISTA, anti-LAG3, anti-CD25, anti-CD27, anti-CD28, anti-CD137, anti-OX40, anti-GITR, anti-ICOS, anti-TIGIT, or IDO. Blockade of CTLA-4 or PD-1 / PD-L1 with an antibody can enhance the patient's immune response to cancerous cells. In particular, CTLA-4 blockade has been shown to be effective when used in conjunction with a vaccination protocol.

[0193] The optimal amount of each peptide to be included in the vaccine composition and the optimal dosing regimen can be determined by one of skill in the art without undue experimentation. For example, the peptide or its variant may be prepared for intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, or intramuscular (im) injection. Preferred methods of peptide injection include sc, id, ip, im, and iv. Preferred methods of DNA injection include id, im, sc, ip, and iv. For example, a dose of 1 to 500 mg, 50 μg to 1.5 mg, preferably 10 μg to 500 μg of peptide or DNA may be administered, depending on the respective peptide or DNA. Doses in this range have been used successfully in previous clinical trials (Brunsvig PF, et al., Cancer Immunol Immunother. 2006; 55(12):1553-1564; M. Staehler, et al., ASCO meeting 2007; Abstract No. 3017). Other methods of administering vaccine compositions are known to those skilled in the art.

[0194] The vaccines of the present invention may be tailored so that the selection, number, and / or amount of peptides present in the composition are tissue-, cancer-, and / or patient-specific. For example, the 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 state of the disease, the early treatment regimen, the patient's immune status, and, of course, the patient's HLA-haplotype. Furthermore, the vaccines of the present invention may contain individual components tailored to the individual needs of a particular patient. Examples include varying the amount of peptides according to the expression of relevant neoantigens in a particular patient, unwanted side effects due to individual allergies or other treatments, and adjustments for secondary treatments after a first round or regimen of treatment.

[0195] Such vaccines may be administered to individuals already suffering from cancer. In therapeutic applications, such vaccines are administered to patients in an amount sufficient to elicit an effective CTL response against tumor antigens and to cure or partially halt at least symptoms and / or complications. An amount sufficient to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend, for example, on the peptide composition, the mode of administration, the stage and severity of the disease being treated, the patient's weight and general health, and the judgment of the prescribing physician, but generally range from about 1.0 μg to about 50,000 μg of peptide for an initial immunization (whether for therapeutic or prophylactic administration), followed by about 1.0 μg to about 10,000 μg of peptide according to a boosting dosage or boosting regimen, for a 70 kg patient, over several weeks to months, depending on the patient's response and condition, and optionally by measuring specific CTL activity in the patient's blood. It should be noted that the peptides and compositions of the present invention can generally be used in severe disease states, i.e., life-threatening or potentially life-threatening situations, especially when the cancer has metastasized. For therapeutic use, administration should be initiated as soon as possible after tumor detection or surgical removal. This is followed by increasing the dose at least until symptoms are substantially alleviated and thereafter. Pharmaceutical compositions (e.g., vaccine compositions) for therapeutic treatment are for parenteral, topical, nasal, oral, or local administration. Preferably, the pharmaceutical composition is administered parenterally, for example, intravenously, subcutaneously, intradermally, or intramuscularly. The composition may also be administered at the surgical resection site to induce a local immune response to the tumor.

[0196] Such embodiments are not limited to a particular type of adjuvant. Generally, an adjuvant is any substance whose incorporation into a vaccine composition increases or otherwise modifies the immune response to a variant peptide. The carrier is a scaffold structure, such as a polypeptide or polysaccharide, to which an antigenic peptide (e.g., a neoantigenic peptide) can be associated. Optionally, the adjuvant is covalently or noncovalently conjugated to the peptide or polypeptide of the present invention.

[0197] The ability of an adjuvant to increase the immune response to an antigen is usually manifested by a significant increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity is usually manifested by a significant increase in the titer of antigen-induced antibodies, and an increase in T cell activity is usually manifested by an increase in cell proliferation, cytotoxicity, or cytokine secretion. Adjuvants can also modify the immune response, for example, by changing a predominantly humoral or Th2 response to a predominantly cellular or Th1 response.

[0198] Suitable adjuvants include 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide These include, but are not limited to, ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, vector systems, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF traps, R848, β-glucan, Pam3Cys, Aquila's QS21 Stimulon (Aquila Biotech, Worcester, Massachusetts, USA) derived from saponins, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox.Quil or Superfos. Some immunological adjuvants specific for dendritic cells (e.g., MF59) and their preparation have been previously described (Dupuis M, et al., Cell Immunol. 1998;186(1):18-27; Allison AC; Dev Biol Stand. 1998;92:3-11). Cytokines may also be used. Some cytokines have been directly implicated in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), promoting the maturation of dendritic cells into effective antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (see, inter alia, U.S. Pat. No. 5,849,589, incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI, et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418).Toll-like receptors (TLRs), sometimes used as adjuvants, are important members of a family of pattern recognition receptors (PRRs) that recognize conserved motifs shared by many microorganisms, called "pathogen-associated molecular patterns" (PAMPS).

[0199] Recognition of these "danger signals" activates multiple components of the innate and adaptive immune systems. TLRs are expressed by cells of the innate and adaptive immune systems, such as dendritic cells (DCs), macrophages, T and B cells, mast cells, and granulocytes, and are localized in different cellular compartments, such as the plasma membrane, lysosomes, endosomes, and endolysosomes. Different TLRs recognize distinct PAMPs. For example, TLR4 is activated by LPS contained in bacterial cell walls, TLR9 is activated by unmethylated bacterial or viral CpG DNA, and TLR3 is activated by double-stranded RNA. TLR ligand binding leads to the activation of one or more intracellular signaling pathways, ultimately resulting in the production of many important molecules related to inflammation and immunity, particularly the transcription factor NF-κB and type I interferons. TLR-mediated DC activation leads to DC activation, phagocytosis, activation, and upregulation of costimulatory markers such as CD80, CD83, and CD86, enhanced expression of CCR7, which enables DC migration to lymph nodes and promotes antigen presentation to T cells, and increased secretion of cytokines such as type I interferon, IL-12, and IL-6. All of these downstream events are important for the induction of adaptive immune responses.

[0200] Other receptors that can be targeted include Toll-like receptors (TLRs). TLRs recognize and bind to pathogen-associated molecular patterns (PAMPs). PAMPs target TLRs on the surface of dendritic cells and signal internally, potentially increasing DC antigen uptake, maturation, and T cell stimulatory capacity. PAMPs conjugated or co-encapsulated on particle surfaces include unmethylated CpG DNA (bacterial), double-stranded RNA (viral), lipopolysaccharide (bacterial), peptidoglycan (bacterial), lipoarabinomannan (bacterial), zymosan (yeast), mycoplasma lipoproteins such as MALP-2 (bacterial), flagellin (bacterial), poly(inosinic-cytidylic) acid (bacterial), lipoteichoic acid (bacterial), or imidazoquinoline (synthetic).

[0201] 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. In preclinical studies, poly-ICLC appears to be the most potent TLR adjuvant compared to LPS and CpG due to its lack of induction of proinflammatory cytokines and stimulation of IL-10 in DCs and the maintenance of high levels of costimulatory molecules. Furthermore, poly-ICLC was recently directly compared with CpG in non-human primates (rhesus macaques) as an adjuvant for a protein vaccine consisting of human papillomavirus (HPV) 16 capsomers (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)).

[0202] In some embodiments, the adjuvant is a dendritic cell targeting molecule (DC). DCs are potent and involved in the initiation of antigen-specific immune responses. One biological characteristic of DCs is their ability to sense antigen-encounter conditions and initiate the process of "DC maturation." Using receptors for various microorganisms and inflammatory products, DCs respond to antigen exposure in different ways depending on the nature of the pathogen (virus, bacteria, protozoan) they encounter. This information is conveyed to T cells by altering the cytokine release pattern upon antigen presentation in lymph nodes, altering the type of T cell response elicited. Therefore, targeting DCs generally not only quantitatively enhances antigen delivery and antigen responses, but also provides an opportunity to qualitatively control the nature of the immune response depending on the desired vaccination outcome.

[0203] Dendritic cells express multiple cell surface receptors that can mediate endocytosis of bound antigens. Targeting exogenous antigens to internalizing surface molecules on antigen-presenting cells distributed throughout the body facilitates antigen uptake, thus overcoming a major rate-limiting step in immunization and, therefore, vaccination.

[0204] Dendritic cell targeting molecules include monoclonal or polyclonal antibodies or fragments thereof that recognize and bind to epitopes displayed on the surface of dendritic cells. Dendritic cell targeting molecules also include ligands that bind to cell surface receptors on dendritic cells. One such receptor, the lectin DEC-205, has been used in vitro and in mice to increase both humoral (antibody-based) and cellular (CD8 T cell) responses by two to four orders of magnitude (see, e.g., Hawiger, et al., J. Exp. Med., 194(6):769-79 (2001); Bonifaz, et al., J. Exp. Med., 196(12):1627-38 (2002); Bonifaz, et al., J. Exp. Med., 199(6):815-24 (2004)).

[0205] A variety of other endocytic receptors, including mannose-specific lectin (mannose receptor) and IgGFc receptors, have been targeted in this manner with similar enhancements in antigen presentation efficiency. Other suitable receptors that can be targeted include, but are not limited to, DC-SIGN, 33D1, SIGLEC-H, DCIR, CD11c, heat shock protein receptors, and scavenger receptors.

[0206] In some embodiments, the adjuvant is CpG. CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in vaccine settings. Without wishing to be bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cellular vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, it enhances the maturation and differentiation of dendritic cells, leading to enhanced Th1 cell activation and the generation of potent cytotoxic T lymphocytes (CTLs), even in the absence of CD4 T cells. The Th1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote a Th2 bias. CpG oligonucleotides further exhibit greater adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, which are particularly necessary to induce strong responses when the antigen is relatively weak. They also enhance immune responses, allowing the antigen dose to be reduced by approximately two orders of magnitude in some experiments with equivalent antibody responses to a total dose of vaccine without CpG (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, June 2006, 471-484). U.S. Patent No. 6,406,705 B1 describes the combination of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce antigen-specific immune responses. A commercially available CpG TLR9 antagonist is dSLIM (double stem-loop immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR binding molecules, such as RNA-binding TLR7, TLR8, and / or TLR9, may also be used.

[0207] For example, xanthenone derivatives such as Vadimezan (also known as 5,6-dimethylxanthenone-4-acetic acid (DMXAA)) or AsA404 may be used as adjuvants according to embodiments of the present invention. Alternatively, such derivatives may be administered in parallel with the vaccines of the present invention, e.g., via systemic or intratumoral delivery, to stimulate immunity at the tumor site. Without being bound by theory, it is believed that such xanthenone derivatives act by stimulating interferon (IFN) production via the stimulator of IFN genes (ISTING) receptor (see, e.g., Conlon et al. (2013) Mouse, but not Human STING, Binds and Signals in Response to the Vascular Disrupting Agent 5,6-Dimethylxanthenone-4-Acetic Acid, Journal of Immunology, 190:5216-25 and Kim et al. (2013) Anticancer Flavonoids are Mouse-Selective (See STING Agonists, 8:1396-1401). Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), Poly(I:C) (e.g., polyi:CI2U), non-CpG bacterial DNA or RNA, as well as immunologically active small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may act therapeutically and / or as an adjuvant. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by one of ordinary skill in the art without undue experimentation. Further adjuvants include colony stimulating factors, such as granulocyte macrophage colony stimulating factor (GM-CSF, sarlamostim).

[0208] Poly-ICLC is a synthetically prepared double-stranded RNA consisting of a poly(I) strand and a poly(C) strand with an average length of approximately 5,000 nucleotides, stabilized against heat denaturation and hydrolysis by serum nucleases by the addition of polylysine and carboxymethylcellulose. This compound activates the TLR3 and RNA helicase domains of MDA5, a member of the PAMP family, leading to the activation of DCs and natural killer (NK) cells and the production of a "natural mixture" of type I interferons, cytokines, and chemokines. Furthermore, poly-ICLC exerts more direct and broader host-targeted anti-infective and anti-tumor effects, likely 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.

[0209] Such methods are not limited to producing sHDL nanoparticles conjugated with an antigen and an adjuvant (e.g., a dendritic cell targeting molecule). In some embodiments, the antigen and adjuvant are conjugated to the outer surface of the sHDL nanoparticle.

[0210] In some embodiments, sHDL nanoparticles are synthesized with thiol-reactive phospholipids, which enable reduction-sensitive conjugation of antigens and / or adjuvants. In some embodiments, loading of DCs into sHDL nanoparticles is facilitated by cholesterol modification of DC molecules. In some embodiments, a lyophilization method is used to prepare homogeneous sHDL. In some embodiments, phospholipids and ApoA-mimetic peptides are dissolved in glacial acetic acid and lyophilized. In some embodiments, antigenic peptides are incubated with sHDL in a buffer (e.g., sodium phosphate buffer (pH 7.4)) (e.g., at room temperature for 3 hours) to allow conjugation of the antigenic peptide. In some embodiments, unconjugated antigenic peptides are removed using a desalting column (MWCO = 7000 Da). In some embodiments, incorporation of cholesterol-modified DCs (Cho-DCs) into sHDL involves incubation with sHDL for approximately 30 minutes at room temperature.

[0211] Such embodiments are not limited to a particular technique for characterizing sHDL conjugated with antigen and DC. In some embodiments, the morphology of sHDL is observed by TEM. In some embodiments, the size distribution of sHDL is analyzed by dynamic light scattering (DLS) using a Malven Nanosizer instrument and a GPC assay.

[0212] sHDL nanoparticles (e.g., sHDL-αGalCer) configured to activate an immune response (e.g., Ag / DC-sHDL) are useful for activating T cells in subjects for prophylactic and therapeutic applications. Activation of T cells with the nanoparticle vaccine composition increases their proliferation, cytokine production, differentiation, effector function, and / or survival. Methods for measuring these are well known to those skilled in the art. T cells activated by the nanoparticle vaccine composition can be any cells that express T cell receptors, including α / β and γ / δ T cell receptors. T cells include all cells that express CD3, including T cell subsets that also express CD4 and CD8. T cells include both naive and memory cells, as well as effector cells such as CTLs. T cells also include regulatory cells such as Th1, Tc1, Th2, Tc2, Th3, Treg, and Tr1 cells. T cells also include NKT cells and similar unique classes of T cell lineages. In some embodiments, activated T cells include CD8 + T cells.

[0213] In general, compositions comprising sHDL nanoparticles (e.g., sHDL-αGalCer) configured to activate an immune response (e.g., Ag / DC-sHDL) are useful for treating subjects with or susceptible to any disease or disorder in which the subject's immune system mounts an immune response. The compositions are useful as prophylactic vaccines, which confer resistance to subsequent exposure to infectious agents. The compositions are also useful as therapeutic vaccines that can be used to initiate or enhance a subject's immune response to pre-existing antigens, such as tumor antigens in subjects with cancer or viral antigens in subjects infected with viruses. The compositions are also useful as desensitization vaccines, which function to "tolerize" individuals to environmental antigens, such as allergens.

[0214] The ability of these compositions to target professional antigen-presenting cells, such as dendritic cells, and to induce T cell-mediated immune responses by causing cross-presentation of antigens makes them particularly useful for eliciting cell-mediated responses against disease-associated antigens to attack disease. Thus, in some embodiments, the type of disease to be treated or prevented is a malignancy or chronic infection caused by bacteria, viruses, protozoa, helminths, or other microbial pathogens that invade intracellularly, i.e., are attacked by cytotoxic T lymphocytes.

[0215] The desired outcome of a preventative, therapeutic, or desensitized immune response may vary depending on the disease, according to principles well known in the art. For example, an immune response against an infectious agent may completely prevent colonization and replication of the infectious agent, affecting "sterile immunity" and the absence of any 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 population with symptoms; or reduces transmission of the infectious agent. Similarly, an immune response against cancer, an allergen, or an infectious agent may completely treat the disease, alleviate symptoms, or be one aspect of an overall therapeutic intervention against the disease. For example, stimulating an immune response against cancer may be combined with surgical, chemotherapeutic, radiological, hormonal, and other immunological approaches to affect treatment.

[0216] Subjects with or exposed to an infectious agent can be treated therapeutically or prophylactically with sHDL nanoparticles (e.g., sHDL-αGalCer) (e.g., Ag / DC-sHDL) configured to activate an immune response as disclosed herein. Infectious agents include bacteria, viruses, and parasites. In some cases, subjects can be treated prophylactically, for example, if they may be at risk of developing a disease from the infectious agent. Individuals who travel to or live in areas with endemic infectious diseases are considered at risk and at risk and may be considered candidates for vaccination against a particular infectious agent. Prophylactic treatment can be applied to any number of diseases where there is a known relationship between the particular disease and certain risk factors, such as geographic location or work environment.

[0217] Subjects with or at risk of developing malignant tumors can be treated therapeutically or prophylactically with sHDL nanoparticles (e.g., sHDL-αGalCer) (e.g., Ag / DC-sHDL) configured to activate an immune response as disclosed herein. In mature animals, a balance between cell renewal and cell death is normally maintained in most organs and tissues. Various types of mature cells in the body have a given lifespan: when these cells die, new cells are generated by the proliferation and differentiation of various types of stem cells. Under normal circumstances, the production of new cells is regulated, so the number of cells of any particular type remains constant. However, occasionally, cells arise that no longer respond to normal growth control mechanisms. These cells give rise to clones of cells that can expand to a significant size and produce tumors or neoplasms. Tumors that cannot grow indefinitely and do not extensively invade healthy surrounding tissue are benign. Tumors that continue to grow and become gradually 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 leave the tumor, invade the blood or lymphatic vessels, and are transported to other tissues where they continue to grow. In this way, a primary tumor at one site can give rise to a secondary tumor at another site. sHDL nanoparticles configured to activate an immune response (e.g., sHDL-αGalCer) (e.g., Ag / DC-sHDL) as disclosed herein are useful for treating subjects with malignant tumors.

[0218] Malignant tumors that can be treated are classified herein according to the embryonic origin of the tissue from which they originate. Carcinomas are tumors that arise from endodermal or ectodermal tissues, such as the skin or the epithelial lining of internal organs and glands. Melanoma is a type of carcinoma of the skin for which the present invention is particularly useful. Sarcomas, which occur less frequently, originate from mesodermal connective tissues, such as bone, fat, and cartilage. Leukemia and lymphoma are malignant tumors of the hematopoietic cells of the bone marrow. Leukemias grow as single cells, while lymphomas tend to grow as tumor masses. Malignant tumors can appear in multiple organs or tissues of the body to establish cancer.

[0219] Cancer types that can be treated with the provided sHDL nanoparticles (e.g., sHDL-αGalCer) (e.g., Ag / DC-sHDL) configured to activate an immune response include, but are not limited to, the following: bladder, brain, breast, cervix, colorectal, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, etc. Administration is not limited to treating existing tumors or infections, but can also be used to prevent or reduce the risk of developing such diseases in individuals, i.e., for prophylactic use. Potential candidates for prophylactic vaccination include individuals at high risk of developing cancer, i.e., who have a personal or family history of a particular type of cancer.

[0220] Subjects with or at risk of exposure to allergens can be treated therapeutically or prophylactically with sHDL nanoparticles (e.g., sHDL-αGalCer) configured to activate an immune response as disclosed herein (e.g., Ag / DC-sHDL). Such sHDL nanoparticles may be administered to a subject for the purpose of preventing and / or mitigating an allergic reaction, such as an allergic reaction that leads to anaphylaxis. An allergic reaction is a T cell response to an antigen that results in the presence of IgE antibodies. H It may be characterized by two responses. H Stimulation of the immune response and production of IgG antibodies can alleviate allergic diseases. Thus, sHDL nanoparticles configured to activate the immune response (e.g., sHDL-αGalCer) (e.g., Ag / DC-sHDL) as disclosed herein are useful for producing antibodies that prevent and / or reduce allergic reactions in subjects exposed to allergens.

[0221] Subjects with or at risk of immunosuppressive conditions can be treated therapeutically or prophylactically with sHDL nanoparticles (e.g., sHDL-αGalCer) configured to activate an immune response (e.g., Ag / DC-sHDL) as disclosed herein. The sHDL nanoparticle vaccines disclosed herein can be used for the treatment of conditions characterized by immunosuppression, including, but not limited to, AIDS or AIDS-related complex diseases, idiopathic immunosuppression, drug-induced immunosuppression, other viral or environmentally induced conditions, and certain congenital immune deficiencies. Such sHDL nanoparticle vaccine compositions can also be used to increase immune function impaired by the use of immunosuppressive drugs (e.g., certain chemotherapeutic drugs) or radiation therapy and, therefore, may be particularly useful when used in conjunction with such drugs or radiation therapy.

[0222] Subjects at risk for or at risk of coronary heart disease and / or elevated LDL-C levels can be treated therapeutically or prophylactically with sHDL nanoparticles configured to activate an immune response as disclosed herein. While the efficacy of mAb therapy against PCSK9 has been established (see, e.g., Banerjee, Y.; et al., New England Journal of Medicine 2012, 366(25), 2425-2426; Stein, EA; et al., Circulation 2013, 128(19), 2113-2120), the development of a more durable PCSK9 vaccine is needed. In addition, one of the challenges of PCSK9 vaccines is that self-antigens such as PCSK9 peptides are not immunogenic unless they are coupled to a vaccine / adjuvant system that can efficiently co-deliver the antigen and immune stimulatory molecules to immune cells (e.g., Krishnamachari, Y.; et al., Advanced Drug Delivery Reviews 2009, 61(3), 205-217; Hamdy, S.; et al., Advanced Drug See Delivery Reviews 2011,63(10-11),943-955).

[0223] Embodiments of the present invention in which sHDL nanoparticles are conjugated with a PCSK9 antigen and a CpG adjuvant (PCSK9-Ag / CpG-sHDL) address this need. Indeed, vaccination against PCSK9 with PCSK9-Ag / CpG-sHDL embodiments effectively inhibits the interaction between PCSK9 and LDLR while avoiding the need for repeated injections of expensive mAbs (see, e.g., Fattori, E.; et al., Journal of Lipid Research 2012, 53(8), 1654-1661; Gergana Galabova, et al., PLOS ONE 2014, 9(12)). Furthermore, such PCSK9-Ag / CpG-sHDL nanoparticles have a sufficiently small size (e.g., 10-45 nm) to allow efficient excretion to lymph nodes compared to larger particles (see, e.g., Bachmann, MF; et al., Nature Reviews Immunology 2010, 10(11), 787-796).

[0224] Generally, methods of administering vaccines as disclosed herein (e.g., sHDL nanoparticles (e.g., sHDL-αGalCer) (e.g., Ag / DC-sHDL) configured to activate an immune response) are well known in the art. Any acceptable method known to one of skill in the art may be used to administer the formulation to a subject. Administration may be local (i.e., to a specific region, physiological system, tissue, organ, or cell type) or systemic. Vaccines can be administered by a number of routes, including, but not limited to, oral, inhalation (intranasal or pulmonary), intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means. Injections can be, for example, intravenous, intradermal, subcutaneous, intramuscular, or intraperitoneal injections. In some embodiments, injections can be administered at multiple locations.

[0225] Administration of the formulation may be achieved by any acceptable method that allows an effective amount of vaccine to reach the target. The specific mode selected will depend on factors such as the specific formulation, the severity of the condition of the subject being treated, and the dosage required to induce an effective immune response. As generally used herein, an "effective amount" is an amount that can induce an immune response in the treated subject. The actual effective amount of the vaccine may vary depending on the specific antigen or combinations thereof used, the specific composition formulated, the mode of administration, and the age, weight, condition, and route of administration and disease or disorder of the individual being vaccinated.

[0226] In certain embodiments, glycolipids encapsulated within sHDL nanoparticles are used as stimulators of natural killer T cell-mediated immune responses.

[0227] Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. Many of these cells recognize non-polymorphic CD1d molecules, antigen-presenting molecules that bind to self and foreign lipids and glycolipids. NKT cells comprise only approximately 0.1% of all peripheral blood T cells. NKT cells are a subset of T cells that co-express the αβ T cell receptor but also express various molecular markers typically associated with NK cells, such as NK1.1. The best-known NKT cells differ from conventional αβ T cells in that they have a much more limited diversity of T cell receptors ("invariant" or "type 1" NKT). These and other CD1d-restricted T cells ("type 2" NKT) recognize lipids and glycolipids presented by CD1d molecules, members of the CD1 family of antigen-presenting molecules, instead of peptide major histocompatibility complex (MHC). NKT cells express NK1.1 + and NK1.1 - Both CD4 + , CD4 - , CD8 + , and CD8 - Contains cells.

[0228] In some embodiments, the glycolipid is the synthetic glycolipid α-galactosylceramide (αGalCer). Dendritic cells presenting antigens in the context of CD1d can result in rapid natural and prolonged production of cytokines such as interferon and IL-4 by natural killer T cells (NKT cells). CD1d is a major histocompatibility complex class I-like molecule that presents glycolipid antigens to a subset of NKT cells. Advantageously, αGalCer is non-toxic to humans and has been shown to act as an adjuvant to prime both antigen-specific CD4+ and CD8+ T cell responses. For example, it has been shown that αGalCer in combination with a malaria vaccine can result in a cytotoxic response against infected cells, an ideal scenario for a vaccine against infectious diseases. In addition to αGalCer, other glycolipids that function as adjuvants to activate NKT cell-mediated immune responses can be used.

[0229] The present invention is not limited to a specific method for producing sHDL nanoparticles with encapsulated αGalCer. For example, in some embodiments, a lyophilization method is used to prepare homogeneous sHDL. In some embodiments, phospholipids and ApoA-mimetic peptides are dissolved in glacial acetic acid and lyophilized. In some embodiments, loading of αGalCer into sHDL nanoparticles is facilitated by hydrophobic interactions between αGalCer and sHDL. In some embodiments, lyophilized phospholipids and ApoA-mimetic peptides are hydrated (e.g., in PBS (pH 7.4)), thermally cycled above and below the transition temperature (Tm) at which the phospholipids form blank sHDL, and then incubated with αGalCer at room temperature for an optimal time (e.g., 5, 10, 20, 25, 30, 35, 50, 80, 120, or 360 minutes) to form sHDL containing encapsulated αGalCer.

[0230] Such embodiments are not limited to a particular manner of characterizing sHDL containing encapsulated αGalCer. In some embodiments, the morphology of sHDL-αGalCer is observed by TEM. In some embodiments, the size distribution of sHDL-αGalCer is analyzed by dynamic light scattering (DLS) using a Malven Nanosizer instrument and a GPC assay.

[0231] Such embodiments are not limited to any particular technique for assessing the delivery profile of αGalCer in vitro and in vivo, in some embodiments, labeling the molecule with an imaging agent (e.g., the fluorescent dye Cy3) allows visualization of the biodistribution of the αGalCer molecule at the organ level, as well as the intracellular delivery profile.

[0232] In certain embodiments, the present invention provides methods for inducing a natural killer T cell-mediated immune response in cells, the methods comprising exposing the cells to a composition comprising an αGalCer glycolipid encapsulated in sHDL nanoparticles, wherein such exposure results in the induction of a killer T cell-mediated immune response. In some embodiments, the cells are in vivo cells. In some embodiments, the cells are in vitro cells. In some embodiments, the cells are ex vivo cells.

[0233] In certain embodiments, the present invention provides a method for inducing a natural killer T cell-mediated immune response in a subject (e.g., a human patient), the method comprising administering to the patient a pharmaceutical composition comprising αGalCer glycolipid encapsulated within sHDL nanoparticles, wherein such administration results in the induction of a natural killer T cell-mediated immune response. Further embodiments In certain embodiments, sHDL nanoparticles as described herein (e.g., configured for RNA interference) (e.g., configured to activate an immune response) encapsulate one or more therapeutic agents. Such embodiments are not limited to a particular type or variety of therapeutic agents.

[0234] In some embodiments, the therapeutic agent is configured to treat and / or prevent cancer. Examples of such therapeutic agents include, but are not limited to, chemotherapeutic agents, anti-carcinogenic agents, anti-angiogenic agents, tumor suppressors, anti-microbial agents, etc.

[0235] In some embodiments, the therapeutic agent is configured to treat and / or prevent an autoimmune disorder and / or an inflammatory disorder. Examples of such therapeutic agents include disease-modifying antirheumatic drugs (e.g., leflunomide, methotrexate, sulfasalazine, hydroxychloroquine), biologic 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), immunomodulatory agents (e.g., anakinra, abatacept), glucocorticoids (e.g., prednisone, methylprednisone), TNF-α inhibitors (e.g., adalimumab, cetulizumab 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.

[0236] In some embodiments, the therapeutic agent is configured for treating and / or preventing cardiovascular-related disorders (e.g., atherosclerosis, heart failure, arrhythmias, atrial fibrillation, hypertension, coronary artery disease, angina pectoris, etc.). Examples of therapeutic agents known to be useful for treating and / or preventing cardiovascular-related disorders include angiotensin-converting enzyme (ACE) inhibitors (e.g., benazepril, enalapril, lisinopril, perindopril, ramipril), adenosine, alpha-blockers (alpha-adrenergic antagonist drugs) (e.g., clonidine, guanabenz, labetalol, phenoxybenzamine, terazosin, doxazosin, guanfacine, methyldopa, prazosin), angiotensin II receptor blockers (e.g., benzodiazepines, benzocaine ... Anticoagulants (ARBs) (e.g., candesartan, irbesartan, olmesartan medoxomil, telmisartan, eprosartan, losartan, tasosartan, valsartan), anticoagulants (e.g., heparin, fondaparinux, warfarin, ardeparin, enoxaparin, reviparin, dalteparin, nadroparin, tinzaparin), antiplatelet agents (e.g., abciximab, clopidogrel, eptifibatide, ticlopidine, cilostazol, dipyridamole, sulfinpyrazone, tirofloxacin, thiazolinone), beta-blockers (e.g., acebutolol, betaxolol, carteolol, metoprolol, penbutolol, propranolol, atenolol, bisoprolol, esmolol, nadolol, pindolol, timolol), calcium channel blockers (e.g., amlopidine, felodipine, isradipine, nifedipine, verapamil, diltiazem, nicardipine, nimodipine, nisoldipine), diuretics, aldosterone blockers, loop diuretics (e.g., bumetanide, furosemide, ethacrynic acid, torasemide), potassium-sparing diuretics, thiazide diuretics (e.g., chlorothiazide, chlorthalidone, hydrochlorothiazide, hydroflumeazide, methyclothiazide, metolazone, polythiazide, quinatazone, trichlormethiazide), cardiac inotropes, bile acid sequestrants (e.g., cholestyramine, coletipol, colesevelam), fibrates (e.g., clofibrate, gemfibrozil, fenofibrate), statins (e.g.,atorvastatin, lovastatin, simvastatin, fluvastatin, pravastatin), selective cholesterol absorption inhibitors (e.g., ezetimibe), potassium channel blockers (e.g., amidarone, ibutilide, dofetilide), sodium channel blockers (e.g., disopyramide, mexiletine, procainamide, quinidine, flecainide, moricizine, propafenone), thrombolytic agents (e.g., alteplase, reteplase, tenecteplase, anistreplase, streptokinase, urokinase), vasoconstrictors, vasodilators (e.g., hydralazine, minoxidil, mecamylamine, isorbidiol dintrate, isorbidiol mononitrate, nitroglycerin).

[0237] Generally, the sHDL nanoparticles so formed are spherical and have a diameter of about 5 nm to about 20 nm (e.g., 4-75 nm, 4-60 nm, 4-50 nm, 4-22 nm, 6-18 nm, 8-15 nm, 8-10 nm, etc.). In some embodiments, the sHDL nanoparticles are subjected to size exclusion chromatography to obtain a more homogeneous preparation.

[0238] In some embodiments, the sHDL nanoparticles further encapsulate an agent useful for determining the location of the administered particle. Agents useful for this purpose include fluorescent tags, radionuclides, and imaging agents.

[0239] Suitable imaging agents include fluorescent molecules such as those described in Molecular Probes (Handbook of fluorescent probes and research products), such as rhodamine, fluorescein, Texas red, acridine orange, Alexa Fluor (various), allophycocyanin, 7-aminoactinomycin D, BOBO-1, BODIPY (various), Calcien, Calcium Crimson, Calcium green, Calcium Orange, 6-carboxyrhodamine 6G, Cascade blue, Cascade yellow, DAPI, DiA, DID, Di1, DiO, DiR, ELF 97, Eosin, ER Tracker Blue-White, EthD-1, ethidium bromide, Fluo-3, Fluo-4, FM1-43, FM4-64, Fura-2, Fura Red, Hoechst 33258, Hoechst 33342, 7-hydroxy-4-methylcoumarin, Indo-1, JC-1, JC-9, JOE dye, Lissamine rhodamine B, Lucifer Yellow CH, LysoSensor Blue DND-167, LysoSensor Green, LysoSensor Yellow / Blu, Lysotracker Green FM, Magnesium Green, Marina Blue, Mitotracker Green FM, Mitotracker Orange CMTMRos, MitoTracker Red CMxRos, Monobromobimane, NBD amines, NeruoTrace 500 / 525 green, Nile red, Oregon Green, Pacific Blue, POP-1, propidium iodide, rhodamine 110, rhodamine red, R-phycocyanin, Resorfin, RH414, Rhodamine-2, rhodamine green, rhodamine 123, ROX dye, sodium green, SYTO blue (various), SYTO green (various), SYTO orange (various), SYTOX blue, SYTOX green, SYTOX orange, tetramethylrhodamine B, TOT-1, TOT-3, X-rhod-1, YOYO-1, and YOYO-3. In some embodiments, ceramide is provided as an imaging agent. In some embodiments, the S1P agonist is provided as an imaging agent.

[0240] Additionally, radionuclides can be used as imaging agents. Suitable radionuclides include, but are not limited to, Fe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(II), indium, gallium, and technetium radioactive species. Other suitable imaging agents include metal ions commonly used for chelation in paramagnetic T1-type MIR contrast agents, including divalent and trivalent cations such as copper, chromium, iron, gadolinium, manganese, erbium, europium, dysprosium, and holmium. Metal ions that can be chelated and used for radionuclide imaging include, but are not limited to, metals such as gallium, germanium, cobalt, calcium, indium, iridium, rubidium, yttrium, ruthenium, yttrium, technetium, rhenium, platinum, thallium, and samarium. Additionally, metal ions known to be useful in neutron capture radiotherapy include boron and other metals with large nuclear cross sections. Additionally, metal ions useful in ultrasound and x-ray contrast compositions are also suitable.

[0241] Other examples of suitable contrast agents include radiopaque gases or gas-releasing compounds.

[0242] In some embodiments, the sHDL nanoparticles further encapsulate a targeting agent. In some embodiments, the targeting agent is used to aid in the delivery of the sHDL-TA nanoparticles to a desired body region (e.g., a body region affected by a cardiovascular-related disorder). Examples of targeting agents include, but are not limited to, antibodies, receptor ligands, hormones, vitamins, and antigens, and the present invention is not limited by the nature of the targeting agent. In some embodiments, the antibody is specific for 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, glycoproteins, and VEGFR. In some embodiments, the receptor ligand is folate.

[0243] In some embodiments, the sHDL nanoparticles of the present invention may be delivered to a localized site in a patient via a medical device. Medical devices suitable for use in the present invention include known devices for localized delivery of therapeutic agents. Such devices include, but are not limited to, catheters, e.g., injection catheters, balloon catheters, double-balloon catheters, microporous balloon catheters, channel balloon catheters, infusion catheters, and perfusion catheters (which may be coated with, e.g., a therapeutic agent or through which a drug is administered); needle injection devices such as hypodermic needles and needle injection catheters; needleless injection devices such as jet injectors; coated stents, bifurcated stents, vascular grafts, stent grafts, and the like; and vascular occlusion devices such as wire coils.

[0244] Exemplary devices are described in U.S. Patent Nos. 5,935,114; 5,908,413; 5,792,105; 5,693,014; 5,674,192; 5,876,445; 5,913,894; 5,868,719; 5,851,228; 5,843,089; 5,800,5 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 the present application include the RADIUS (SCIMED LIFE SYSTEMS, Inc.), the SYMPHONY (Boston Scientific Corporation), the Wallstent (Schneider Inc.), the PRECEDENT II (Boston Scientific Corporation), and NIR (Medinol Inc.). Such devices are delivered and / or implanted to target locations within the body using known techniques.

[0245] In some embodiments, the present invention also provides kits comprising the sHDL nanoparticles described herein, hi some embodiments, the kits include one or more of the reagents and tools necessary to generate sHDL nanoparticles, as well as methods for using such sHDL nanoparticles.

[0246] The sHDL nanoparticles of the present invention may be characterized for size and uniformity by any suitable analytical technique, including atomic force microscopy (AFM), electrospray ionization mass spectrometry, MALDI-TOF mass spectrometry, 13 Analytical methods include, but are not limited to, C nuclear magnetic resonance spectroscopy, high-performance liquid chromatography (HPLC), size-exclusion chromatography (SEC) (equipped with multi-angle laser light scattering, dual UV, and refractive index detectors), capillary electrophoresis, and gel electrophoresis. These analytical methods ensure the homogeneity of the sHDL nanoparticle population and are important in manufacturing quality control for final use in vivo.

[0247] In some embodiments, gel permeation chromatography (GPC), which can separate sHDL nanoparticles from liposomes and free ApoA-I mimetic peptides, is used to analyze sHDL-TA nanoparticles. In some embodiments, size distribution and zeta potential are measured by dynamic light scattering (DLS), for example, using a Malven Nanosizer instrument.

[0248] When clinical use is contemplated, in some embodiments of the present invention, the sHDL nanoparticles are prepared as part of a pharmaceutical composition in a form appropriate for the intended use. Generally, this requires preparing 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, neat sHDL nanoparticle formulations may be administered using one or more of the routes described herein.

[0249] In a preferred embodiment, sHDL nanoparticles are used with appropriate salts and buffers to deliver the composition in a stable manner that allows uptake by target cells. A buffer is also used when the sHDL nanoparticles are introduced into a patient. Aqueous compositions contain a cell-effective amount of sHDL nanoparticles dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions are also called inocula. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Use of any conventional media or agents in therapeutic compositions is contemplated except insofar as they are incompatible with the vectors or cells of the present invention. Supplementary active ingredients may also be incorporated into the compositions.

[0250] In some embodiments of the present invention, the active compositions comprise classical pharmaceutical formulations. Administration of these compositions according to the present invention is via any common route, so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal, or topical administration. Alternatively, administration may be via orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection.

[0251] Active sHDL nanoparticles may be administered parenterally, intraperitoneally, or intratumorally. A solution of the active compound as a free base or pharmacologically acceptable salt is prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under normal storage and use conditions, these preparations contain a preservative to prevent the growth of microorganisms.

[0252] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents delaying absorption, for example, aluminum monostearate and gelatin, in the compositions.

[0253] Sterile injection solution is prepared by incorporating the active sHDL nanoparticles in the required amount in a suitable solvent with various other ingredients listed above as needed, followed by filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the other ingredients listed above as needed.For the preparation of sterile powder for sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces a powder of active ingredient and any additional desired ingredients from the solution that has been previously sterile-filtered.

[0254] When formulated, sHDL nanoparticles are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug-release capsules, and the like. For parenteral administration in aqueous solution, for example, the solution should be suitably buffered, and, if necessary, the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. For example, one dosage can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion solution or injected at the proposed infusion site (see, e.g., "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). In some embodiments of the invention, the active particles or agents are formulated in the therapeutic mixture to contain about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0, or about 10 milligrams per dose. Multiple doses may be administered.

[0255] Additional formulations suitable for other modes of administration include vaginal suppositories and pessaries. Rectal pessaries or suppositories may also be used. Suppositories are solid dosage forms of various weights and shapes that are typically dispensed for insertion into the rectum, vagina, or urethra. After insertion, the suppository softens, melts, or dissolves in the fluids of the body cavity. In general, for suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides, and such suppositories may be formed from mixtures containing 0.5% to 10%, preferably 1% to 2%, of the active ingredient. Vaginal suppositories or pessaries are usually spherical or oval, each weighing approximately 5 g. Vaginal medications are available in various physical forms, such as creams, gels, or liquids, which deviate from the classic concept of a suppository. sHDL nanoparticles may also be formulated as inhalants.

[0256] The present invention also includes methods comprising co-administration of the sHDL nanoparticles described herein with one or more additional active agents. Indeed, it is a further aspect of the present invention to provide a method for enhancing prior art therapies and / or pharmaceutical compositions by co-administering the sHDL nanoparticles of the present invention. In co-administration procedures, the agents may be administered simultaneously or sequentially. In some embodiments, the sHDL nanoparticles described herein are administered before the other active agent(s). The agent(s) to be co-administered will depend on the type of condition being treated.

[0257] The present disclosure further provides compositions comprising sHDL nanoparticles as described herein or kits containing the components necessary to synthesize sHDL nanoparticles as described herein. In some embodiments, the kits contain all of the components necessary, sufficient, or useful for administering such sHDL nanoparticles. [Example]

[0258] The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting its scope. Example 1 This example describes materials and methods for the synthesis of biopolymer-loaded sHDL. material 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and rhodamine (RHOD)-labeled DOPE (DOPE-RHOD) were all purchased from Avanti Polar Lipids (Alabaster, AL). Dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP) was also synthesized. All peptides used, including the HDL-mimetic peptide (22A; SEQ ID NO: 4), SIINFEKL (SEQ ID NO: 341), CSSSIINFEKL (SEQ ID NO: 342), and FITC-labeled CSSSIINFEK(FITC)L, were customized using GenScript. The oligodeoxynucleotide TLR9 ligand CpG1826 (5'-tccatgacgttcctgacgtt-3', lowercase letters represent the phosphorothioate backbone) (SEQ ID NO: 343) and cholesterol-modified CpG1826 (5'-tccatgacgttcctgacgtt-3'-TEG-cholesterol) were ordered from Integrated DNA Technologies. HPLC-grade solvents, such as methanol and acetonitrile, were purchased from Fisher Scientific. Fetal bovine serum (FBS), penicillin-streptomycin, β-mercaptoethanol, and ACK lysis buffer were purchased from Life Technologies (Grand Island, NY). Granulocyte-macrophage colony-stimulating factor (GM-CSF) was from PeproTech (Rocky Hill, NJ). Rat anti-mouse CD16 / 32, CD86-PE, CD40-APC, SIINFEKL H-2K b Anti-mouse CD8-PE and MHC class II-FITC were obtained from eBioscience (San Diego, CA). Rat anti-mouse CD8-APC, hamster anti-mouse CD11c-PE, and streptavidin-Cy7 were obtained from BD Bioscience (San Jose, CA). iTAg tetramer / PE-H-2Kb OVA (SIINFEKL) was purchased from Beckman Coulter (Brea, CA). Preparation of peptide-, nucleic acid-, or glycolipid-loaded sHDL nanoparticles.

[0259] DMPC and DOPE-PDP (weight ratio = 4:0.25) were dissolved in chloroform. The mixture was dried under a nitrogen stream for 5 minutes and then placed in a vacuum oven for 1 hour. The resulting lipid film was then dissolved in 10 mM sodium phosphate buffer (0.3117 g / L NaHPO 4· HO and 2.0747 g / L NaHPO 4· The lipids were hydrated in 7H2O (22A) and sonicated in a bath sonicator for 10 minutes, followed by probe sonication for an additional 2.5 minutes. 22A peptide dissolved in endotoxin-free water was added to the above mixture (22A:lipid = 1:2, weight ratio), which was then heated (50°C) for 3 minutes and cooled (ice water) for 3 minutes, for a total of 3 cycles, to obtain sHDL.

[0260] To load sHDL with tumor antigen peptides, cysteine-terminal tumor antigen peptides dissolved in endotoxin-free water were added to the sHDL (antigen peptide:DOPE-PDP = 2.5:1 molar ratio) and incubated at room temperature with gentle shaking on an orbital shaker for 3 hours. Unreacted tumor antigen peptides were removed using a Zeba Spin Desalting column (Pierce) with a MWCO = 7000 Da cutoff according to the manufacturer's instructions. The conjugation efficiency of tumor antigen peptides was calculated based on the reduction of DOPE-PDP measured by HPLC. Briefly, 200 μl of sHDL preparation was lyophilized and reconstituted in 300 μl of methanol. The mixture was filtered through a 220 nm PTFE filter, and 20 μl was injected into a Shimadzu HPLC system equipped with a Vydac 219TP diphenyl column (4.6 mm x 250 mm internal diameter). The two solvents used for HPLC analysis consisted of water:trifluoroacetic acid = 100:0.5 (mobile phase A) and methanol:acetonitrile:trifluoroacetic acid = 50:50:0.05 (mobile phase B). The solvent system gradient program consisted of 25% mobile phase B, linearly increasing to 100% B over 75 min, linearly decreasing to 25% B in 80 min, and maintaining at 25% B between 80 and 90 min for equilibration before the next analysis. The flow rate was 1 mL / min, and the detection wavelength was 220 nm. The tumor antigen peptide loading efficiency was also determined by measuring the fluorescence intensity of the sHDL formulation at Ex = 490 nm and Em = 520 nm using FITC-labeled peptides after dissolving the formulation in PBS containing 1% Triton X-100.

[0261] To load CpG into sHDL, different concentrations of cholesterol-modified CpG (Cho-CpG) were incubated with sHDL at room temperature for 30 minutes with gentle shaking on an orbital shaker. The amounts of CpG incorporated into sHDL and free CpG were analyzed by gel permeation chromatography (GPC). Briefly, sHDL preparations were diluted with PBS to a 22A peptide concentration of 0.5 mg / mL. After filtering the preparations through a 220 nm filter, 40 μl of the sample was injected into a Shimadzu HPLC system equipped with a TSKgel G2000SWxl column (7.8 mm ID x 30 cm, Tosoh Bioscience LLC). The flow rate of the mobile phase, PBS (pH 7.4), was set to 0.7 mL / min, and the detection wavelength for CpG was set to 260 nm.

[0262] To load sHDL with α-galactosylceramide (aGC), a freeze-drying-based method for generating sHDL was developed. Briefly, phospholipids, aGC, and ApoA mimetic peptides were dissolved in glacial acetic acid and freeze-dried. The resulting powder was hydrated in PBS (pH 7.4) and cycled above and below the transition temperature (Tm) of the phospholipid to form aGC-sHDL. A similar protocol was used to load siRNA into sHDL. Cholesterol-modified PCSK9 siRNA was incubated with blank sHDL for 30 minutes at room temperature to form PCSK9 siRNA-sHDL. Measurement of sHDL morphology and size. The sHDL preparations were diluted to 0.5 mg / mL 22A in PBS, and the size was measured by dynamic light scattering (DLS, Zetasizer Nano ZSP, Malvern, UK). After appropriate dilution of the original sample, the morphology of the sHDL was observed by transmission electron microscopy (TEM). Preparation of bone marrow-derived dendritic cells (BMDCs) BMDCs were prepared. Briefly, mouse femurs and tibiae were harvested, washed, and ground in BMDC culture medium (RPMI 1640 supplemented with 10% FBS, 1% penicillin-streptomycin, 50 μM β-mercaptoethanol, and 20 ng / ml GM-CSF). The cell suspension was passed through a cell strainer (mesh size = 40 μm) followed by centrifugation to collect the cells. The cells were collected at a concentration of 2 × 10 5 BMDCs were seeded at a density of 1000 cells / ml onto non-tissue culture treated Petri dishes and cultured at 37°C in 5% CO. Culture medium was refreshed on days 3, 6, and 8, and BMDCs were used from days 8 to 12. Upregulation of activation markers on BMDCs Immature BMDCs were plated in 12-well plates at 1 x 10 6 BMDCs were seeded at 1000 x 1000 cells / well. The old medium was aspirated, and the BMDCs were washed once with PBS before being incubated with 0.5 μg / mL of different CpG-containing formulations or 0.5 μg / mL of LPS (positive control) for 24 hours at 37°C. BMDCs were harvested, washed once with FACS buffer (1% BSA in PBS), incubated with anti-CD16 / 32 for 10 minutes at room temperature, and then stained with fluorescent probe-conjugated antibodies against CD11c, CD40, CD80, CD86, and MHC class II for 30 minutes at room temperature. Finally, the cells were washed twice with FACS buffer, resuspended in 2 μg / mL DAPI solution, and analyzed by flow cytometry (Cyan 5, Beckman Coulter, USA). Antigen presentation by BMDCs Immature BMDCs were plated in 12-well plates at 1 x 10 6BMDCs were seeded at 1000 cells / well. The old medium was aspirated, and the BMDCs were washed once with PBS and then incubated with 0.5 μg / mL CpG and / or 0.5 μg / mL antigen peptide-containing formulations in complete medium at 37°C for different times (2, 6, 24, and 48 hours). BMDCs were harvested, washed once with FACS buffer, and incubated with anti-CD16 / 32 for 10 minutes at room temperature, followed by PE-tagged anti-mouse SIINFEKLH-2K for 30 minutes at room temperature. b The cells were stained with monoclonal antibody 25-D1.16. Finally, the cells were washed twice with FACS buffer, resuspended in 2 μg / ml DAPI solution, and analyzed by flow cytometry (Cyan 5, Beckman Coulter, USA). Imaging the intracellular delivery of sHDL-based peptide vaccines using CLSM 1 x 10 cells in 2 mL of complete medium 6 JAWS II cells were seeded onto 35 mm Petri dishes (MatTek) pre-equilibrated with the same medium and allowed to settle overnight. To understand the intracellular delivery profile of sHDL itself, DOPE-Rhod was used to label the lipids of sHDL, and Texas Red®-X, succinimidyl ester (Life Technologies). The 22A peptide of sHDL was labeled by incubating the sHDL with LysoTracker® Green DND-26 (Life Technologies) and then passed through a desalting column to remove unreacted dye. These labeled sHDL were incubated with JAWS II cells for 24 hours at 37°C. After incubation, the cells were washed three times with PBS and incubated with serum-free medium containing phenol and 500 nM LysoTracker® Green DND-26 (Life Technologies) and 2 μg / mL Hoechst for 30 minutes at 37°C to stain lysosomes and nuclei, respectively, followed by imaging using a confocal microscope (Nikon A1). To determine the intracellular delivery profile of the antigen peptide, free CSSSIINFEK (FITC) L + CpG or sHDL-CSSSIINFEK (FITC) L / CpG was incubated with JAWS II cells for different times (6, 24, and 48 hours). After incubation, cells were washed three times with PBS and incubated for 30 minutes at 37°C with serum-free medium containing phenol and 50 nM LysoTracker® Red DND-99 (Life Technologies) and 2 μg / mL Hoechst to stain lysosomes and nuclei, respectively, and then imaged using a confocal microscope (Nikon A1). B3Z T cell activation in vitro BMDCs were plated in a U-bottom 96-well plate at 5 x 10 4 BMDCs were seeded at 10 x 10 cells / well and grown overnight. The old medium was aspirated, and the BMDCs were washed once with PBS and then incubated with different concentrations (0.02, 0.1, and 0.5 μg / mL) of SIINFEKL and CpG containing formulations for 24 or 48 hours at 37°C. After incubation, the cells were carefully washed three times with PBS and plated at 10 x 10 cells / well. 4B3Z T cells / well were added and co-cultured for an additional 24 hours in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 55 μM β-mercaptoethanol, 1 mM pyruvate, and 100 U / mL penicillin and 100 μg / mL streptomycin. Cells were then pelleted by centrifugation (1500 rcf, 7 min). The medium was carefully aspirated, and 150 μL of CPRG / lysis buffer (PBS containing 0.15 mM chlorophenol red β-D-galactopyranoside (CPRG), 0.1% Triton-X100, 9 mM MgCl2, and 100 μM mercaptoethanol) was added. The plate was incubated at 37°C in the dark for 90 minutes, after which the absorbance of released chlorophenol red was measured at 570 nm using a plate reader. Lymph node excretion of antigenic peptides sHDL-CSSIINFEK(FITC)L was prepared as described above. Six- to eight-week-old female C57BL / 6 mice were purchased from Harlan Laboratories. C57BL / 6 mice were subcutaneously injected with free CSSIINFEK(FITC)L or sHDL-CSSIINFEK(FITC)L. Twenty-four hours after injection, mice were euthanized by carbon dioxide inhalation, and axillary and inguinal lymph nodes were collected and imaged using an IVIS optical imaging system (Caliper Lifesciences). Analysis of cytotoxic T cell responses in the setting of in vivo vaccination and prevention and treatment of melanoma tumor growth C57BL / 6 mice were immunized subcutaneously at the base of the tail with different formulations containing SIINFEKL (15 μg / mouse) and CpG (15 μg / mouse) according to a predetermined schedule. The percentage of tumor antigen-specific CD8+ T cells was measured 7 days after each vaccination using a tetramer staining assay. Briefly, 100 μl of blood was collected from each mouse. The blood samples were lysed with ACK lysis buffer and subsequently centrifuged to collect pellets. These were then washed once with FACS buffer, blocked with CD16 / 32 blocking antibodies, and incubated with PE-labeled SIINFEKL tetramers for 30 minutes at room temperature. The samples were then incubated with anti-CD8 APCs for 20 minutes on ice. Cells were washed twice with FACS buffer and resuspended in 2 μg / ml DAPI solution for analysis by flow cytometry (Cyan 5, Beckman Coulter, USA). To examine the effect of T cell responses on tumor growth, mice were challenged with 200,000 B16.OVA / mouse by subcutaneous injection in the right flank one day after the final tetramer staining. Tumor development was monitored every other day, and tumor volume was calculated by the following formula: tumor volume = length × width. 2 × 0.52. To examine the effect of sHDL vaccination on established tumors, C57BL / 6 mice were inoculated with 200,000 B16.OVA / mouse by subcutaneous injection into the right flank on day 0. On days 4 and 11, mice were immunized with different formulations containing tumor antigen peptide (15 μg / mouse) and CpG (15 μg / mouse). The percentage of tumor antigen-specific CD8+ T cells was measured on days 10 and 17 by tetramer staining assay as described above. Tumor burden was monitored every other day. aGC-CD1d presentation assay JAWS II cells were seeded at a density of 200,000 cells / well in 12-well plates. After 48 hours, the medium was replaced with fresh medium containing different formulations of aGC at 2000 ng / mL. After 20-24 hours of incubation with the formulations, cells were trypsinized into FACS tubes, washed twice with FACS buffer, and then incubated with CD16 / 32 blocking reagent for 10 minutes at room temperature. Next, cells were incubated with anti-mouse aGC-CD1d-PE for 30 minutes at room temperature, washed twice with FACS buffer, and suspended in 0.3 mL of FACS buffer containing DAPI for flow cytometry. Characterization of PCSK9 siRNA-loaded sHDL To quantify the amount of PCSK9 siRNA molecules loaded into sHDL, various concentrations of PCSK9 siRNA were incubated with sHDL. The concentration of PCSK9 siRNA associated with sHDL relative to the free form will be measured at 260 nm using gel permeation chromatography (GPC). PCSK9 knockdown in HepG2 cells Different formulations of PCSK9 siRNA were incubated with HepG2 cells for 48 hours. After incubation, cells were washed twice with PBS, and cell lysates were prepared. PCSK9 protein levels were analyzed by Western blot assay. Biodistribution of sHDL To examine the biodistribution of sHDL, DiD-loaded sHDL was intravenously injected into C57BL / 6 mice. 24 hours after injection, the mice were euthanized, and the distribution of sHDL in major organs (heart, liver, spleen, lungs, and kidneys) was analyzed using an IVIS optical imaging system. Example II. This example demonstrates that PCSK9 siRNA incorporated into sHDL can efficiently accumulate in the liver, deliver its cargo to SR-BI-positive cells, and knockdown PCSK9 in HepG2 cells. A rapid and inexpensive freeze-drying method for the preparation of homogeneous sHDL nanoparticles was implemented. The homogeneity of the sHDL was confirmed by transmission electron microscopy (TEM), dynamic laser scattering (DLS), and gel permeation chromatography (GPC) (Figure 1A). sHDL was labeled with the fluorescent dye DiR and intravenously injected into mice. The majority of the DiR signal was detected in the liver, with little or no signal detected in other organs (Figure 1B). sHDL also efficiently delivered the fluorescent dye DiO to SR-BI-positive cells (BHK-SR-BI), but not to SR-BI-negative cells (BHK-vector), and uptake by SR-BI-positive cells was blocked by excess blank sHDL (Figure 1C). Furthermore, preliminary data showed that cholesterol-modified PCSK9 siRNA (PCSK9 Cho-siRNA) could be quantitatively incorporated into sHDL. Although free PCSK9 Cho-siRNA could knockdown PCSK9 in HepG2 cells due to increased siRNA uptake induced by cholesterol conjugation, PCSK9 siRNA-sHDL still sufficiently knocked down PCSK9 protein in HepG2 cells in vitro (Figure 1D-F). Example III. This example demonstrates that colocalized delivery of antigen and adjuvant by sHDL results in a potent immune response. Figure 4A shows a schematic diagram of antigen- and adjuvant-loaded sHDL. When an MHC class I antigen peptide (ovalbumin-derived CD8+ T cell epitope peptide SIINFEKL) was incubated with functional lipid-containing sHDL, the antigen peptide was quantitatively conjugated to the functional lipids of sHDL, as evidenced by the disappearance of the functional lipid and the appearance of lipid-peptide conjugates (Figure 2B). It was also shown that cholesterol-modified CpG (Cho-CpG) was quantitatively incorporated into sHDL (Figure 2C). After one primary dose and two booster doses, antigen and CpG-loaded sHDL (sHDL-Ag / CpG) elicited a stronger immune response than a mixture of antigen and CpG in Montanide (CpG + Montanide is one of the most potent experimental adjuvants currently undergoing clinical evaluation) (Figure 2D).

[0263] Figure 3 shows a schematic diagram of the synthesis of sHDL-CSSIINFEK(FITC)L / CpG.

[0264] FIG. 4 shows the homogeneous particle size of sHDL-Ag / CpG analyzed by cryoEM and dynamic light scattering.

[0265] Figures 5A and 5B show that antigen delivery by sHDL significantly prolongs antigen presentation by dendritic cells compared to the free antigen form.

[0266] FIG. 6 shows that sHDL-Ag / CpG significantly enhances the induction of antigen-specific CD8+ T cells compared to vaccination with free antigen mixed with conventional adjuvants.

[0267] FIG. 7 shows that sHDL-Ag / CpG vaccination induces strong CD8+ T cell responses and reduces tumor growth in tumor-bearing mice. Example IV. This example demonstrates that sHDL delivers α-galactosylceramide, a glycolipid ligand to CD1-d, to activate the induction of natural killer T cells.

[0268] FIG. 8 shows that α-GalCer delivered via sHDL significantly enhanced CD1d presentation on antigen-presenting cells compared to the free soluble form.

[0269] FIG. 9 shows that lyophilization provides a convenient method for large-scale synthesis of α-GalCer-loaded sHDL. Example V This example demonstrates that preformed high-density lipoprotein-mimetic nanodiscs can be readily conjugated with antigen (Ag) peptides and adjuvants to produce stable, ultrasmall nanoparticles that significantly improve Ag / adjuvant co-delivery to lymphoid organs and achieve sustained Ag presentation on dendritic cells.

[0270] We first identified lipids and peptides that trigger Nanodisc formation. DMPC lipid films were hydrated, and a series of ApoA-I mimetic peptides were added, followed by thermocycling from 50°C to 4°C. We identified a subset of peptides containing 22A and the D-amino acid of 22A that produced clear sHDL suspensions that were stable for 1 month when stored at 4°C (Figure 13a). In addition, the use of phospholipids with transition temperatures (Tm) below RT (e.g., POPC and DOPC, Tm = -2°C and -17°C, respectively) produced turbid liposome suspensions, whereas lipids with higher Tm (e.g., DPPC and DMPC, Tm = 41°C and 24°C, respectively) formed clear sHDL suspensions in the presence of 22A (Figure 13b), demonstrating flexibility in material design. Based on their size, homogeneity, and long-term stability, 22A and DMPC, the main components of Nanodisc vaccines, were selected for further investigation.

[0271] To achieve intracellular release of Ag within APCs via reduction-sensitive conjugation of Ag onto sHDL, we synthesized dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (PDP, Figure 14) and incorporated PDP into sHDL (4 mol%). When incubated at room temperature for 30 minutes with Ag peptides modified with a cysteine-serine-serine (CSS) linker (see, e.g., Hirosue, S., et al., Vaccine 28, 7897-7906 (2010)), sHDL nanodiscs were able to bind various Ag peptides (e.g., OVA). 257-264 , a model CD8α+ T cell epitope of ovalbumin Ag;gp100 25-33 Efficient surface modification with Cho-CpG (Adgpk, a melanoma-associated Ag; and Adgpk, a neoantigen in MC-38) followed by incubation with Cho-CpG at room temperature for 30 min resulted in nearly complete (approximately 98%) insertion of CpG into sHDL, generating Ag and CpG co-loaded nanodiscs (termed sHDL-Ag / CpG, with approximately 6.5 Ag peptides and approximately 1 CpG molecule per nanodisc, Figure 15; Table 3). sHDL-Ag / CpG exhibited a uniform discoidal morphology with a mean diameter of 10.5 ± 0.5 nm and a polydispersity index of 0.20 ± 0.02 (Figures 16a and 16b). Importantly, sHDL-Ag / CpG could be easily sterile filtered and thawed at 37 °C after frozen storage at -20 °C for at least 8 weeks without adversely affecting its homogeneity (Figure 16c).

[0272] [Table 3]

[0273] Next, we examined the effect of nanodiscs on Ag presentation. Bone marrow-derived dendritic cells (BMDCs) pulsed with sHDL-CSSSIINFEKL / CpG for 24 hours presented SIINFEKL-H-2K. bOVA more efficiently than BMDCs treated with free Ag peptide mixed with CpG or sHDL-CSSSIINFEKL, as measured by staining DCs with 25-D1.16 mAb against the complex. 257-264 DCs pulsed with free SIINFEKL+CpG presented Ag efficiently during the first 6 hours of incubation, but Ag presentation declined sharply after 6 hours (Figs. 16e and 16f; Fig. 17c), suggesting initial direct Ag binding to MHC-I molecules followed by rapid Ag degradation or dissociation. In contrast, compared with free SIINFEKL+CpG, Ag presentation using sHDL-Ag / CpG gradually increased over time, reaching approximately 9-fold higher levels at 24 hours and remaining approximately 4-fold higher at 48 hours.

[0274] Aiming to extend Ag presentation, CSS-SIINFEK (FITC) The process of nanodisc uptake and Ag localization was investigated using L. SIINFEKL, in which the ε-amino group of the lysine residue was modified with FITC, was used to investigate the uptake of H-2K. bIt is known that sHDL-Ag(FITC) / CpG retains its ability to bind to molecules (see, e.g., Saini, SK et al. Proc. Natl. Acad. Sci. USA 110, 15383-15388 (2013)). JAWS II cells (immortalized immature DCs) incubated with free Ag(FITC)+CpG showed a weak fluorescent signal at the plasma membrane at 6 hours, and only dim fluorescence was observed by 24 hours (Figure 16g; Figure 18). In sharp contrast, sHDL-Ag(FITC) / CpG treatment resulted in a strong FITC signal that colocalized with endosomes / lysosomes by 6 hours, and a strong Ag(FITC) signal was detected on the plasma membrane by 24 hours and persisted for up to 48 hours. In addition, nanodiscs containing Rh-PE or Texas Red-labeled-22A were detected primarily within endosomes / lysosomes, indicating cellular uptake of whole, intact nanodiscs (Figure 19). To assess the effect of prolonged Ag presentation on T cell cross-priming, BMDCs were treated with free Ag peptide + CpG or sHDL-Ag / CpG for 24 or 48 hours, followed by SIINFEKL-specific H-2K expression. b The BMDCs were then treated with a B3Z T cell-restricting hybridoma. sHDL-Ag / CpG-pulsed BMDCs promoted robust B3Z T cell activation even after 48 h of incubation, whereas free Ag peptide + CpG induced minimal B3Z T cell activation over a 24 h period (Figure 16h). Furthermore, sHDL-Ag / CpG potently stimulated DC maturation (Figure 20). Overall, free Ag peptide was rapidly loaded and dissociated from MHC-I molecules on the cell membrane, whereas Nanodiscs promoted intracellular delivery of Ag / CpG and mediated its sustained release within endosomes / lysosomes, thereby promoting sustained Ag presentation, APC maturation, and cross-priming of CD8α+ T cells in vitro.

[0275] Next, the effect of Nanodiscs on the lymphatic delivery of Ag / CpG in vivo and the induction of CTL responses (see, e.g., Reddy, ST et al. Nat. Biotechnol. 25, 1159-1164 (2007)) was investigated. (FITC) C57BL / 6 mice injected subcutaneously at the base of the tail with L showed significant reductions in IL-1 expression, potentially due to systemic dissemination of small MW Ag peptides or direct Ag binding on non-APCs at the injection site (e.g., Melief, CJ & van der Burg, SH Nat. Rev. Cancer 8, 351-360 (2008)), and had minimal FITC signal in the inguinal dLN after 1 day (see, e.g., Figure 21a). In contrast, the sHDL-Ag group showed a significant increase in FITC signal in the dLN (p<0.01, Figure 21a), and Ag (FITC) and Cy5-tagged 22A colocalized within the dLN (Figure 22). Similarly, injection of 2.3 nmol of Cy5-tagged Cho-CpG into sHDL increased LN accumulation compared with injection of the free soluble form (p<0.01, Figure 21b). These results indicated that sHDL nanodiscs facilitate the simultaneous delivery of Ag and CpG to the dLN. Next, C57BL / 6 mice were immunized with 15.5 nmol of Ag and 2.3 nmol of CpG (non-fluorophore-tagged), and peripheral blood mononuclear cells (PBMCs) were analyzed for the frequency of SIINFEKL-MHC-I tetramer+CD8α+ T cells. Mixtures of free Ag peptide (SIINFEKL or CSS-SIINFEKL) and CpG induced 1-3% Ag-specific CTLs after the third immunization (Figures 21c and 21d). As a benchmark, animals were also vaccinated with equivalent doses of Ag and CpG emulsified in water-in-oil Montanide (see, e.g., Speiser, DE et al. J. Clin. Invest. 115, 739-746 (2005); Fourcade, J. et al. J. Immunother. 31, 781-791 (2008)). Ag+CpG+Montanide induced approximately 2% Ag-specific CTLs after priming, but no further T cell expansion was observed even after the third immunization, consistent with recent studies reporting dysfunction and loss of high-avidity T cells after repeated immunization with depot-forming water-in-oil adjuvants (see, e.g., Rezvani, K. et al. Haematologica 96, 432-440 (2011); Hailemichael, Y. et al. Nat. Med. 19, 465-472 (2013)).In marked contrast, the sHDL-Ag / CpG group induced approximately 21% of the peak frequency of Ag-specific CD8α+ T cells after the third vaccination (29-fold greater than soluble SIINFEKL+CpG and 9-fold greater than Ag+CpG+Montanide, P<0.0001, Figures 21c and 21d). 5 When administered B16OVA cells, mice immunized with sHDL-Ag / CpG had no detectable tumor masses by 28 days, and 40% of the animals survived for over 200 days, whereas mice immunized with free Ag peptide + CpG or Ag + CpG + Montanide all died of tumors, resulting in the lowest survival benefit (Figures 2e and 2f). Importantly, throughout these experiments, no signs of toxicity or autoimmunity were observed in animals immunized multiple times with sHDL-Ag / CpG.

[0276] Experiments were performed to rule out the possibility that CSS-modified peptides or Cho-CpG dissociated from sHDL-Ag / CpG in vivo were responsible for the potent CTL response. Introducing a CSS linker into SIINFEKL and replacing free CpG with the free, soluble form of Cho-CpG resulted in minimal T cell responses, and a physical mixture of Ag, CpG, and sHDL also elicited a weak CTL response (Figure 21g). In contrast, sHDL-Ag / CpG nanodiscs significantly improved the CTL response, eliciting a remarkable 41-fold greater frequency of Ag-specific CD8α+ T cells than the CSSSINFEKL+Cho-CpG group (day 35, p<0.0001, Figure 21g). CTLs were primarily CD44 high CD62L low Effector phenotype and potent IFN-γ + ELISPOT responses were shown (Fig. 21h; Fig. 23).

[0277] The antitumor effect of sHDL was evaluated in tumor-bearing mice. Therapeutic sHDL vaccination in B16OVA melanoma-bearing mice resulted in a strong Ag-specific CTL response, significantly slowed tumor growth, and prolonged animal survival (Figure 24). Next, we tested the nanodisc vaccine using the non-immunogenic B16F10 melanoma as a more clinically relevant model. gp100 25-33 After confirming the incorporation of sHDL-gp100 / CpG into Nanodiscs with Cho-CpG (Figure 15; Table 3), mice were treated with 15.5 nmol Ag and 2.3 nmol CpG on days 4 and 11 after subcutaneous inoculation of B16F10 cells. Vaccination with sHDL-gp100 / CpG elicited potent CTL responses, resulting in a 22-fold higher frequency of gp100-specific CTLs than with free gp100 + CpG (day 17, p<0.0001, Figure 25a; Figure 26), leading to a significant delay in tumor growth and prolonged animal survival compared with the ineffective free gp100 + CpG group (Figures 25b and 25c).

[0278] Finally, to demonstrate the utility of our platform technology for vaccination against neoantigens, we recently identified a single epitope mutation within the Adpgk protein.

[0279] [ka]

[0280] Using the murine MC-38 colon carcinoma model, which has been reported to have b The Adpgk neoantigen mutation in MC-38 cells was confirmed by cDNA sequencing (Fig. 25d; Fig. 27). sHDL-Adpgk / CpG was synthesized by mixing nanodiscs with neoepitopes modified with CSS-linkers and Cho-CpG. C57BL / 6 mice were infected with 10 5MC-38 cells were subcutaneously inoculated and treated with 15.5 nmol of the Adpgk mutant peptide and 2.3 nmol of CpG. Mice treated with free Adpgk Ag+CpG had similar levels of Adpgk-specific CD8α+ T cells as non-immunized MC-38-bearing mice, whereas sHDL-Adpgk / CpG significantly enhanced the CTL response (day 23, p<0.001, Fig. 25e). In addition, sHDL-Adpgk / CpG significantly enhanced the CTL response by upregulating the multifunctional IFN-γ + and IFN-γ + TNF-alpha + Adpgk-specific CD8α T cells were induced (2.5-fold and 7-fold greater than the free Adpgk+CpG group, p<0.05 and p<0.001, respectively, Fig. 25f). Importantly, therapeutic treatment with sHDL-Adpgk / CpG substantially slowed MC-38 tumor growth and prolonged animal survival (median survival: 54 days vs. 33 days, p<0.01, Fig. 25g and Fig. 25h, respectively), in contrast to the conventional soluble Adpgk+CpG vaccine, which had no statistically significant effect on tumor growth or survival. Example VI This example relates to the materials and methods of Example V. material 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and rhodamine (RHOD)-labeled DOPE (DOPE-RHOD) were purchased from Avanti Polar Lipids (Alabaster, AL). ApoA1 mimetic peptide (22A), OVA, 257-264 SIINFEKL, CSSSIINFEKL, CSSSIINFEK(FITC)L, hgp100 25-33KVPRNQDWL, CSSSKVPRNQDWL, and the Adpgk mutant peptide ASMTNMELM were synthesized by GenScript Corp. (Piscataway, NJ). CSSASMENMELM was synthesized by AnaSpec (Fremont, CA). The oligodeoxynucleotide TLR9 ligand CpG 1826 (5'-tccatgacgttcctgacgtt-3', lowercase letters indicate a phosphorothioate backbone), CpG 1826 modified with cholesterol at the 3' end (Cho-CpG), and Cy5-modified Cho-CpG were synthesized by Integrated DNA Technologies (Coralville, IA). HPLC-grade methanol and acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA). Fetal bovine serum (FBS), penicillin-streptomycin, β-mercaptoethanol, and ACK lysis buffer were purchased from Life Technologies (Grand Island, NY). Granulocyte-macrophage colony-stimulating factor (GM-CSF) was obtained from GenScript Corp. (Piscataway, NJ). Anti-mouse CD16 / 32, CD86-PE, CD40-APC, CD62L-PECy7, and SIINFEKL / H-2K antibodies were used. b 25-D1.16 mAb-PE against β-glucan was obtained from eBioscience (San Diego, CA). Anti-mouse CD8α-APC, CD44-FITC, TNF-α-FITC, IFN-γ-PE, and CD11c-PE Cy7 were obtained from BD Bioscience (San Jose, CA). Tetramer H-2K b -SIINFEKL-PE and Tetramer H-2D b -KVPRNQDWL-PE was purchased from Beckman Coulter (Brea, CA). Tetramer / H-2D b-ASMTNMELM-PE was kindly provided by the NIH Tetramer Core Facility (Atlanta, GA). B3Z CD8α+ T cell hybridoma was obtained from Dr. N. Shastri (University of California, Berkeley), B16OVA from Dr. Kenneth Rock (University of Massachusetts, Amherst, MA); MC-38 cells were obtained from Dr. Weiping Zou (University of Michigan, Ann Arbor, MI). method Synthesis and characterization of DOPE-PDP Dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP) was synthesized as previously reported with slight modifications (see, for example, Kuai, R., et al. Mol. Pharm. 7, 1816-1826 (2010)). Briefly, DOPE, SPDP (succinimidyl 3-(2-pyridyldithio)propionate), and triethylamine (1:1:1.5 molar ratio) were dissolved in chloroform. The mixture was reacted in the dark for 5 hours. The progress of the reaction was monitored by thin-layer chromatography (TLC) using the following mixture as the developing solvent: chloroform / methanol / water = 65 / 25 / 4 (volume ratio). After TLC showed the disappearance of the starting material and the appearance of a faster-running spot, the reaction mixture was dried by rotary evaporation and purified on a silica gel column. Synthesis of sHDL co-loaded with antigenic peptides and CpG DMPC and DOPE-PDP (molar ratio = 96:4) were dissolved in chloroform. The mixture was dried with a stream of nitrogen and placed under vacuum for at least 1 h. The resulting lipid film was then dissolved in 10 mM sodium phosphate buffer (0.3117 g / L NaHPO 4· HO and 2.0747 g / L NaHPO 4·The lipids were hydrated with 7H2O (pH 7.4) and sonicated in a bath sonicator for 10 minutes, followed by probe sonication for an additional 2.5 minutes. ApoA1 mimetic peptide 22A dissolved in endotoxin-free water was added to the above mixture (22A:lipid = 1:7.5, molar ratio), which was then heated (50 °C) for 3 minutes and cooled (ice water) for 3 minutes, for a total of 3 cycles to obtain sHDL.

[0281] To conjugate tumor antigen peptides to sHDL, cysteine-terminal tumor antigen peptides dissolved in endotoxin-free water were added to the sHDL (antigen peptide:DOPE-PDP = 2.5:1 molar ratio) and incubated at room temperature with gentle shaking on an orbital shaker. Unreacted tumor antigen peptides were removed using a Zeba Spin Desalting column (Pierce) according to the manufacturer's instructions. The conjugation efficiency of tumor antigen peptides was calculated based on the decrease in absorbance signal associated with DOPE-PDP as measured by HPLC. Briefly, 200 μl of sHDL preparation was lyophilized and reconstituted in 300 μl of methanol. The mixture was filtered through a 0.22 μm PTFE filter and analyzed on a Shimadzu HPLC system using a Vydac 219TP diphenyl column (4.6 mm x 250 mm ID). The two solvents used for HPLC analysis were water:trifluoroacetic acid = 100:0.5 (mobile phase A) and methanol:acetonitrile:trifluoroacetic acid = 50:50:0.05 (mobile phase B) (0-75 min, 15-100%). The flow rate was 0.4 mL / min, and the detection wavelength was 220 nm. After dissolving the formulation in PBS containing 1% Triton X-100, the loading efficiency of tumor antigen peptides in sHDL was confirmed by measuring the fluorescence intensity of the sHDL formulation at Ex = 490 nm and Em = 520 nm using FITC-labeled peptides.

[0282] To load sHDL with CpG, different concentrations (0–200 μg / mL) of cholesterol-modified CpG (Cho-CpG) were incubated with sHDL at room temperature while gently shaking on an orbital shaker. The amounts of CpG incorporated into sHDL and free CpG were analyzed by gel permeation chromatography (GPC). Briefly, sHDL preparations were diluted in PBS to a 22A peptide concentration of 0.5 mg / mL. The preparations were filtered through a 0.22 μm filter and analyzed on a Shimadzu HPLC system equipped with a TSKgel G2000SWxl column (7.8 mm i.d. x 30 cm, Tosoh Bioscience LLC). The flow rate of the mobile phase (PBS, pH 7.4) was set at 0.7 mL / min, and the detection wavelength for CpG was set at 260 nm. Characterization of peptide / CpG-loaded sHDL formulations The sHDL preparation was diluted to 0.5 mg / mL 22A with PBS, and particle size was measured by dynamic light scattering (DLS, Zetasizer Nano ZSP, Malvern, UK). After appropriate dilution of the original sample, the morphology of the sHDL was observed by transmission electron microscopy (TEM). Briefly, 3 μL of sample solution was deposited on a 400-mesh copper grid (Electron Microscopy Sciences) coated with a carbon film and allowed to dry for 1 min. The sample was then negatively stained with five drops of 1% uranyl acetate solution, and excess solution was wiped off the grid. The grid was then allowed to dry before TEM observation. All images were acquired with a JEM 1200EX electron microscope (JEOL USA, Woburn, MA) equipped with an AMT XR-60 digital camera (Advanced Microscopy Techniques Corp., Woburn, MA). Preparation of BMDCs BMDCs were prepared as previously described (see, e.g., Lutz, MB, et al. J. Immunol. Methods 223, 77-92 (1999)). Briefly, femurs and tibias were aseptically collected from C57BL / 6 mice, and bone marrow was flushed into a Petri dish using a 5 mL syringe (26G needle) loaded with BMDC medium (RPMI 1640 supplemented with 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μM β-mercaptoethanol, and 20 ng / mL GM-CSF). The cell suspension was passed through a cell strainer (mesh size = 40 μm) followed by centrifugation to collect cells. Cells were collected at a concentration of 2 × 10 5 BMDCs were seeded at a density of 1000 cells / ml onto non-tissue culture treated Petri dishes and cultured at 37°C and 5% CO. The medium was refreshed on days 3, 6, 8, and 10, and BMDCs were used for subsequent assays on days 8–12. BMDC activation Immature BMDCs were plated in a 12-well plate at 1 x 10 6 BMDCs were seeded at 1000 cells / well. After 24 hours, BMDCs were washed once with PBS and incubated with different formulations of 75 nM CpG or 0.5 μg / mL LPS (positive control) for 24 hours at 37°C and 5% CO2. BMDCs were harvested, washed with FACS buffer (1% BSA in PBS), incubated with anti-CD16 / 32 for at least 10 minutes at room temperature, and then stained with fluorophore-conjugated antibodies against CD11c, CD40, CD80, and CD86 for 30 minutes at room temperature. Finally, cells were washed twice with FACS buffer, resuspended in 2 μg / mL DAPI solution, and analyzed by flow cytometry (Cyan 5, Beckman Coulter, USA). Antigen presentation on BMDCs Immature BMDCs were plated in 12-well plates at 1 x 10 6BMDCs were seeded at 1000 cells / well. BMDCs were washed with PBS and incubated with various formulations of 75 nM CpG and / or 500 nM antigen peptide in complete medium for different times (2, 6, 24, and 48 hours). BMDCs were then harvested, washed with FACS buffer, and incubated with anti-CD16 / 32 for at least 10 minutes at room temperature, followed by PE-conjugated anti-mouse SIINFEKL / H-2K for 30 minutes at room temperature. B The cells were stained with mAb 25-D1.16, then washed, resuspended in 2 μg / ml DAPI solution, and analyzed by flow cytometry (Cyan 5, Beckman Coulter, USA). Confocal microscopic imaging of intracellular trafficking of sHDL JAWSII cells (ATCC, Manassas, VA) were plated at 1 × 10 in 35-mm Petri dishes (MatTek Corp., Ashland, MA) pre-equilibrated with complete cell culture medium. 6Cells were seeded and cultured overnight. To investigate the intracellular delivery profile of the antigenic peptide, JAWS II cells were incubated with a physical mixture of free CSSSIINFEK(FITC)L and CpG or sHDL-CSSSIINFEK(FITC)L / CpG for different times (6, 24, and 48 hours). Cells were then washed three times with PBS and incubated in phenol / serum-free medium with 50 nM LysoTracker® Red DND-99 (Invitrogen) and 2 μg / mL Hoechst for 30 minutes at 37°C to stain lysosomes and nuclei, respectively. In parallel, to examine the intracellular delivery profile of sHDL structural components, the lipid layer of sHDL was incorporated with DOPE-Rhod by adding 0.5 mol% DOPE-Rhod to the initial lipid film. The 22A peptide of sHDL was labeled by incubating preformed sHDL with Texas Red®-X succinimidyl ester (Life Technologies) and passing the Texas Red-labeled sHDL through a desalting column to remove unreacted dye. The resulting fluorophore-tagged sHDL preparation was incubated with JAWS II cells at 37°C and 5% CO2. After 24 h of incubation, cells were washed three times with PBS and then incubated with 500 nM LysoTracker® Green DND-26 (Invitrogen) and 2 μg / mL Hoechst in phenol / serum-free medium for 30 min at 37°C to stain lysosomes and nuclei, respectively. JAWSII cells were then imaged using a confocal microscope (Nikon A1). Activation of B3Z CD8+ T hybridoma cells with sHDL BMDCs were plated at 5 x 10 in a U-bottom 96-well plate. 4 After overnight culture, BMDCs were washed with PBS and incubated with different formulations of SIINFEKL (20, 100, and 500 nM) and CpG (3, 15, and 75 nM) for 24 or 48 hours at 37°C. The cells were then carefully washed three times with PBS and incubated for 10 min at 37°C. 5B3Z CD8+ T hybridoma cells / well were added to RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 55 μM β-mercaptoethanol, 1 mM pyruvate, and 100 U / mL penicillin and 100 μg / mL streptomycin. After 24 h of incubation, cells were pelleted by centrifugation (1500 rcf, 7 min), the medium was carefully aspirated, and 150 μL of CPRG / lysis buffer (0.15 mM chlorophenol red β-D-galactopyranoside (CPRG), 0.1% Triton-X100, 9 mM MgCl, 100 μM mercaptoethanol in PBS) was added. The plates were incubated at 37°C in the dark for 90 minutes, after which the absorbance of released chlorophenol red was measured at 570 nm using a microplate reader. In vivo immunoassay Animals were cared for in accordance with federal, state, and local guidelines. All studies performed on animals were approved by the University Committee on Use and Care of Animals (UCUCA) at the University of Michigan, Ann Arbor. Six- to eight-week-old female C57BL / 6 mice (Harlan Laboratories) were immunized subcutaneously at the base of the tail with 100 μl of different formulations containing antigen peptide (15.5 nmol / mouse) and CpG (2.3 nmol / mouse) at the indicated time points. In some studies, antigen peptide and CpG emulsified in Montanide served as a positive control (see, e.g., Speiser, DE, et al. J. Clin. Invest. 115, 739-746 (2005); Fourcade, J., et al. J. Immunother. 31, 781-791 (2008); Karbach, J., et al. Int. J. Cancer 126, 909-918 (2010)). Briefly, antigen peptide (155 nmol) and CpG (23 nmol) in 0.5 mL of PBS were thoroughly emulsified in 0.5 mL of Montanide until the mixture was homogeneous.

[0283] For lymph node drainage studies, C57BL / 6 mice were injected with free CSSSIINFEK(FITC)L, sHDL-CSSSIINFEK(FITC)L, free Cho-CpG(Cy5), or sHDL-Cho-CpG(Cy5). 24 hours later, inguinal lymph nodes were harvested, and FITC or Cy5 fluorescent signals were measured using an IVIS optical imaging system (Caliper Life Sciences).

[0284] In the prophylactic tumor challenge study, vaccinated animals received 2 x 10 5 B16OVA cells / mouse were administered by subcutaneous injection. Tumor growth was monitored every other day, and throughout the study, tumor volume was calculated using the following formula (see, e.g., Gorrin-Rivas, MJ, et al. Clin. Cancer Res. 6, 1647-1654 (2000)): tumor volume = length x width. 2 × 0.52. Animals were euthanized when tumor masses reached 1.5 cm in diameter or when the animals became moribund with severe weight loss or ulceration.

[0285] In the therapeutic tumor vaccination study, C57BL / 6 mice received tumor cells (2 × 10 per mouse) by subcutaneous injection on day 0 in the right flank. 5 2 x 10 B16OVA cells 5 B16F10 cells, or 1 x 10 5 In the B16OVA and B16F10 studies, mice were vaccinated with different formulations containing 15.5 nmol of tumor antigen peptide (SIINFEKL and hgp100, respectively) and 2.3 nmol of CpG on days 4 and 11. In the MC-38 study, mice were vaccinated with 15.5 nmol of ASMTNMELM in either sHDL or free soluble form and 2.3 nmol of CpG on days 10, 17, and 24. Tumor growth was monitored as described above. Peptide-MHC tetramer assay Immunized mice were analyzed for the percentage of tumor antigen-specific CD8α T cells among peripheral blood mononuclear cells (PBMCs) using a tetramer staining assay as previously described (see, e.g., Ochyl, LJ & Moon, JJJ Vis. Exp. e52771 (2015)). Briefly, 100 μl of blood was collected from each mouse at the indicated time points by submandibular bleeding, and red blood cells were lysed with ACK lysis buffer. PBMCs were then washed with FACS buffer, blocked with anti-CD16 / 32 antibody, and incubated for 30 minutes at room temperature with PE-tagged peptide-MHC tetramers (e.g., H-2K). b Restraint SIINFEKL, H-2D b Restrictive KVPRNQDWL, or H-2D b The cells were then incubated with anti-CD8α-APC (restrictive ASMTNMELM) for 20 minutes on ice. The cells were washed twice with FACS buffer and resuspended in 2 μg / ml DAPI solution for analysis by flow cytometry (Cyan 5, Beckman Coulter, USA). ELISPOT and intracellular cytokine staining assays For the ELISPOT assay, spleens from immunized mice were aseptically collected, processed into single cell suspensions for each mouse, and plated at 3 × 10 cells per well in a 96-well PVDF plate (EMD Millipore) preincubated overnight with IFN-γ coating Ab (R&D Systems). 5 Splenocytes were seeded with 1000 splenocytes. Splenocytes were incubated with antigen peptide (2.5 μg / ml) or control for 24 hours. The assay was completed using sequential incubations with biotinylated secondary Ab, streptavidin-alkaline phosphatase (Sigma Chemical), and NBT / BCIP substrate (Surmodics). The assay was performed using an AID iSpot Reader (Autoimmune The number of expressed spots was counted using a ELISA kit (Diagnostika GmbH, Germany). For the intracellular cytokine staining (ICS) assay, 100–150 μL of peripheral blood collected from vaccinated mice was lysed with ACK lysis buffer, washed with PBS, and seeded at approximately 10 million cells / mL in 50 μL of T cell medium (RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 55 μM β-mercaptoethanol, 1 mM pyruvate, 100 U / mL penicillin, 100 μg / mL streptomycin, HEPES, and non-essential amino acids) in a 96-well U-bottom plate. Cells were pulsed with 10 μg / mL of antigen peptide for 6 hours, and the protein transport inhibitor brefeldin A (BD Biosciences) was added during the final 4 hours of incubation. Cells were then washed twice with ice-cold FACS buffer (1% BSA in PBS) and subsequently incubated with anti-CD16 / 32 on ice for at least 10 minutes and anti-CD8α on ice for 20 minutes. Cells were then fixed / permeabilized on ice for 20 minutes and then stained with anti-IFN-γ-PE and anti-TNF-α-FITC on ice for 30 minutes. After extensive washing, cells were analyzed by flow cytometry. cDNA sequencing of neoepitope (Adpgk) in MC-38 cells Total RNA was extracted from MC-38 cells using the RNeasy® mini Kit (QIAGEN) according to the manufacturer's instructions. First-strand cDNA was synthesized using 1 μg of total RNA using the SuperScript™ III First-Strand Synthesis SuperMix Kit (Invitrogen). Adpgk cDNAs of 250 bp and 485 bp in length were selectively amplified using the following two sets of sequence-specific primers: Primer 1: TGCCAACCGCTTCATCTTCT (forward primer) and GGTAGACCAGCGTGTGGAAA (reverse primer). Primer 2: CTCCAACGGGGCCATGAATA (forward primer) and CGTGGAAAGACCTGCTGAT (reverse primer). Amplification was performed using the SuperScript One Step RT-PCR System (Invitrogen). The final cDNA product was visualized on a 1.5% agarose gel containing ethidium bromide, and the Adpgk cDNA band was excised and purified using the PureLink® Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen). The purified cDNA was sequenced by the University of Michigan DNA Sequencing Core using the Sanger sequencing method (see, e.g., Sanger, F., Nicklen, S. & Coulson, AR DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977)). Example VII. This example describes neoantigen vaccination using sHDL, liposomes, and other nanoparticles, including gold nanoparticles (Figure 29), as well as the generation of multivalent neoantigen vaccination using multiple neoantigen peptides (Figure 28). Preparation of multivalent neoantigen-loaded sHDL To prepare Nanodisc-based multivalent peptide vaccines, multiple neoantigen peptides (M30 and M27) ​​modified with a CSS linker at the N-terminus were conjugated to DOPE-PDP in dimethylformamide for 3 hours at room temperature, followed by dilution with 10x water and lyophilization to obtain lipid-peptide conjugates. The conjugates were mixed with DMPC and 22A in acetic acid and lyophilized. The resulting powders were then heated (50°C) for 3 minutes and cooled (ice water) for 3 minutes, for a total of three cycles, to obtain sHDL loaded with different neoantigens (sHDL-M30 / M27). Alternatively, the conjugates were dissolved in DMSO and incubated with preformed sHDL to obtain sHDL loaded with different neoantigens (sHDL-M30 / M27). Any unincorporated neoantigen peptides were removed by passage through a desalting column. The loading efficiency was analyzed by HPLC. Cholesterol-CpG was incubated with the above sHDL for 30 min at room temperature to obtain a nanodisc-based multivalent peptide vaccine (sHDL-M30 / M27 / CpG). Preparation of neoantigen-loaded liposomes To prepare the liposome-based neoantigen vaccine, DMPC and DOPE-PDP (molar ratio = 92:8) were dissolved in chloroform. The mixture was dried under nitrogen and placed under vacuum for at least 1 hour. The resulting lipid film was hydrated in 10 mM sodium phosphate buffer (0.3117 g / L NaH2PO4.H2O and 2.0747 g / L Na2HPO4.7H2O, pH 7.4) and sonicated in a bath sonicator for 10 minutes, followed by probe sonication for an additional 2.5 minutes to obtain liposomes. The CSS-modified Adpgk peptide was incubated with PDP-presenting liposomes, followed by desalting column-based separation of the unconjugated peptide. The neoantigen peptide Adpgk was conjugated to the liposomes. The conjugation efficiency was analyzed by HPLC. Cholesterol-CpG was incubated with the above liposomes at room temperature for 30 minutes to obtain a liposome-based neoantigen peptide vaccine (lip-Adpgk / CpG). Preparation of spike-shaped gold nanoparticle-based neoantigen peptide vaccines To obtain spike-shaped gold nanoparticles (AuNPs), citrate gold nanoparticles were first prepared by boiling an aqueous solution of HAuCl4 with sodium citrate. Then, AuNPs were formed by the seed-seed growth method by sequentially adding HAuCl4, HCl, AgNO3, and ascorbic acid at room temperature under vigorous stirring. The as-synthesized AuNPs were purified and concentrated by centrifugation using 0.01% SDS. AuNP-based peptide vaccines were prepared by thiol-mediated surface decoration of neoantigen peptides on AuNPs, followed by loading of polyIC and CpG layers by electrostatic complexation. Briefly, the peptide vaccine was surface-conjugated to AuNPs by incubating AuNPs with the CSS-modified neoantigen peptide, CSS-ASMTNMELM, overnight. Any unreacted peptide was removed from the AuNP conjugates by centrifugation. Polyethylene glycol (average Mn 6,000)-modified polyethyleneimine (branched, average Mw approximately 25,000) (PEG-PEI) was used to load polyIC and CpG via electrostatic interactions. Peptide-conjugated AuNPs were mixed with PEG-PEI for 10 min, purified from excess PEG-PEG by centrifugation, and added to a polyIC and CpG mixed solution in 10 mM NaCl. After 5 min, the mixture was transferred to a 10 mM NaCl PEG-PEI solution, and the salt concentration was increased stepwise to 150 mM NaCl in 50 mM increments every 5 min. Finally, the crude mixture was centrifuged with 0.01% Tween 20 to remove any unbound polyIC and CpG. Intracellular cytokine staining C57BL / 6 mice were vaccinated with a nanodisc-based multivalent neoantigen peptide vaccine (sHDL-M30 / M27 / CpG) on days 0, 7, and 14. Seven days after the last vaccination, 100–150 μL of peripheral blood collected from vaccinated mice was lysed in ACK lysis buffer, washed with PBS, and seeded at approximately 10 million cells / mL in 50 μL of T cell medium (RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 55 μM β-mercaptoethanol, 1 mM pyruvate, and 100 U / mL penicillin and 100 μg / mL streptomycin, HEPES, and non-essential amino acids) in 96-well U-bottom plates. Cells were co-cultured with 50,000 BMDCs / well and pulsed with 20 μg / mL M30 or M27 peptide for 6 hours. The protein transport inhibitor brefeldin A (BD Biosciences) was added during the final 4 hours of incubation. Cells were then washed twice with ice-cold FACS buffer (1% BSA in PBS) and subsequently incubated with anti-CD16 / 32 on ice for at least 10 minutes, and with anti-CD8α and anti-CD4 on ice for 20 minutes. Cells were then fixed / permeabilized on ice for 20 minutes and then stained with anti-IFN-γ-PE on ice for 30 minutes. After extensive washing, cells were analyzed by flow cytometry. The results shown in Figure 28 demonstrate that sHDL-M30 / M27 / CpG generated high frequencies of CD4+ T cells directed against neoantigen M30 (Figure 28A) and CD8+ T cells directed against neoantigen M27 (Figure 28B). Therapeutic trials In the therapeutic tumor vaccination study, C57BL / 6 mice received tumor cells (1 × 10 per mouse) by subcutaneous injection on day 0 in the right flank. 5MC38 cells). Mice were vaccinated with 15.5 nmol of ASMTNMELM and 2.3 nmol of CpG (or 15 μg of polyIC / mouse) formulated in either liposomal or soluble form on days 10 and 17. For the AuNP-immunized mouse group, intratumoral administration of AuNPs modified with Adpgk and adjuvant was performed on days 10 (both with and without laser groups) and 16 (only for the without laser group) with 12 nmol of ASMTNMELM, 5.2 nmol of CpG, and 83 μg of polyIC per mouse. An 808 nm CW diode laser was used to irradiate the tumor with a laser at 1.2 W / cm. 2 The tumor tissue was directly irradiated with light for 5 minutes.

[0286] At the indicated time points, PBMCs were collected and stained for Adpgk-specific CD8+ T cells in PBMCs by tetramer staining followed by cytometry analysis. Tetramer staining of PBMCs showed that Adpgk-containing liposomes and AuNPs all generated stronger neoantigen-specific CD8+ T cell responses compared to vaccination with soluble peptide and adjuvant (Figure 29A). In addition, tumor growth was monitored every other day, and tumor volume was calculated throughout the study using the following formula: tumor volume = length × width. 2 × 0.52. Animals were euthanized when tumor masses reached 1.5 cm in diameter or when they became moribund due to severe weight loss or ulcers. The results showed that Adpgk-containing nanoparticles, including liposomes and AuNPs, delayed tumor progression compared with vaccination with soluble peptides and CpG (Figure 29B). Incorporation by Reference The entire disclosure of each patent document and scientific article referenced herein is incorporated by reference for all purposes. equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The above-described embodiments, therefore, are to be considered in all respects as illustrative rather than limiting of the invention described herein. The scope of the invention is, therefore, indicated by the appended claims rather than the foregoing specification, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. 1. A composition comprising sHDL nanoparticles, The sHDL nanoparticles are (i) a phospholipid selected from dipalmitoylphosphatidylcholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); (ii) an ApoA-I mimetic having the sequence of any one of SEQ ID NOs: 1, 4, 15, 28, 35, 54, and 79; and (iii) comprising a thiol-reactive phospholipid; The composition, wherein the thiol-reactive phospholipid is dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide].

2. The composition of claim 1 , wherein the phospholipid is DMPC.

3. The composition of claim 1 , wherein the phospholipid is DPPC.

4. The composition of any one of claims 1 to 3, wherein the thiol-reactive phospholipid is dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (DOPE-PDP).

5. The composition according to any one of claims 1 to 4, wherein the nanoparticles have an average particle size of 6 to 500 nm.

6. The composition of any one of claims 1 to 5, wherein an imaging agent is encapsulated within the nanoparticles.

7. the sHDL nanoparticles encapsulate siRNA; the siRNA is capable of inhibiting a target gene by RNA interference; The composition of any one of claims 1 to 6, wherein the siRNA comprises two RNA strands that are complementary to each other.

8. The composition of claim 7 , wherein the siRNA is cholesterol-modified on the 3′ sense strand.

9. 8. The composition of claim 6 or 7 for use in inhibiting a target gene in a cell.

10. The composition of claim 9 , wherein the cell is in vitro, in vivo, or ex vivo.

11. The composition of claim 9 , wherein the cell is in a human.

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