Targeting multiple T cell types using a spherical nucleic acid vaccine architecture

Abstract

The present disclosure generally relates to spherical nucleic acids (SNAs), which are nanostructures having a core surrounded by radial presentation of oligonucleotides that can target multiple classes of immune cells. Methods of making and using nanoparticles are also provided herein. In some aspects, the present disclosure provides spherical nucleic acids (SNAs), comprising: (a) a nanoparticle core; (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 167,977, filed March 30, 2021, and U.S. Provisional Patent Application No. 63 / 222,869, filed July 16, 2021, each of which is incorporated by reference in its entirety herein.

[0002] Statement regarding government support This invention was made with Government support under Grant Nos. CA208783 and CA199091-03 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Submitted Materials A sequence listing, which is part of this disclosure, is submitted as a text file concurrently with the specification. The name of the text file containing the sequence listing is "2021-087R_Seqlisting.txt", which was created on March 30, 2022, and is 9,503 bytes in size. The contents of the sequence listing are incorporated herein by reference.

[0004] The present disclosure generally relates to spherical nucleic acids (SNAs), which are nanostructures having a core surrounded by radial presentation of oligonucleotides capable of targeting multiple classes of immune cells. Methods of making and using the nanoparticles are also provided herein. Summary of the Invention [Problem to be solved by the invention]

[0005] Spherical nucleic acids (SNAs) are potent immunotherapeutic agents capable of activating the immune system against specific targets. SNAs, which contain a dense shell of oligonucleotides radially conjugated to a nanoparticle core, demonstrate that vaccine structure directly impacts function and the success of the resulting therapy. This is a powerful concept known as rational vaccinology, which allows for the same clinically employed targets to be utilized to boost immune responses, but with an architecture that is exploited for efficacy. The immune system is complex, and thus vaccines must activate multiple different types of cells for a robust response and eventual tumor rejection. This disclosure describes the synthesis of SNAs with defined positioning of multiple different targets that activate multiple different immune cell types, can enhance responses, and shows that the structure and positioning of the targets within the SNA strongly dictate the efficacy of the vaccine.

[0006] Applications of the technology described herein include, but are not limited to: Vaccine design Cancer immunotherapy Nanomedicine Treatment of immune-related disorders (eg, autoimmune disorders).

[0007] Advantages of the technology described herein include, but are not limited to: Structural control of the vaccine leading to controlled presentation of components to cells, Defined location of immune targets within nanoparticles and control over intracellular trafficking and release kinetics following uptake by immune cells; The presentation enhances serum stability, cellular uptake, and codelivery of immune activating and targeting components. Platform technologies with modularity for altering targets (e.g., cancer targets); Enhanced immune response [Means for solving the problem]

[0008] Thus, in some aspects, the present disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen. In some embodiments, the first antigen is encapsulated in the nanoparticle core. In some embodiments, the second antigen is bound to one or more oligonucleotides in the shell of the oligonucleotides through a linker. In further embodiments, the second antigen is bound to an oligonucleotide in the shell of the oligonucleotides bound to the nanoparticle core through a linker. In yet further embodiments, the second antigen is bound to an oligonucleotide that hybridizes to an oligonucleotide in the shell of the oligonucleotides bound to the nanoparticle core through a linker. In some embodiments, the second antigen is bound to the outer surface of the nanoparticle core through a linker. In some embodiments, the second antigen is encapsulated in the nanoparticle core. In further embodiments, the first antigen is attached to one or more oligonucleotides in the shell of the oligonucleotide through a linker. In some embodiments, the first antigen is attached to an oligonucleotide in the shell of the oligonucleotide attached to the nanoparticle core through a linker. In further embodiments, the first antigen is attached to an oligonucleotide hybridized to an oligonucleotide in the shell of the oligonucleotide attached to the nanoparticle core through a linker. In still further embodiments, the first antigen is attached to the exterior surface of the nanoparticle core through a linker. In some embodiments, the SNA of the present disclosure includes a third antigen that is a major histocompatibility complex type I (MHC-I) antigen. In further embodiments, the SNA of the present disclosure includes a fourth antigen that is a major histocompatibility complex type II (MHC-II) antigen. In some embodiments, the third antigen is encapsulated in the nanoparticle core.In further embodiments, the fourth antigen is attached to one or more oligonucleotides in the shell of the oligonucleotide through a linker. In still further embodiments, the fourth antigen is attached to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core through a linker. In still further embodiments, the fourth antigen is attached to an oligonucleotide hybridized to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core through a linker. In some embodiments, the fourth antigen is attached to the exterior surface of the nanoparticle core through a linker. In some embodiments, the third antigen is attached to one or more oligonucleotides in the shell of the oligonucleotide bound to the nanoparticle core through a linker. In some embodiments, the third antigen is attached to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core through a linker. In still further embodiments, the third antigen is attached to an oligonucleotide hybridized to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core through a linker. In still further embodiments, the third antigen is attached to the exterior surface of the nanoparticle core through a linker. In some embodiments, the fourth antigen is encapsulated in the nanoparticle core. In some embodiments, the first antigen and the third antigen are the same. In some embodiments, the first antigen and the third antigen are different. In some embodiments, the second antigen and the fourth antigen are the same. In further embodiments, the second antigen and the fourth antigen are different.In various embodiments, the MHC-I antigen is selected from the group consisting of OVA257-264 (OVA1) (SEQ ID NO: 7), GP100(25-33) (KVPRNQDWL (SEQ ID NO: 11)), TC-1 E6(49-58) (VYDFAFRDLC (SEQ ID NO: 12)), TC-1 E7(49-57) (RAHYNIVTF (SEQ ID NO: 13)), PSMA(634-642) (SAVKNFTEI (SEQ ID NO: 14)), SPAS-1 (SNC9-H8) (STHVNHLHC (SEQ ID NO: 15)), SIMS2(237-245) (SLDLKLIFL (SEQ ID NO: 16)), PAP(115-123) (SAMTNLAAL (SEQ ID NO: 17)), B16 MART-1(M27)(LCPGNKYEM (SEQ ID NO:9)), TRP-1(252-260)(ATGKNVCDV (SEQ ID NO:18)), TRP-1(252V260M)(ATGKNVCDM (SEQ ID NO:19)), TRP-1(455-463)(TAPDNLGYA (SEQ ID NO:20)), TRP-1(455A463M)(TAPDNLGYM (SEQ ID NO:21)), TRP-2(180-188)(SVYDFFVWL (SEQ ID NO:22)), Melan-A / MART-(127-135), tyrokinase(1-9), tyrokinase(369-377D), MC38 Adpgk (ASMTNMELM (SEQ ID NO: 23)), Irgq-minimal (AALLNSAVL (SEQ ID NO: 24)), Irgq-long peptide (KARDETAALLNSAVLGAAPLFVPPAD (SEQ ID NO: 25)), or a combination thereof.In various embodiments, the MHC-II antigen is selected from the group consisting of OVA323-339 (OVA2) (SEQ ID NO: 8), GP100:(46-58) (RQLYPEWTEAQRL (SEQ ID NO: 26)), TC-1 E6(43-57) (QLLRREVYDFAFRDL (SEQ ID NO: 27)), SIMS2(240-254) (LKLIFLDSRVTEVTG (SEQ ID NO: 28)), PAP(114-128) (MSAMTNLAALFPPEG (SEQ ID NO: 29)), B16 MART-1 (M30) (VDWENVSPELNSTDQ (SEQ ID NO: 30)), TRP-1 (113-127) (CRPGWRGAACNQKIL (SEQ ID NO: 31)), TRP-1 (106-130) (SGHNCGTCRPGWRGAACNQKILTVR (SEQ ID NO: 32)), Li-Key (77-92) (LRMKLPKPPKPVSQMR (SEQ ID NO: 33)), tyrokinase (56-70), GP100 (44-59), GP100 (167-189), Melan-A / MART-1 (102-111) (PAY EKLSAEQSPPPY (SEQ ID NO: 34), Melan-A / MART-1(27-40) (AAGIGILTVILGVL (SEQ ID NO: 35)), Melan-A / MART-1(51-70) (RNGYRALMDKSLHVGTQCAL (SEQ ID NO: 36)), Melan-A / MART-1(51-73) (RNGYRALMDKSLHVGTQCALTRR (SEQ ID NO: 37)), Melan-A / MART-1(43-57) (IGCWYCRRRNGYRAL (SEQ ID NO: 38)), or a combination thereof. In some embodiments, at least one of the one or more immunostimulatory oligonucleotides is a toll-like receptor (TLR) agonist. In further embodiments, each of the one or more immunostimulatory oligonucleotides is a toll-like receptor (TLR) agonist.In still further embodiments, the TLR is selected from the group consisting of toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 5 (TLR5), toll-like receptor 6 (TLR6), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), toll-like receptor 9 (TLR9), toll-like receptor 10 (TLR10), toll-like receptor 11 (TLR11), toll-like receptor 12 (TLR12), and toll-like receptor 13 (TLR13). In some embodiments, the TLR is TLR9. In some embodiments, the immunostimulatory oligonucleotide comprises a CpG nucleotide sequence. In further embodiments, one or more oligonucleotides within the shell of the oligonucleotide comprises or consists of the sequence 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 39). In some embodiments, one or more oligonucleotides within the oligonucleotide shell comprise or consist of the sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 40). In some embodiments, one or more oligonucleotides within the oligonucleotide shell comprise or consist of the sequence 5'-TCCATGACGTTCCTGACGTT(spacer-18(hexaethylene glycol))2cholesterol-3' (SEQ ID NO: 41). In further embodiments, one or more oligonucleotides within the oligonucleotide shell comprise or consist of the sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT(spacer-18(hexaethylene glycol))2cholesterol-3' (SEQ ID NO: 6). In various embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the oligonucleotides within the oligonucleotide shell are immunostimulatory oligonucleotides. In various embodiments, the linker is a carbamate alkylene disulfide linker, a thiol linker, a disulfide linker, an amido alkylene disulfide linker, an amido alkylene thio-succinimidyl linker, or a combination thereof.In further embodiments, the nanoparticle core is a micelle, liposome, polymer, lipid nanoparticle (LNP), or a combination thereof. In various embodiments, the polymer is polylactide, polylactide-polyglycolide copolymer, polycaprolactone, polyacrylate, alginate, albumin, silica, polypyrrole, polythiophene, polyaniline, polyethyleneimine, poly(methyl methacrylate), or chitosan. In some embodiments, the polymer is poly(lactic-co-glycolic acid) (PLGA). In some embodiments, the nanoparticle core is a liposome. In various embodiments, the liposomes may be selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac- The lipid comprises a lipid selected from the group consisting of distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), and cholesterol. In some embodiments, one or more oligonucleotides in the oligonucleotide shell are attached to the exterior surface of the nanoparticle core through a lipid anchor group. In further embodiments, the lipid anchor group is attached to the 5' or 3' terminus of one or more oligonucleotides. In still further embodiments, the lipid anchor group is tocopherol or cholesterol. In some embodiments, one or more oligonucleotides in the oligonucleotide shell are modified at their 5' and / or 3' terminus with dibenzocyclooctyl (DBCO). In a further embodiment, one or more of the oligonucleotides within the shell of oligonucleotides is modified with a thiol at its 5' and / or 3' terminus.In various embodiments, an oligonucleotide. The oligonucleotide shell comprises DNA oligonucleotides, RNA oligonucleotides, or combinations thereof. In some embodiments, the oligonucleotide shell comprises DNA oligonucleotides and RNA oligonucleotides. In further embodiments, the oligonucleotide shell comprises single stranded DNA, double stranded DNA, single stranded RNA, double stranded RNA, or combinations thereof. In some embodiments, one or more of the oligonucleotides in the oligonucleotide shell are modified oligonucleotides. In various embodiments, the oligonucleotide shell comprises about 2 to about 200 oligonucleotides. In some embodiments, the oligonucleotide shell comprises about 2 to about 100 oligonucleotides. In further embodiments, the oligonucleotide shell comprises about 150 oligonucleotides. In still further embodiments, the oligonucleotide shell comprises about 200 oligonucleotides. In various embodiments, the oligonucleotide shell comprises about 10 to about 80 oligonucleotides. In some embodiments, the oligonucleotide shell comprises about 75 oligonucleotides. In various embodiments, each oligonucleotide in the oligonucleotide shell is about 5 to about 1000 nucleotides in length. In some embodiments, each oligonucleotide in the oligonucleotide shell is about 10 to about 50 nucleotides in length. In some embodiments, each oligonucleotide within the oligonucleotide shell is about 20 to about 30 nucleotides in length. In further embodiments, the diameter of the SNA is about 1 nanometer (nm) to about 500 nm. In some embodiments, the diameter of the SNA is about 80 nanometers or less. In some embodiments, the diameter of the SNA is about 50 nanometers or less. In various embodiments, the oligonucleotide shell comprises a targeting oligonucleotide, an inhibitory oligonucleotide, a non-targeting oligonucleotide, or a combination thereof. In further embodiments, the inhibitory oligonucleotide is an antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme.

[0009] In some aspects, the present disclosure provides a composition comprising a plurality of SNAs as described herein. In some embodiments, at least two SNAs in the plurality comprise different nanoparticle cores.

[0010] In some aspects, the disclosure provides a pharmaceutical formulation comprising a plurality of SNAs or compositions described herein and a pharma- ceutically acceptable carrier or diluent.

[0011] In a further aspect, the disclosure provides a vaccine comprising the SNA, composition, or pharmaceutical formulation of the disclosure. In some embodiments, the vaccine comprises an adjuvant or an additional adjuvant.

[0012] In some aspects, the disclosure provides an antigenic composition comprising a SNA of the disclosure or a pharmaceutical formulation of the disclosure in a pharma- ceutically acceptable carrier, diluent, stabilizer, or preservative, capable of generating an immune response in a subject, including antibody generation, activation of cytotoxic T cells, activation of helper T cells, or a protective immune response. In some embodiments, the immune response comprises an antibody response. In further embodiments, the antibody response is a neutralizing antibody response or a protective antibody response.

[0013] In some aspects, the disclosure provides methods of inhibiting expression of a gene product comprising hybridizing a polynucleotide encoding the gene product to an inhibitory oligonucleotide described herein, wherein hybridization between the polynucleotide and the inhibitory oligonucleotide occurs over a length of the polynucleotide that has a sufficient degree of complementarity to inhibit expression of the gene product. In various embodiments, expression of the gene product is inhibited in vivo or in vitro.

[0014] In some aspects, a method of generating an immune response in a subject comprises administering to the subject an effective amount of each SNA, composition, pharmaceutical formulation, vaccine, or antigenic composition described herein, thereby generating an immune response in the subject. In some embodiments, the immune response comprises an antibody response. In further embodiments, the antibody response is a total antigen-specific antibody response. In some embodiments, the antibody response is a neutralizing antibody response or a protective antibody response.

[0015] In some aspects, the disclosure provides a method of immunizing a subject against one or more antigens, comprising administering to the subject an effective amount of each SNA, composition, pharmaceutical formulation, vaccine, or antigenic composition described herein, thereby immunizing the subject against the one or more antigens. In some embodiments, the composition or vaccine is a cancer vaccine.

[0016] In some aspects, the disclosure provides a method of treating cancer, comprising administering to a subject an effective amount of each SNA, composition, pharmaceutical formulation, vaccine, or antigenic composition described herein, thereby treating the cancer in the subject. In various embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, glioblastoma, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, lymphoma, non-Hodgkin's lymphoma, osteocarcinoma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and human papillomavirus-induced cancer, or a combination thereof. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is colon cancer. In further embodiments, the cancer is lymphoma. In some embodiments, the method of the disclosure further comprises administering an additional agent. In various embodiments, the additional agent is an anti-programmed cell death protein 1 (PD-1) antibody, an anti-programmed death-ligand 1 (PD-L1) antibody, a cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody, a T-cell immunoglobulin and ITIM domain (TIGIT) antibody, or a combination thereof. [Brief description of the drawings]

[0017] [Figure 1] Spherical nucleic acid (SNA) structures incorporating two antigen classes are shown. (Left) In dual antigen SNA (DA-SNA)1, MHC-I (killer CD8+ T cell) antigens are encapsulated in the liposomal core, while MHC-II (helper CD4+ T cell) antigens hybridize to the adjuvant DNA shell. (Right) In DA-SNA2, the opposite is true. [Diagram 2] The SNA construct demonstrates enhanced properties and shows its potential use as a tool in immunotherapy. [Diagram 3] We present additional demonstrated and enhanced properties of the SNA structure that are particularly relevant to its use as a tool in immunotherapy. [Figure 4] 1 shows an SNA structure incorporating two classes of antigens that are uniquely positioned. [Diagram 5] FIG. 1 shows the results of an experiment in which different dual antigen SNAs activated cytokine production in different T cells. [Figure 6] Results of experiments in which expression of memory markers in T cells differed based on DA-SNA structure are shown. [Figure 7] 1 shows the results of an experiment demonstrating that circulating antigen-specific immune cells were different based on dual-antigen spherical nucleic acid (DA-SNA) treatment. [Figure 8] 1 shows the results of experiments demonstrating circulating immune cell memory profiling cells from DA-SNA treatment. [Figure 9] We show that structural changes in the way antigens are used to activate both helper and cytotoxic T cells lead to differences in efficacy. DA-SNA2 dramatically altered vaccine potency and antitumor efficacy, stalling tumors. [Figure 10] Cartoon showing how spherical nucleic acids (SNAs) enable elucidation of the influence of structural configuration on immune responses. [Figure 11]Figure 11 shows that structural SNA changes altered tumor growth. C57BL6 female mice were injected subcutaneously into the right flank with 500,000 EG.7-OVA tumor cells. Weekly treatment of mice began on day 3. A total of four vaccines were administered and tumors were measured every 2-3 days. N=9 per group. Statistics were calculated using one-way ANOVA on day 24 followed by Tukey multiple comparison post-hoc test *p<0.05, **p<0.01. Related to Figure 9, Figure 11 additionally includes data showing the added benefit provided by an SNA containing both MHC-I and MHC-II antigens versus only containing MHC-I antigens. The presence of OVA1 antigen without OVA2 antigen ("OVA1 only SNA") did not stall tumor growth over the long term as did DA-SNA2. [Figure 12] Figure 1 shows that immune cell spleen populations are altered as a result of vaccination. EG7.OVA tumor-bearing C57BL6 female mice were injected weekly with SNA for a total of four times, after which spleens were harvested and processed to identify cell populations. (Left) The percentage of the population that is CD8+ (cytotoxic) T cells or (right) the relative ratio of CD8 T cells to CD4 (helper) T cells are shown. Statistics were calculated using one-way ANOVA on day 24 followed by Tukey's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 13] We show that simultaneous DC activation with MHC I and II antigens leads to increased T cell proliferation. Using the OVA system with identified helper (OVA2) and cytotoxic (OVA1) antigens, enhanced immune activation was observed when both classes of antigens were delivered to the same cells in one SNA. Delivery of different antigen types to the same cells (combination) induced an approximately two-fold enhancement in T cell proliferation compared to delivery of each antigen type on a separate SNA (separate). *p<0.05, **p<0.01. [Figure 14]Gene expression profiles of both CD8+ and CD4+ T cells are shown to be unique based on vaccination condition. Principal component analysis (PCA) of the transcriptomes of CD8 and CD4 T cell populations isolated from splenocytes following in vivo vaccination with different treatments. PCA reduces the dimensionality of a genomic dataset consisting of a large number of interrelated variables while preserving as much of the variation present in the dataset as possible. Grouping of like colors (treatments) indicates that the treatments caused similar gene-level changes. The furthest apart on the x-axis (PC1) indicate the groups that were least similar at the genetic level (i.e., the admixture on the left and SNA containing only OVA1H, and the admixture on the right and all three SNA groups). The next level of variance is the separation between groups on the y-axis (PC2). [Figure 15] Figure 1 shows that gene expression profiles of CD8+ T cells differed based on vaccination condition. Gene expression heatmaps for CD8+ T cell populations represent gene pathways that were uniquely activated based on vaccination treatment condition. Black boxes highlight genes with substantial variability across treatment groups. [Figure 16-1] Figure 1 shows that gene expression profiles of CD4+ T cells differed based on vaccination condition. Gene expression heatmaps for CD4+ T cell populations represent gene pathways that were uniquely activated based on vaccination treatment condition. Black boxes highlight genes with substantial variability across treatment groups. [Figure 16-2] Figure 1 shows that gene expression profiles of CD4+ T cells differed based on vaccination condition. Gene expression heatmaps for CD4+ T cell populations represent gene pathways that were uniquely activated based on vaccination treatment condition. Black boxes highlight genes with substantial variability across treatment groups. [Figure 17]We show that DA-SNA2 (see, e.g., FIG. 4 for a description of DA-SNA-1 and DA-SNA-2) generated robust CD8+ T cell memory. The positioning of the antigen within the dual antigen SNA (DA-SNA) influenced the immune response. DA-SNA2 elicited higher levels of CD8+ effector function compared to other vaccination treatments. Both DA-SNAs enhanced CD4+ effector function. [Figure 18] Figure 1 shows that DA-SNA2 vaccination increased IFN-γ secretion upon antigen stimulation. Representative counts and images of IFN-γ-secreting splenic T cells upon different ex vivo stimulations. [Figure 19] Figure 18 shows that DA-SNA2 vaccination increased IFN-γ secretion upon antigen stimulation. Enumeration of IFN-γ-secreting splenic T cells upon different ex vivo stimulations. (Representative images shown in Figure 18). [Figure 20] Figure 1 shows that IFN-γ and CD107a production was increased in CD8+ cytotoxic T cells in the treatment groups. Intracellular IFN-γ (left and right) and CD107a (center) levels in either CD8+ (left) or CD4+ (right) T cells demonstrate enhanced IFN-γ in T cells vaccinated with DA-SNA2 upon different ex vivo stimulations. *P<0.05. [Figure 21] Figure 1 shows that OVA1-specific T cell memory was enhanced due to SNA treatment. Different groups (n=6 per group) of tumor-bearing C57BL6 mice treated (following the same schedule as in Figure 11) were harvested at d17 for evaluation of memory immune responses throughout the course of treatment. (Left) The amount of CD8-antigen-specific (OVA1) T cells measured by dimer is shown. (Right) Of these specific T cells, the effector memory state is most enhanced in DA-SNA2-immunized mice. The stars immediately above the bars are the comparisons to the PBS group. Otherwise, specific comparisons are indicated. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 22]Figure 2 shows that OVA2-specific T cell memory was enhanced due to SNA treatment. Different groups (following the same schedule as in Figure 21) of tumor-bearing C57BL6 mice (n=6 per group) treated were harvested at d17 for evaluation of memory immune responses throughout the course of treatment. (Left) The amount of CD4-antigen-specific (OVA2) T cells measured by tetramer is shown. (Right) Among these specific T cells, the effector memory state is most enhanced in DA-SNA1 immunized mice, with slightly lower levels versus the DA-SNA2 and OVA1H-only SNA groups. *P<0.05. [Figure 23] We show that DA-SNA stalled tumor growth when applied to clinically relevant melanoma tumors. C57BL6 female mice were injected subcutaneously into the right flank with 100,000 B16.F10 melanoma tumor cells. On day 3, mice were treated weekly. A total of four vaccines were administered and tumors were measured every 2-3 days. N=6 per group. Statistics were calculated using one-way ANOVA on day 20 followed by Tukey's multiple comparison post-hoc test. *p<0.05, **p<0.01. [Figure 24] We show that combination therapy with anti-PD-1 enhanced the anti-tumor properties of structure-driven SNAs. C57BL6 female mice were injected subcutaneously into the right flank with 100,000 B16.F10 melanoma tumor cells. On day 3, mice were started on weekly treatment. A total of four vaccines were administered with anti-PD-1 checkpoint inhibitors injected 3 and 6 days after vaccine injection. Tumors were measured every 2-3 days. N=6 per group. Statistics were calculated using one-way ANOVA comparing groups with anti-PD-1 followed by Tukey's multiple comparison post-hoc test. *p<0.05; **p<0.01. [Diagram 25]The results show that the combination therapy also had a positive effect on animal survival. C57BL6 female mice were injected subcutaneously into the right flank with 100,000 B16.F10 melanoma tumor cells. On day 3, weekly treatment of the mice began. A total of four vaccines were administered with the anti-PD-1 checkpoint inhibitor injected 3 and 6 days after vaccine injection. Tumors were measured every 2-3 days and animals were sacrificed when tumor size reached 1500mm^3. N=6 per group. Statistics for tumor volume were calculated using one-way ANOVA comparing groups with anti-PD-1 followed by Tukey multiple comparison post-hoc test. Survival statistics were compared using Gehan-Breslow-Wilcoxon test. *p<0.05, **p<0.01. [Figure 26-1]Delivery of two classes of antigens from a spherical nucleic acid (SNA) vaccine alters how antigens are processed in vitro. a) Dual antigen SNA (DA-SNA) vaccines synthesized to alter the positioning of MHC-I and MHC-II restricted antigens within the same nanoparticle structure. b) Delivery of the two antigen classes on either separate nanoparticles (dashed lines, separate) or a single DA-SNA (solid lines, combined) does not affect the fold change in the population of CD86 or CD80 expressing CD11c+DCs. c) CD8+ T cells specific for OVA1 antigen (left) or CD4+ T cells specific for OVA2 antigen elicited from co-culture of naive splenic T cells with therapeutically pulsed DCs. (Left) T cells versus separate and combined OVA1 (encapsulated) & OVA2 (hybridized) (P = 0.0025 and 0.0016, respectively), and separate and combined OVA2 (encapsulated) & OVA1 (hybridized) (P = 0.0017 and 0.0094, respectively). (Right) T cells versus combined OVA1 (encapsulated) & OVA2 (hybridized) (P = 0.0268), and OVA2 (encapsulated) & OVA1 (hybridized) (P = 0.0285). d) Median fluorescence intensity (MFI) of CD69 activation marker signal within a population of antigen-specific CD8+ (left) or CD4+ (right) T cells. (Right) T cells versus combined OVA2 (encapsulated) & OVA1 (hybridized) (P = 0.0047). e) Fold change of T cell proliferation from OT1 spleen cells specific for OVA1 antigen after co-culture with therapeutic pulsed DCs. (Left) Separate vs. combined OVA1(encapsulated) & OVA2(hybridized) (P=0.0036), (Right) Separate vs. combined OVA2(encapsulated) & OVA1(hybridized) (P=0.0306). For all panels, mean ± sem shown with statistical significance between relevant comparisons. Significance was calculated using one-way ANOVA with Sidak's multiple comparison test, n=3-4 replicates per group. ns=not significant. *p<0.05, **p<0.01. [Figure 26-2]Delivery of two classes of antigens from a spherical nucleic acid (SNA) vaccine alters how antigens are processed in vitro. a) Dual antigen SNA (DA-SNA) vaccines synthesized to alter the positioning of MHC-I and MHC-II restricted antigens within the same nanoparticle structure. b) Delivery of the two antigen classes on either separate nanoparticles (dashed lines, separate) or a single DA-SNA (solid lines, combined) does not affect the fold change in the population of CD86 or CD80 expressing CD11c+DCs. c) CD8+ T cells specific for OVA1 antigen (left) or CD4+ T cells specific for OVA2 antigen elicited from co-culture of naive splenic T cells with therapeutically pulsed DCs. (Left) T cells versus separate and combined OVA1 (encapsulated) & OVA2 (hybridized) (P = 0.0025 and 0.0016, respectively), and separate and combined OVA2 (encapsulated) & OVA1 (hybridized) (P = 0.0017 and 0.0094, respectively). (Right) T cells versus combined OVA1 (encapsulated) & OVA2 (hybridized) (P = 0.0268), and OVA2 (encapsulated) & OVA1 (hybridized) (P = 0.0285). d) Median fluorescence intensity (MFI) of CD69 activation marker signal within a population of antigen-specific CD8+ (left) or CD4+ (right) T cells. (Right) T cells versus combined OVA2 (encapsulated) & OVA1 (hybridized) (P = 0.0047). e) Fold change of T cell proliferation from OT1 spleen cells specific for OVA1 antigen after co-culture with therapeutic pulsed DCs. (Left) Separate vs. combined OVA1(encapsulated) & OVA2(hybridized) (P=0.0036), (Right) Separate vs. combined OVA2(encapsulated) & OVA1(hybridized) (P=0.0306). For all panels, mean ± sem shown with statistical significance between relevant comparisons. Significance was calculated using one-way ANOVA with Sidak's multiple comparison test, n=3-4 replicates per group. ns=not significant. *p<0.05, **p<0.01. [Figure 27] Schematic of separate SNA combinations administered as a side-by-side comparison to DA-SNA treatment to assess the impact of antigen distribution. Separate nanoparticles were prepared with a single antigen hybridized on the surface and encapsulated within the liposome core. [Figure 28] An example of a standard curve from a peptide assay used to quantify the amount of peptide encapsulated within liposomes is shown. Data points were fitted to a linear regression with an R2>0.98 consistently. [Figure 29] An example of a standard curve from a phosphatidylcholine (PC) assay used to quantify the concentration of liposomes through analysis of lipid content using manufacturer-supplied standards is shown. Data points were fitted to a linear regression with an R2 > 0.98 at all times. [Diagram 30] The ESI of the four purified peptide-DNA conjugates used in Example 2: M27, M30, OVA1, OVA2 are shown along with their expected and confirmed masses. [Diagram 31] Dynamic light scattering (DLS) of liposomes and SNA is shown. A size shift between encapsulated or DOPC liposomes to either DA-SNA1 or DA-SNA2 structures with statistical significance is shown, indicating SNA formation. Data show mean ± SEM from 5 independent measurements. ***p<0.001, **p<0.0001. [Diagram 32] The gating strategy for FIG. 26C is shown to quantify the percentage of viable CD19− splenocytes that are double positive for either CD8+OVA1-H-2kb or CD4+OVA2-H-2-Iad. [Figure 33-1]Figure 1 shows that antigen positioning within the DA-SNA influenced the immune response after immunization. a) Biweekly immunization schedule for C57BL / 6 mice. Dose: 6 nmol of each antigen, 6 nmol of adjuvant. b) Changes in CD8+ (left) or CD4+ (right) cell populations in the spleen following a vaccination scheme. (left) Admixture vs. DA-SNA2 (P=0.0414). c) Intracellular production of IFN-γ proinflammatory cytokine (left) or CD107a degranulation marker (center) was assessed upon ex vivo restimulation with peptide antigen. Polyfunctional T cells (double positive for both markers) were quantified (right). DA-SNA2 significantly elevated production of all markers in CD8+ T cells, whereas differences between production in CD4+ T cells were more subtle, with both DA-SNAs elevating levels significantly more than mice immunized with the admixture vaccine. (Top) Admixture vs. DA-SNA2, and DA-SNA1 vs. DA-SNA2 (all left: P=0.0102 and 0.0124, center: P=0.0188 and 0.0266, right: P=0.01 and 0.0119, respectively). d) Effector memory phenotype measured through CD44+CD62L-markers was increased by DA-SNA2 for CD8+ T cells. CD4+ effector function was most elevated upon DA-SNA1 immunization. (Left) Admixture vs. DA-SNA2 and DA-SNA1 vs. DA-SNA2 (P=0.0016 and 0.0380, respectively). e) Representative counts and images of IFN-γ-secreting splenic T cells upon different ex vivo stimulations (left) along with total spot forming cells (SFC) measured by ELISpot assay (right). Admix vs. DA-SNA2 and DA-SNA1 vs. DA-SNA2 for OVA1 ex vivo stimulation (P=<0.0001 and 0.0047, respectively). Admix vs. DA-SNA2 and DA-SNA1 vs. DA-SNA2 for OVA2 ex vivo stimulation (P=<0.0001 and 0.0004, respectively). Means ± SEM shown. n=3 mice per group. Statistical significance between relevant comparisons is indicated. For all panels, significance was calculated using one-way ANOVA with Sidak's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 33-2]Figure 1 shows that antigen positioning within the DA-SNA influenced the immune response after immunization. a) Biweekly immunization schedule for C57BL / 6 mice. Dose: 6 nmol of each antigen, 6 nmol of adjuvant. b) Changes in CD8+ (left) or CD4+ (right) cell populations in the spleen following a vaccination scheme. (left) Admixture vs. DA-SNA2 (P=0.0414). c) Intracellular production of IFN-γ proinflammatory cytokine (left) or CD107a degranulation marker (center) was assessed upon ex vivo restimulation with peptide antigen. Polyfunctional T cells (double positive for both markers) were quantified (right). DA-SNA2 significantly elevated production of all markers in CD8+ T cells, whereas differences between production in CD4+ T cells were more subtle, with both DA-SNAs elevating levels significantly more than mice immunized with the admixture vaccine. (Top) Admixture vs. DA-SNA2, and DA-SNA1 vs. DA-SNA2 (all left: P=0.0102 and 0.0124, center: P=0.0188 and 0.0266, right: P=0.01 and 0.0119, respectively). d) Effector memory phenotype measured through CD44+CD62L-markers was increased by DA-SNA2 for CD8+ T cells. CD4+ effector function was most elevated upon DA-SNA1 immunization. (Left) Admixture vs. DA-SNA2 and DA-SNA1 vs. DA-SNA2 (P=0.0016 and 0.0380, respectively). e) Representative counts and images of IFN-γ-secreting splenic T cells upon different ex vivo stimulations (left) along with total spot forming cells (SFC) measured by ELISpot assay (right). Admix vs. DA-SNA2 and DA-SNA1 vs. DA-SNA2 for OVA1 ex vivo stimulation (P=<0.0001 and 0.0047, respectively). Admix vs. DA-SNA2 and DA-SNA1 vs. DA-SNA2 for OVA2 ex vivo stimulation (P=<0.0001 and 0.0004, respectively). Means ± SEM shown. n=3 mice per group. Statistical significance between relevant comparisons is indicated. For all panels, significance was calculated using one-way ANOVA with Sidak's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 34-1] Figure 1 shows that immunization of mice with differently structured vaccines induced specific differences in gene expression between CD8+ and CD4+ T cells. a) Principal component analysis (PCA) plots from the complete transcriptome of CD8+ (left) and CD4+ (right) T cells. b) Gene expression changes represented by subsets of log fold changes (LFC) for CD8+ (left) and CD4+ (right) T cell populations as a result of different treatments. c) Selection of significantly enriched pathways calculated using GSEA analysis. The color of the boxes corresponds to the enrichment score for each pathway as a result of different treatments for CD8+ and CD4+ (right) T cells. d) Gene signatures for CD8+ (top) and CD4+ (bottom) T cells. The colors refer to normalized (z-scored) gene expression levels. Selection of associated genes labeled. e) Volcano plots of CD8+ (left) and CD4+ (right) T cells between pairwise comparisons of DA-SNA2 and DA-SNA1. Red dots indicate significantly expressed genes, with positive LFCs indicating upregulation of DA-SNA2 relative to DA-SNA1 and negative LFCs indicating downregulation of DA-SNA2 relative to DA-SNA1. [Figure 34-2]Figure 1 shows that immunization of mice with differently structured vaccines induced specific differences in gene expression between CD8+ and CD4+ T cells. a) Principal component analysis (PCA) plots from the complete transcriptome of CD8+ (left) and CD4+ (right) T cells. b) Gene expression changes represented by subsets of log fold changes (LFC) for CD8+ (left) and CD4+ (right) T cell populations as a result of different treatments. c) Selection of significantly enriched pathways calculated using GSEA analysis. The color of the boxes corresponds to the enrichment score for each pathway as a result of different treatments for CD8+ and CD4+ (right) T cells. d) Gene signatures for CD8+ (top) and CD4+ (bottom) T cells. The colors refer to normalized (z-scored) gene expression levels. Selection of associated genes labeled. e) Volcano plots of CD8+ (left) and CD4+ (right) T cells between pairwise comparisons of DA-SNA2 and DA-SNA1. Red dots indicate significantly expressed genes, with positive LFCs indicating upregulation of DA-SNA2 relative to DA-SNA1 and negative LFCs indicating downregulation of DA-SNA2 relative to DA-SNA1. [Figure 35-1]Figure 1 shows DA-SNA immune activation for enhanced tumor suppression. a-c) C57BL / 6 mice were inoculated subcutaneously with E.G7-OVA cells (5x105) in the right flank and provided weekly immunizations starting on day 3 for a total of four vaccinations (6 nmol adjuvant, 6 nmol of each antigen). Mean tumor growth curves and animal survival rates are shown. Day 24 tumor volumes for DA-SNA2 compared to DA-SNA1 and admixture (P=0.0121 and P=0.0084, respectively). Animal survival rates comparing DA-SNA1 vs. DA-SNA2 (P=0.0219), DA-SNA2 vs. PBS and admixture (P=0.0030 and P=0.0029, respectively). d) Tumor weights (day 15) following the treatment schedule shown in a. Tumor weight comparing PBS vs. DA-SNA1 and DA-SNA2 (P=0.0064 and P=0.0034, respectively). e) Evaluation of immune CD8+ T cells in the spleen at the end of the experiment (left). CD8+ / CD4+ T cell ratio (right). T cells in the spleen of DA-SNA2 vs. PBS (P=0.0297), admixture (P=0.0013), and DA-SNA1 (P=0.0002). f-i) Flow cytometric analysis of PBMCs on day 15 isolated from tumor-bearing mice under the schedules shown in af) CD8+ T cells specific for the OVA1 antigen. DA-SNA2 vs. admixture (P=0.0401). g) Effector memory CD8+ T cells (CD44+ / CD62L-) within this antigen-specific T cell subset. DA-SNA2 vs. admixture (P=0.0020). h) CD4+ T cells specific for OVA2 antigen. Admixture vs. DA-SNA1 (P=0.0229) and DA-SNA2 (P=0.0436). i) Effector memory CD4+ T cells (CD44+ / CD62L-). DA-SNA1 vs. Admixture (P=0.0001) and DA-SNA2 (P=0.0012). Data show mean ± sem from two independent experiments (n=7-9 per experiment). For panels b, d-g, i, significance was calculated using one-way ANOVA with Tukey's multiple comparison test. For panel h, due to significant differences between groups in standard deviations, Welch ANOVA followed by Dunnett's multiple comparison test was used. Panel c was analyzed using the log-rank test.*p<0.05、**p<0.01、***p<0.001。 [Figure 35-2]Figure 1 shows DA-SNA immune activation for enhanced tumor suppression. a-c) C57BL / 6 mice were inoculated subcutaneously with E.G7-OVA cells (5x105) in the right flank and provided weekly immunizations starting on day 3 for a total of four vaccinations (6 nmol adjuvant, 6 nmol of each antigen). Mean tumor growth curves and animal survival rates are shown. Day 24 tumor volumes for DA-SNA2 compared to DA-SNA1 and admixture (P=0.0121 and P=0.0084, respectively). Animal survival rates comparing DA-SNA1 vs. DA-SNA2 (P=0.0219), DA-SNA2 vs. PBS and admixture (P=0.0030 and P=0.0029, respectively). d) Tumor weights (day 15) following the treatment schedule shown in a. Tumor weight comparing PBS vs. DA-SNA1 and DA-SNA2 (P=0.0064 and P=0.0034, respectively). e) Evaluation of immune CD8+ T cells in the spleen at the end of the experiment (left). CD8+ / CD4+ T cell ratio (right). T cells in the spleen of DA-SNA2 vs. PBS (P=0.0297), admixture (P=0.0013), and DA-SNA1 (P=0.0002). f-i) Flow cytometric analysis of PBMCs on day 15 isolated from tumor-bearing mice under the schedules shown in af) CD8+ T cells specific for the OVA1 antigen. DA-SNA2 vs. admixture (P=0.0401). g) Effector memory CD8+ T cells (CD44+ / CD62L-) within this antigen-specific T cell subset. DA-SNA2 vs. admixture (P=0.0020). h) CD4+ T cells specific for OVA2 antigen. Admixture vs. DA-SNA1 (P=0.0229) and DA-SNA2 (P=0.0436). i) Effector memory CD4+ T cells (CD44+ / CD62L-). DA-SNA1 vs. Admixture (P=0.0001) and DA-SNA2 (P=0.0012). Data show mean ± sem from two independent experiments (n=7-9 per experiment). For panels b, d-g, i, significance was calculated using one-way ANOVA with Tukey's multiple comparison test. For panel h, due to significant differences between groups in standard deviations, Welch ANOVA followed by Dunnett's multiple comparison test was used. Panel c was analyzed using the log-rank test.*p<0.05, **p<0.01, ***p<0.001. [Diagram 36] E.G7-OVA tumor growth curves from individual animals per treatment growth are shown. Mean tumor growth values ​​are shown in Figure 35B. A line was drawn at day 21 for easy comparison between spider plots of different groups. [Figure 37] Shown are gross images of E.G7-OVA tumors used to calculate tumor weight on day 15. Dotted white circles represent tumor-free mice. Scale bar: 1 mm. [Figure 38-1]Tumor inhibition utilizing dual antigen immunotherapy with immune checkpoint inhibitors. a-b) C57BL / 6 mice were inoculated subcutaneously with B16-F10 cells (105) in the right flank and provided with weekly subcutaneous immunizations starting on day 3 for a total of four vaccinations (9 nmol adjuvant, 9 nmol of each antigen). Mean tumor growth curves and animal survival are shown. c) CD8+ T cells specific for M27 antigen and memory CD8+ T cell marker 44+ / 62- in isolated PBMCs. PBS vs. DA-SNA1 (P=0.0060) and DA-SNA2 (P=0.0001). d-e) B16-F10 tumor-bearing mice receiving weekly subcutaneous immunizations of DA-SNA in combination with anti-PD-1 immune checkpoint inhibitors administered intraperitoneally 3 and 6 days after DA-SNA immunization. Mean tumor growth curves and animal survival are shown. Tumor growth comparing anti-PD-1 vs. DA-SNA2 + anti-PD-1 on days 17 (P=0.0354), 20 (P=0.0319), and 22 (P=0.0475). Animal survival comparing DA-SNA2 + anti-PD-1 vs. PBS (P<0.0001) and anti-PD-1 (P<0.0001). f-k) Flow cytometry analysis of PBMCs on day 17 isolated from tumor-bearing mice receiving the indicated schedules for evaluation of circulating CD8+ T cells in df), and total effector memory CD8+ T cells (CD44+ / 62L-) in g). For f: DA-SNA2 + anti-PD-1 vs. PBS (P=0.0001), anti-PD-1 (P=0.0009), and DA-SNA1 + anti-PD-1 (P<0.0001). For g: DA-SNA2 + anti-PD-1 vs. PBS (P=<0.0001), anti-PD-1 (P<0.0001), and DA-SNA1 + anti-PD-1 (P=0.0090). DA-SNA1 + anti-PD-1 vs. PBS (P=0.0023) and anti-PD-1 (P=0.0203). h) M27-specific CD8+ / CD19- T cells. DA-SNA2 + anti-PD-1 vs. PBS (P=0.0093), anti-PD-1 (P=0.0138), and DA-SNA1 + anti-PD-1 (P=0.0052). i) Quantification of circulating CD4+ T cells, and j) Evaluation of effector memory CD4+ T cells (CD44+ / 62L-). For i: DA-SNA2 + anti-PD-1 vs. DA-SNA1 + anti-PD-1 (P = 0.0372).For j: PBS vs. DA-SNA1 + anti-PD-1 (P = 0.0268) and DA-SNA2 + anti-PD-1 (P = 0.0349), anti-PD-1 vs. DA-SNA1 + anti-PD-1 (P = 0.0067), and DA-SNA2 + anti-PD-1 (P = 0.0089). k) M30-specific CD4+ / CD19- T cells. Data show mean ± sem from two independent experiments (n = 9-15). For all panels except b and e, significance was calculated using one-way ANOVA with Tukey's multiple comparison test. Animal survival was analyzed using the log-rank test. Asterisks in d indicate statistically significant differences between DA-SNA2 and anti-PD-1 treatment groups. ns=not significant; nd=not detected; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 38-2]Tumor inhibition utilizing dual antigen immunotherapy with immune checkpoint inhibitors. a-b) C57BL / 6 mice were inoculated subcutaneously with B16-F10 cells (105) in the right flank and provided with weekly subcutaneous immunizations starting on day 3 for a total of four vaccinations (9 nmol adjuvant, 9 nmol of each antigen). Mean tumor growth curves and animal survival are shown. c) CD8+ T cells specific for M27 antigen and memory CD8+ T cell marker 44+ / 62- in isolated PBMCs. PBS vs. DA-SNA1 (P=0.0060) and DA-SNA2 (P=0.0001). d-e) B16-F10 tumor-bearing mice receiving weekly subcutaneous immunizations of DA-SNA in combination with anti-PD-1 immune checkpoint inhibitors administered intraperitoneally 3 and 6 days after DA-SNA immunization. Mean tumor growth curves and animal survival are shown. Tumor growth comparing anti-PD-1 vs. DA-SNA2 + anti-PD-1 on days 17 (P=0.0354), 20 (P=0.0319), and 22 (P=0.0475). Animal survival comparing DA-SNA2 + anti-PD-1 vs. PBS (P<0.0001) and anti-PD-1 (P<0.0001). f-k) Flow cytometry analysis of PBMCs on day 17 isolated from tumor-bearing mice receiving the indicated schedules for evaluation of circulating CD8+ T cells in df), and total effector memory CD8+ T cells (CD44+ / 62L-) in g). For f: DA-SNA2 + anti-PD-1 vs. PBS (P=0.0001), anti-PD-1 (P=0.0009), and DA-SNA1 + anti-PD-1 (P<0.0001). For g: DA-SNA2 + anti-PD-1 vs. PBS (P=<0.0001), anti-PD-1 (P<0.0001), and DA-SNA1 + anti-PD-1 (P=0.0090). DA-SNA1 + anti-PD-1 vs. PBS (P=0.0023) and anti-PD-1 (P=0.0203). h) M27-specific CD8+ / CD19- T cells. DA-SNA2 + anti-PD-1 vs. PBS (P=0.0093), anti-PD-1 (P=0.0138), and DA-SNA1 + anti-PD-1 (P=0.0052). i) Quantification of circulating CD4+ T cells, and j) Evaluation of effector memory CD4+ T cells (CD44+ / 62L-). For i: DA-SNA2 + anti-PD-1 vs. DA-SNA1 + anti-PD-1 (P = 0.0372).For j: PBS vs. DA-SNA1 + anti-PD-1 (P = 0.0268) and DA-SNA2 + anti-PD-1 (P = 0.0349), anti-PD-1 vs. DA-SNA1 + anti-PD-1 (P = 0.0067), and DA-SNA2 + anti-PD-1 (P = 0.0089). k) M30-specific CD4+ / CD19- T cells. Data show mean ± sem from two independent experiments (n = 9-15). For all panels except b and e, significance was calculated using one-way ANOVA with Tukey's multiple comparison test. Animal survival was analyzed using the log-rank test. Asterisks in d indicate statistically significant differences between DA-SNA2 and anti-PD-1 treatment groups. ns=not significant; nd=not detected; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 39] B16-F10 tumor growth curves from individual animals per treatment group are shown, with mean tumor growth values ​​shown in Figure 38D. [Diagram 40] A cartoon showing the heterogeneous tumor populations that vaccine design must combat. CD4+ T cells can provide antitumor responses in the absence of CD8+ T cells by secreting cytokines such as interferon-γ (Mumberg et al., 1999; Qin and Blankenstein, 2000) or by activating and recruiting effector cells such as macrophages and eosinophils (Greenberg, 1991; Hung et al., 1998). However, the primary role of CD4+ T cells in the immune response to cancer is to prime CD8+ cells and sustain their proliferation. [Diagram 41] A cartoon showing that vaccine design must combat heterogeneous tumor populations. [Diagram 42] We show that antigen-targeting strategies that elicit additional immune cells improve vaccine efficacy. [Diagram 43] Cartoon showing that potent vaccine delivery stimulates multiple classes of cells. [Diagram 44]a-b) Results of an experiment in which C57BL / 6 mice were inoculated subcutaneously with MC38 cells (5x105) in the right flank and provided with weekly immunizations starting on day 3 for a total of 4 vaccinations (6 nmol adjuvant, 6 nmol of each antigen). Mean tumor growth curves and animal survival are shown. c) Mean tumor volume on day 24. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In various aspects, the present disclosure provides spherical nucleic acids (SNAs), which are nanostructures with a core surrounded by a high-density radial presentation of oligonucleotides that target multiple classes of immune cells (e.g., T cells) through the incorporation and individual structural positioning of different classes of antigens. Antigens can be incorporated at locations including, but not limited to, encapsulated in the nanoparticle core, anchored on the surface, or conjugated to complementary oligonucleotide strands hybridized to the adjuvant oligonucleotide SNA shell. In various embodiments, the multi-antigen targeted SNAs of the present disclosure provide a defined structural presentation of vaccine components that optimally activate multiple different types of immune cells for a synergistic immune response.

[0019] Spherical nucleic acids (SNAs) are nanomaterials that can improve the delivery and efficacy of vaccine components. Comprised of a nanoparticle core with a dense surface layer of radially oriented oligonucleotides (e.g., unmethylated cytosine-phosphate-guanine (CpG) motif DNA that activates toll-like receptor 9 (TLR9) in antigen-presenting cells (APCs)), SNAs are modular structures that have several advantages over conventional vaccines. They have high affinity target binding, rapid cellular uptake without the need for transfection reagents, high biocompatibility, reduced nuclease degradation, and easy drainage into lymph nodes upon subcutaneous injection (Cutler, JI; Auyeung, E.; Mirkin, CA Spherical Nucleic Acids. J. Am. Chem. Soc. 2012, 134(3), 1376-1391; Banga, RJ; Chernyak, N.; Narayan, SP; Nguyen, ST; Mirkin, CA Liposomal Spherical Nucleic Acids. Acids.J.Am.Chem.Soc.2014,136(28),9866-9869, Radovic-Moreno,AF;Chernyak,N.;Mader,CC;Nallagatla,S.;Kang,RS;Ha o,L.;Walker,DA;Halo,TL;Merkel,TJ;Rische,CH;Anantatmula,S.;Burkhart,M.;Mirkin,CA;Gryaznov,SMImmunomodulatory Spherical Nucleic Acids.Proc.Natl.Acad.Sci.2015,112(13),3892-3897, Choi, CHJ; Hao, L.; Narayan, SP; Auyeung, E.; Mirkin, CAMechanism for the Endocytosis of Spherical Nucleic Acid Nanoparticle Conjugates.Proc.Natl.Acad.Sci.2013,110(19),7625-7630, Rosi,NLOligonucleotide-Modified Gold Nanoparticles for Intracellular Gene Regulation. Science 2006,312(5776),1027-1030; Wang,S.; Qin,L.; Yamankurt,G.; Skakuj,K.; Huang,Z.; Chen,P.-C.; Dominguez,D.; Lee,A.; Zhang,B.; Mirkin,C. Regulation Vaccinology with Spherical Nucleic Acids. Proc. Natl. Acad. Sci. 2019,116(21),10473-10481). Their ease of synthesis and inherent modularity allows the design of compositionally equivalent structures with diverse arrangements of the two main components - the oligonucleotide adjuvant (immune system activator) shell and the antigen (immune system target). Structural modifications have shown that vaccine architecture can impact function and generate unique immune responses, which has been demonstrated in multiple tumor models, including model ovalbumin (OVA) lymphoma tumors and HPV, as well as in a prostate cancer-related system targeting prostate-specific membrane antigen (PSMA). However, so far, only one class of immune cells has been targeted: the cytotoxic CD8. + It incorporates an antigen that activates only T cells.

[0020] Due to tumor heterogeneity and their ability to evolve and evade the immune system, cytotoxic CD8 + It is important not only to generate enhanced major histocompatibility complex I (MHC-I) targeting of T cell responses, but also to engage synergistic interactions. Specifically, CD8 + and helper CD4 +Both CD4 and CD8 T lymphocytes are required for long-term tumor rejection (Ostroumov, D.; Fekete-Drimusz, N.; Saborowski, M.; Kuhnel, F.; Woller, N. CD4 and CD8 T Lymphocyte Interplay in Controlling Tumor Growth. Cell. Mol. Life Sci. 2018, 75(4), 689-713; Shankaran, V.; Ikeda, H.; Bruce, AT; White, JM; Swanson, PE; Old, LJ; Schreiber, RDIFNγ and Lymphocytes Prevent Primary Tumour Development and Shape Tumour Immunogenicity. Nature 2001, 410(6832), 1107-1111). Thus, the present disclosure provides a method to simultaneously target CD8 T lymphocytes using MHC-I and -II antigens. + T cells and CD4 + The present invention provides SNAs with enhanced vaccine efficacy by priming both T cells and T cells. This is particularly important in melanoma, where traditional treatments such as chemotherapy or radiation are less effective because the tumor has a high genetic mutation load and can therefore easily evade the immune system. However, current clinical alternatives are either ineffective, logistically difficult, or expensive to elicit a broad response that can accommodate mutations, and none consider the impact of structure on the resulting immune response. SNAs can function as robust cancer vaccines by targeting multiple melanoma-associated antigens to control the presentation of immune stimulatory cues and reduce the likelihood of tumor immune evasion. The present disclosure utilizes a rational vaccinology approach to improve vaccine efficacy by presenting multiple epitopes in specific structural configurations to stimulate both cytotoxic T cells and helper T cells.

[0021] Herein, these two classes of antigens (one is CD8 + T cells and the other CD4 +It has been disclosed that the structural positioning of the SNA (targeting T cells) dramatically alters vaccine efficacy by inducing changes at the genetic, cellular, and organismal levels. Using model MHC-I and -II OVA antigens, it was observed that compositionally equivalent SNAs direct distinct immune responses. One particular SNA structure, in which MHC-I antigens hybridized to the shell and MHC-I antigens encapsulated in the core, directed CD8 T cell activation in comparison to the reverse, which had swapped positioning of MHC-I and -II antigens. + The SNA structure induced a two-fold increase in effector cells (Figure 1), as well as a three-fold increase compared to a simple mixture of both antigen and adjuvant. Additionally, the SNA structure enhanced antigen-specific IFN-γ secretion and intracellular IFN-γ production to levels five-fold higher than those from the reverse SNA or the simple mixture. This resulted in an increase in bulk CD8 cells in the spleen after immunization. + and CD4 + These findings were found to be a direct consequence of alterations in the transcriptome, as measured by RNA-seq in T cells. Importantly, these trends were translated to an in vivo melanoma system, where different SNAs induced significant differences in tumor growth.

[0022] This disclosure demonstrates how tumor heterogeneity can be addressed through the rational design of more complex vaccines. By incorporating this approach and considering antigen positioning in vaccine design, it is demonstrated herein that SNA vaccine potency can be altered. The materials and methods of this disclosure are broadly applicable to this field, addressing the opportunity to use nanostructures to present multiple immune stimulatory cues to immune cells and coordinate their processing. For example, the techniques described herein are translatable to other systems and biological knowledge to inform mechanistic understanding of the structural basis for vaccine function. The techniques described herein allow for immune responses that are scalable, controllable, and generated holistically. With the optimized structural presentations incorporating multiple targets described herein, in the case of cancer, there is a more robust response leading to faster and complete remission before tumor immune escape.

[0023] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0024] Language such as "from," "to," "up to," "at least," "greater than," "less than," and the like all refer to ranges that include the recited numbers and can then be broken down into sub-ranges.

[0025] Ranges include each individual member. Thus, for example, a group having 1 to 3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members.

[0026] "About" and "approximately" are generally intended to mean an acceptable degree of error for the quantity measured given the nature or precision of the measurement. Exemplary degrees of error are within 20-25 percent (%), e.g., within 20 percent, within 10 percent, within 5 percent, within 4 percent, within 3 percent, within 2 percent, or within 1 percent of the stated value or range of values.

[0027] A "subject" is a vertebrate organism. The subject can be a non-human mammal (e.g., a mouse, a rat, or a non-human primate), or the subject can be a human subject.

[0028] As used herein, the terms "administering," "administer," "administration," and the like refer to any mode of transferring, delivering, introducing, or transporting an SNA to a subject in need of treatment with such an agent. Such modes include, but are not limited to, oral, topical, intravenous, intraarterial, intraperitoneal, intramuscular, intratumoral, intradermal, intranasal, and subcutaneous administration.

[0029] As used herein, "treating" and "treatment" refer to any reduction in the severity and / or onset of symptoms associated with a disease (e.g., cancer). Thus, "treating" and "treatment" include therapeutic and prophylactic measures. One skilled in the art will appreciate that any degree of protection or recovery from a disease (e.g., cancer) is beneficial to a subject, such as a human patient. The quality of life of a patient is improved by reducing to any degree the severity of symptoms in a subject and / or delaying the onset of symptoms.

[0030] As used herein, a "targeting oligonucleotide" is an oligonucleotide that directs SNA to a specific tissue and / or a specific cell type. In some embodiments, the targeting oligonucleotide is an aptamer. Thus, in some embodiments, the SNA of the present disclosure comprises an aptamer attached to the exterior of the nanoparticle core, and the aptamer is designed to bind to one or more receptors on the surface of a specific cell type.

[0031] As used herein, an "immunostimulatory oligonucleotide" is an oligonucleotide that can stimulate (e.g., induce or enhance) an immune response. Typical examples of immunostimulatory oligonucleotides are CpG motif-containing oligonucleotides, single-stranded RNA oligonucleotides, double-stranded RNA oligonucleotides, and double-stranded DNA oligonucleotides. A "CpG motif" is a cytosine-guanine dinucleotide sequence. In any of the aspects or embodiments of the present disclosure, the immunostimulatory oligonucleotide is a toll-like receptor (TLR) agonist (e.g., a toll-like receptor 9 (TLR9) agonist).

[0032] The term "inhibitory oligonucleotide" refers to an oligonucleotide that reduces the production or expression of a protein, for example, by preventing the translation of mRNA into a protein in a ribosome, or that is sufficiently complementary to either the gene or mRNA encoding one or more of the target proteins to specifically bind (hybridize) to one or more target genes or mRNAs, thereby reducing the expression or biological activity of the target protein. Inhibitory oligonucleotides include, but are not limited to, isolated or synthetic short hairpin RNA (shRNA or DNA), antisense oligonucleotides (e.g., antisense RNA or DNA, chimeric antisense DNA or RNA), miRNA and miRNA mimics, small interfering RNA (siRNA), DNA or RNA inhibitors of innate immune receptors, aptamers, DNAzymes, or aptazymes.

[0033] The term "non-targeting oligonucleotide" refers in some embodiments to oligonucleotides that are not associated with a specific activity (e.g., immunostimulatory activity) but are instead included in the oligonucleotide shell of an SNA, which are used to achieve a certain density of oligonucleotides on the outer surface of the SNA. Non-limiting examples of non-targeting oligonucleotides include scrambled nucleotide sequences and / or homopolymeric oligonucleotides (e.g., polythymidine oligonucleotides (T 20 and the like).

[0034] An "antigenic composition" is a composition of matter suitable for administration to a human or animal subject (e.g., in an experimental or clinical setting) that is capable of eliciting a specific immune response against an antigen, such as, for example, one or more of the antigens described herein. In the context of the present disclosure, the term antigenic composition is understood to encompass compositions intended for administration to a subject or population of subjects for the purposes of eliciting a protective or mitigating immune response against an antigen, such as one or more of the antigens described herein.

[0035] As used herein, the term "dose" refers to a measured amount of any of the SNAs of the disclosure (e.g., SNAs, antigenic compositions, pharmaceutical formulations described herein) taken by a subject (administered to a subject or received by a subject) at any given time.

[0036] An "immune response" is a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus, such as an SNA, as described herein. The immune response can be a B cell response, which results in the production of specific antibodies, such as antigen-specific neutralizing antibodies. An immune response can also be a CD4 + Helper T cell response or CD8 +It may be a T cell response, such as a cytotoxic T cell response. B cell and T cell responses are aspects of a "cellular" immune response. As described herein, an "immune response" may also be a "therapeutic-based" response that is primed while the immune system is actively fighting a tumor. An immune response may also be an antibody-mediated "humoral" immune response. In some cases, the response is specific to a particular antigen (i.e., an "antigen-specific response"). A "protective immune response" is an immune response that inhibits the harmful function or activity of an antigen or reduces symptoms (including death) resulting from an antigen. Protection in this context does not necessarily require that the subject is completely protected against infection. A protective response is achieved when the subject is protected from developing disease symptoms or when the subject experiences less severe disease symptoms. Protective immune responses can be measured by immunoassays that use serum samples from immunized subjects and test the ability of serum antibodies to inhibit pseudovirus binding, such as pseudovirus neutralization assays (or surrogate virus neutralization tests), ELISA neutralization assays, antibody-dependent cell-mediated cytotoxicity assays (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated phagocytosis (ADCP), enzyme-linked immunospot (ELISpot), etc. Additionally, vaccine efficacy can be tested by measuring B or T cell activation after immunization using flow cytometry (FACS) analysis or ELISpot assays. Protective immune responses can be tested by measuring resistance to antigen challenge in vivo in animal models. In humans, protective immune responses can be demonstrated in population studies that compare measures such as symptoms, morbidity, and mortality in treated subjects compared to untreated controls. Exposure of a subject to an immunogenic stimulus, such as the SNAs described herein, induces a primary immune response specific to the stimulus, i.e., the exposure "primes" the immune response. Subsequent exposure to the stimulus (eg, by immunization) can increase or "boost" the magnitude (or duration, or both) of the specific immune response.Thus, for example, "boosting" an existing immune response by administering an antigenic composition of the present disclosure increases the magnitude of the antigen-specific response (e.g., by broadening the breadth of antibodies produced (i.e., when administering a booster that primes the immune system against a variant), by increasing antibody titer and / or affinity, by increasing the frequency of antigen-specific B or T cells, by inducing mature effector function, or a combination thereof). B and T cell "maturity and memory" can also be measured as an indicator of the immune response.

[0037] "Adjuvant" refers to a substance that, when added to a composition containing an antigen, non-specifically enhances or strengthens the immune response to the antigen in a recipient upon exposure. In any of the aspects or embodiments of the present disclosure, the SNA provided herein includes an immunostimulatory oligonucleotide (such as, but not limited to, a Toll-like receptor (TLR) agonist) as an adjuvant and includes an antigen as described herein. Additional adjuvants contemplated for use with the present disclosure include aluminum (e.g., aluminum hydroxide), lipid-based adjuvant AS01B, alum, MF59, in addition to the TLR agonists described herein (e.g., CpG DNA, imiquimod for TLR7, motolimod for TLR8, MPLA4 for TLR4, poly(I:C) for TLR3, or combinations thereof).

[0038] An "effective amount" or "sufficient amount" of a substance is the amount necessary to produce a beneficial or desired result, including a clinical result, and thus "effective amount" depends on the context in which it is applied. In the context of administering the SNA of the present disclosure, for example, an effective amount contains sufficient antigen to induce an immune response. In some embodiments, an effective amount of SNA is an amount sufficient to inhibit gene expression. An effective amount may be administered in one or more doses as further described herein. Efficacy may be demonstrated in experimental or clinical trials, for example, by comparing the results achieved with the substance of interest compared to an experimental control.

[0039] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter cited, and may, in some cases, be incorporated herein in its entirety.

[0040] spherical nucleic acid Spherical nucleic acids (SNAs) comprise densely functionalized and highly oriented polynucleotides on the surface of a nanoparticle core. In various aspects, the present disclosure provides spherical nucleic acids (SNAs) comprising: (a) a nanoparticle core; (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen. In various embodiments, the nanoparticle core is a micelle, a liposome, a polymer, a lipid nanoparticle (LNP), or a combination thereof. In various embodiments, the polymer is polylactide, polylactide-polyglycolide copolymer, polycaprolactone, polyacrylate, alginate, albumin, silica, polypyrrole, polythiophene, polyaniline, polyethyleneimine, poly(methyl methacrylate), or chitosan. In some embodiments, the polymer is poly(lactic-co-glycolic acid) (PLGA). The spherical architecture of the polynucleotide shell provides unique advantages over traditional nucleic acid delivery methods, including entry into nearly all cells independent of transfection agents and resistance to nuclease degradation. Furthermore, SNAs can penetrate biological barriers, including the blood-brain barrier (see, e.g., U.S. Patent Application Publication No. 2015 / 0031745, which is incorporated herein by reference in its entirety) and the blood-tumor barrier, as well as the epidermis (see, e.g., U.S. Patent Application Publication No. 2010 / 0233270, which is incorporated herein by reference in its entirety).

[0041] Liposomes are spherical self-closed structures of various size ranges that contain one or several hydrophobic lipid bilayers with a hydrophilic core. The diameter of these lipid-based carriers ranges from 0.15 to 1 micrometer, which is significantly larger than the effective therapeutic range of 20 to 100 nanometers. Liposomes called small unilamellar vesicles (SUVs) can be synthesized in the size range of 20 to 50 nanometers, but face challenges such as instability and aggregation that lead to interparticle fusion. This interparticle fusion limits the use of SUVs in therapeutics. In some embodiments, liposomal spherical nucleic acids (LSNAs) comprise a liposomal core and a shell of oligonucleotides bound to the outer surface of the liposomal core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides, a first antigen that is a major histocompatibility complex type I (MHC-I) antigen, and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen. Antigens contemplated for use according to the present disclosure are further described herein below.

[0042] Thus, liposomal particles, for example as disclosed in International Patent Application No. PCT / US2014 / 068429 (hereby incorporated by reference in its entirety), are also provided by the present disclosure. The liposomal particles of the present disclosure have at least a substantially spherical shape, an interior and an exterior, and comprise a plurality of lipid groups. In various embodiments, the plurality of lipid groups comprises lipids selected from the group consisting of the phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine families of lipids. Lipids contemplated by the present disclosure include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DSPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DSPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPG), 1,2-dipalmito ... Examples of suitable lipid anchoring groups include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), cardiolipin, lipid A, monophosphoryl lipid A (MPLA), and combinations thereof. In various embodiments, at least one oligonucleotide in the oligonucleotide shell is attached to the exterior of the liposome core via a lipid anchoring group. In various embodiments, each oligonucleotide in the oligonucleotide shell is attached to the exterior of the nanoparticle core through a lipid anchoring group. In further embodiments, a lipid anchoring group is attached to the 5'-terminus or 3'-terminus of at least one oligonucleotide. In still further embodiments, the lipid anchor group is tocopherol or cholesterol.Thus, in various embodiments, at least one of the oligonucleotides in the oligonucleotide shell is an oligonucleotide-lipid conjugate containing a lipid anchor group, and the lipid anchor group is adsorbed to the lipid bilayer. In some embodiments, all of the oligonucleotides in the oligonucleotide shell are oligonucleotide-lipid conjugates containing a lipid anchor group, and the lipid anchor group is adsorbed to the lipid bilayer. In various embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the oligonucleotides in the oligonucleotide shell are bound (e.g., adsorbed) to the outside of the liposome core through lipid anchor groups. The lipid anchor group, in various embodiments, comprises tocopherol, palmitoyl, dipalmitoyl, stearyl, distearyl, or cholesterol. Methods of making oligonucleotides containing lipid anchors are disclosed herein. As an example, first, an oligonucleotide and a phosphoramidite-modified tocopherol are provided, and then the oligonucleotide is exposed to the phosphoramidite-modified tocopherol to create a tocopherol-modified oligonucleotide. The lipid anchor can be attached to the oligonucleotide using any chemistry known to those skilled in the art, including, but not limited to, amide bonds or click chemistry.

[0043] Methods for making liposomal SNAs (LSNAs) are described herein and are generally known (see, e.g., Wang et al., Proc. Natl. Acad. Sci. 2019, 116(21), 10473-10481, which is incorporated by reference in its entirety).

[0044] Lipid nanoparticle spherical nucleic acid (LNP-SNA) is composed of a lipid nanoparticle core decorated with a shell of oligonucleotides. The lipid nanoparticle core comprises an ionizable lipid, a phospholipid, a sterol, a lipid-polyethylene glycol (lipid-PEG) conjugate, and a first antigen that is a major histocompatibility complex type I (MHC-I) antigen, and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen. Antigens contemplated for use according to the present disclosure are further described herein below. A shell of oligonucleotides is attached to the outer surface of the lipid nanoparticle core, and in any of the aspects or embodiments of the present disclosure, the shell of oligonucleotides comprises one or more immunostimulatory oligonucleotides. The spherical architecture of the oligonucleotide shell confers unique advantages over conventional nucleic acid delivery methods, including entry into nearly all cells independent of transfection agents, resistance to nuclease degradation, sequence-based function, targeting, and diagnostics.

[0045] In some embodiments, the ionizable lipid is dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), C12-200, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), similar lipid / lipidoid structures, or combinations thereof. In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dihexadecanoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), monophosphoryl lipid A (MPLA), or combinations thereof. In a further embodiment, the sterol is 3β-hydroxycholest-5-ene (cholesterol), 9,10-secocholesta-5,7,10(19)-trien-3β-ol (vitamin D3), 9,10-secoegosta-5,7,10(19),22-tetraen-3β-ol (vitamin D2), calcipotriol, 24-ethyl-5,22-cholestadien-3β-ol (stigmasterol), 22,23-dihydrostigmasterol (β-sitosterol), 3,28-dihydroxy-lupeol (beta-sitosterol), 5,5-dihydroxy-1,1,2,3,4,4-trimethylol, 5,5-dihydro ... Turin), lupeol, ursolic acid, oleanolic acid, 24α-methylcholesterol (campesterol), 24-ethylcholesta-5,24(28)E-dien-3β-ol (fucosterol), 24-methylcholesta-5,22-dien-3β-ol (brassicasterol), 24-methylcholesta-5,7,22-trien-3β-ol (ergosterol), 9,11-dehydroergosterol, daucosterol, or any of the foregoing sterols modified with one or more amino acids. In some embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate comprises a 2000 Dalton (Da) polyethylene glycol. In further embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate is a lipid-PEG-maleimide.In still further embodiments, the lipid-PEG-maleimide is 1,2-dipalmitryl-sn-glycero-3-phosphoethanolamine (DPPE) conjugated to a 2000 Da polyethylene glycol maleimide, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) conjugated to a 2000 Da polyethylene glycol maleimide, or a combination thereof.

[0046] In any of the aspects or embodiments of the present disclosure, the oligonucleotides are attached to the exterior of the lipid nanoparticle core via covalent attachment to the lipid-polyethylene glycol (lipid-PEG) conjugate of the oligonucleotide. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the oligonucleotides in the oligonucleotide shell are covalently attached to the exterior of the lipid nanoparticle core through the lipid-PEG conjugate. In various embodiments, one or more oligonucleotides in the oligonucleotide shell are attached (or adsorbed) to the exterior of the lipid nanoparticle core through a lipid anchor group as described herein. In various embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the oligonucleotides in the oligonucleotide shell are attached (or adsorbed) to the exterior of the lipid nanoparticle core through a lipid group as described herein. The lipid anchor group is attached to the 5' or 3' end of the oligonucleotide in various embodiments. In various embodiments, the lipid anchor group is tocopherol, palmitoyl, dipalmitoyl, stearyl, distearyl, or cholesterol.

[0047] SNAs may range from about 1 nanometer (nm) to about 500 nm, about 1 nm to about 400 nm, about 1 nm to about 300 nm, about 1 nm to about 200 nm, about 1 nm to about 150 nm, about 1 nm to about 100 nm, about 1 nm to about 90 nm, about 1 nm to about 80 nm in diameter, about 1 nm to about 70 nm in diameter, about 1 nm to about 60 nm in diameter, about 1 nm to about 50 nm in diameter, about 1 nm to about 40 nm in diameter, about 1 nm to about 30 nm in diameter, about 1 nm to about 20 nm in diameter, about 1 nm to about 10 nm, about 10 nm to about 150 nm The size range may be from about 10 nm to about 140 nm in diameter, about 10 nm to about 130 nm in diameter, about 10 nm to about 120 nm in diameter, about 10 nm to about 110 nm in diameter, about 10 nm to about 100 nm in diameter, about 10 nm to about 90 nm in diameter, about 10 nm to about 80 nm in diameter, about 10 nm to about 70 nm in diameter, about 10 nm to about 60 nm in diameter, about 10 nm to about 50 nm in diameter, about 10 nm to about 40 nm in diameter, about 10 nm to about 30 nm in diameter, or about 10 nm to about 20 nm in diameter. In a further aspect, the present disclosure provides a plurality of SNAs, each SNA comprising one or more oligonucleotides attached thereto. Thus, in some embodiments, the size of the plurality of SNAs is about 10 nm to about 150 nm (mean diameter), about 10 nm to about 140 nm mean diameter, about 10 nm to about 130 nm mean diameter, about 10 nm to about 120 nm mean diameter, about 10 nm to about 110 nm mean diameter, about 10 nm to about 100 nm mean diameter, about 10 nm to about 90 nm mean diameter, about 10 nm to about 80 nm mean diameter, about 10 nm to about 70 nm mean diameter, about 10 nm to about 60 nm mean diameter, about 10 nm to about 50 nm mean diameter, about 10 nm to about 40 nm mean diameter, about 10 nm to about 30 nm mean diameter, or about 10 nm to about 20 nm mean diameter. In some embodiments, the diameter of the SNA (or the mean diameter for the plurality of SNAs) is about 10 nm to about 150 nm, about 30 to about 100 nm, or about 40 to about 80 nm. In some embodiments, the size of the nanoparticles used in the methods may vary as required depending on their particular use or application. Size variation is advantageously used to optimize certain physical properties of the SNA, such as the amount of surface area to which oligonucleotides can be attached, as described herein.It will be understood that the aforementioned diameters of the SNA can apply to the diameter of the nanoparticle core itself, or to the diameter of the nanoparticle core and one or more oligonucleotides attached thereto.

[0048] antigen The spherical nucleic acid (SNA) of the present disclosure, in various aspects, comprises: (a) a nanoparticle core; (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen. As described herein, the number and positioning of the MHC-I and MHC-II antigens influence the efficacy of the SNA in its use, for example, as a vaccine. MHC-I antigens bind to CD8 + T cells, whereas MHC-II antigens prime CD4 + In any of the aspects or embodiments of the present disclosure, the SNA comprises at least one MHC-I antigen and at least one MHC-II antigen. MHC-I antigens contemplated by the present disclosure include OVA, 257-264(OVA1) (SEQ ID NO: 7), GP100 (25-33) (KVPRNQDWL (SEQ ID NO: 11)), TC-1 E6 (49-58) (VYDFAFRDLC (SEQ ID NO: 12)), TC-1 E7 (49-57) (RAHYNIVTF (SEQ ID NO: 13)), PSMA (634-642) (SAVKNFTEI (SEQ ID NO: 14)), SPAS-1 (SNC9-H8) (STHVNHLHC (SEQ ID NO: 15)), SIMS2 (237-245) (SLDLKLIFL (SEQ ID NO: 16)), PAP (115-123) (SAMTNLAAL (SEQ ID NO: 17)), B16 MART-1(M27)(LCPGNKYEM (SEQ ID NO:9)), TRP-1(252-260)(ATGKNVCDV (SEQ ID NO:18)), TRP-1(252V260M)(ATGKNVCDM (SEQ ID NO:19)), TRP-1(455-463)(TAPDNLGYA (SEQ ID NO:20)), TRP-1(455A463M)(TAPDNLGYM (SEQ ID NO:21)), TRP-2(180-188)(SVYDFFVWL (SEQ ID NO:22)), Melan-A / MART-(127-135), tyrokinase(1-9), tyrokinase(369-377D), MC38 MHC-II antigens contemplated by the present disclosure include, but are not limited to, Adpgk (ASMTNMELM (SEQ ID NO: 23)), Irgq-minimal (AALLNSAVL (SEQ ID NO: 24)), Irgq-long peptide (KARDETAALLNSAVLGAAPLFVPPAD (SEQ ID NO: 25)), or combinations thereof. 323-339(OVA2) (SEQ ID NO: 8), GP100: (46-58) (RQLYPEWTEAQRL (SEQ ID NO: 26)), TC-1 E6 (43-57) (QLLRREVYDFAFRDL (SEQ ID NO: 27)), SIMS2 (240-254) (LKLIFLDSRVTEVTG (SEQ ID NO: 28)), PAP (114-128) (MSAMTNLAALFPPEG (SEQ ID NO: 29)), B16 MART-1 (M30) (VDWENVSPELNSTDQ (SEQ ID NO: 30)), TRP-1 (113-127) (CRPGWRGAACNQKIL (SEQ ID NO: 31)), TRP-1 (106-130) (SGHNCGTCRPGWRGAACNQKILTVR (SEQ ID NO: 32)), Li-Key (77-92) (LRMKLPKPPKPVSQMR (SEQ ID NO: 33)), Tyrokinase (56-70), GP100 (44-59), GP100 (167-189), Melan-A / MART-1 (102-111) (PAYEKLSAEQS PPPY (SEQ ID NO: 34)), Melan-A / MART-1(27-40) (AAGIGILTVILGVL (SEQ ID NO: 35)), Melan-A / MART-1(51-70) (RNGYRALMDKSLHVGTQCAL (SEQ ID NO: 36)), Melan-A / MART-1(51-73) (RNGYRALMDKSLHVGTQCALTRR (SEQ ID NO: 37)), Melan-A / MART-1(43-57) (IGCWYCRRRNGYRAL (SEQ ID NO: 38)), or a combination thereof.

[0049] The present disclosure contemplates that any configuration and combination of MHC-I and MHC-II antigens may be used in SNA. Thus, in various aspects and embodiments, the present disclosure provides SNAs in which: a) both MHC-I and MHC-II antigens are encapsulated in the nanoparticle core and no antigen is associated with the exterior side of the nanoparticle core; b) both MHC-I and MHC-II antigens are encapsulated in the nanoparticle core and MHC-I and / or MHC-II antigens are additionally associated with the exterior side of the nanoparticle core; c) both MHC-I and MHC-II antigens are on the exterior side of the nanoparticle core and no antigen is encapsulated in the nanoparticle core; d) both MHC-I and MHC-II antigens are on the exterior side of the nanoparticle core and MHC-I and / or MHC-II antigens are additionally encapsulated in the nanoparticle core; e) MHC-I antigens are encapsulated in the nanoparticle core and MHC-II antigens are associated with the exterior side of the nanoparticle core; f) MHC-II antigens are encapsulated in the nanoparticle core and MHC-I antigens are associated with the exterior side of the nanoparticle core. Thus, in various embodiments, the MHC-I antigen(s) and the MHC-II antigen(s) are encapsulated within the nanoparticle core, associated with the nanoparticle core on the exterior side of the nanoparticle core (e.g., through covalent or non-covalent interactions), or any combination thereof. The antigen associated with the nanoparticle core on the exterior side of the nanoparticle core is located distal to the nanoparticle core in any of the aspects or embodiments of the present disclosure. Thus, in some embodiments, the antigen is attached to the terminus of an oligonucleotide in the shell of an oligonucleotide that is not bound to a nanoparticle core (e.g., if an oligonucleotide is bound to a nanoparticle core through its 3' terminus, then the antigen is attached to the 5' terminus of the oligonucleotide). Alternatively, in some embodiments, the antigen is attached to the terminus of an oligonucleotide in the shell of an oligonucleotide that is bound to a nanoparticle core (e.g., if an oligonucleotide is bound to a nanoparticle core through its 3' terminus, then the antigen is attached to the 3' terminus of the oligonucleotide).In further embodiments, the antigen is attached to a terminus of an oligonucleotide proximal to the nanoparticle core, and the oligonucleotide hybridizes to an oligonucleotide attached to the nanoparticle core, or in some embodiments, the antigen is attached to a terminus of an oligonucleotide distal to the nanoparticle core, and the oligonucleotide hybridizes to an oligonucleotide attached to the nanoparticle core.

[0050] As described herein, in some aspects, the disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; (b) an oligonucleotide shell bound to the outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen. In some embodiments, the first antigen is encapsulated in the nanoparticle core. In further embodiments, the second antigen is bound to one or more oligonucleotides in the oligonucleotide shell through a linker. As used herein, an antigen that is "bound to one or more oligonucleotides in the oligonucleotide shell through a linker" may be bound in a variety of ways, including, but not limited to, a) the antigen is directly bound to the oligonucleotide bound to the nanoparticle core, and / or b) the antigen is bound to an oligonucleotide that is hybridized to the oligonucleotide bound to the nanoparticle core. In any of the aspects or embodiments of the present disclosure, when the antigen is directly bound to the oligonucleotide bound to the nanoparticle core, the antigen is bound to the non-targeting oligonucleotide. Thus, in some embodiments, the second antigen is bound to the oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core through a linker. In further embodiments, the second antigen is bound to the oligonucleotide hybridized to the oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core through a linker. In still further embodiments, the second antigen is bound to the outer surface of the nanoparticle core through a linker. In some embodiments, the second antigen is encapsulated in the nanoparticle core. In further embodiments, the first antigen is bound to one or more oligonucleotides in the shell of the oligonucleotide through a linker. In various embodiments, the first antigen is bound to the oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core through a linker.In some embodiments, the first antigen is attached through a linker to an oligonucleotide that hybridizes to an oligonucleotide in the shell of the oligonucleotide that is attached to the nanoparticle core. In further embodiments, the first antigen is attached to the exterior surface of the nanoparticle core through a linker. In some embodiments, the SNA comprises a third antigen that is a major histocompatibility complex type I (MHC-I) antigen. In further embodiments, the SNA comprises a fourth antigen that is a major histocompatibility complex type II (MHC-II) antigen. In some embodiments, the third antigen is encapsulated in the nanoparticle core. In further embodiments, the fourth antigen is attached through a linker to one or more oligonucleotides in the shell of the oligonucleotide. In still further embodiments, the fourth antigen is attached through a linker to an oligonucleotide in the shell of the oligonucleotide that is attached to the nanoparticle core. In some embodiments, the fourth antigen is attached through a linker to an oligonucleotide that hybridizes to an oligonucleotide in the shell of the oligonucleotide that is attached to the nanoparticle core. In some embodiments, the fourth antigen is attached through a linker to an oligonucleotide that hybridizes to an oligonucleotide in the shell of the oligonucleotide that is attached to the nanoparticle core. In some embodiments, the fourth antigen is attached through a linker to the exterior surface of the nanoparticle core. In further embodiments, the third antigen is attached to one or more oligonucleotides in the shell of the oligonucleotide through a linker. In some embodiments, the third antigen is attached to an oligonucleotide in the shell of the oligonucleotide attached to the nanoparticle core through a linker. In further embodiments, the third antigen is attached to an oligonucleotide hybridized to an oligonucleotide in the shell of the oligonucleotide attached to the nanoparticle core through a linker. In still further embodiments, the third antigen is attached to the exterior surface of the nanoparticle core through a linker. In some embodiments, the fourth antigen is encapsulated in the nanoparticle core. In some embodiments, the first antigen and the third antigen are the same. In some embodiments, the first antigen and the third antigen are different. In still further embodiments, the second antigen and the fourth antigen are the same. In some embodiments, the second antigen and the fourth antigen are different.

[0051] In some aspects, the present disclosure provides SNAs comprising a plurality of first antigens that are MHC-I antigens, a plurality of second antigens that are MHC-II antigens, or both. The first antigens and / or the second antigens can be encapsulated in the nanoparticle core, associated with the nanoparticle core on the exterior side of the nanoparticle core, or a combination thereof. In some embodiments, each of the plurality of first antigens is the same. In further embodiments, the plurality of first antigens comprises any combination of the first antigens described herein. In some embodiments, each of the plurality of second antigens is the same. In further embodiments, the plurality of second antigens comprises any combination of the second antigens described herein. Thus, the SNA can comprise any combination of the first and second antigens described herein.

[0052] In some aspects, the disclosure provides an SNA comprising: (a) a nanoparticle core; (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides; and (c) one or more MHC-I antigens encapsulated in the nanoparticle core and one or more MHC-II antigens associated with the nanoparticle core on the outer side of the nanoparticle core. In a further aspect, the disclosure provides an SNA comprising: (a) a nanoparticle core; (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides; and (c) one or more MHC-II antigens encapsulated in the nanoparticle core and one or more MHC-I antigens associated with the nanoparticle core on the outer side of the nanoparticle core. In some embodiments, some of the one or more MHC-I antigens are encapsulated in the nanoparticle core and associated with the nanoparticle core on the outer side of the nanoparticle core. In some embodiments, some of the one or more MHC-II antigens are encapsulated in the nanoparticle core and associated with the nanoparticle core on the outer side of the nanoparticle core. In further embodiments, some of the one or more MHC-I antigens and some of the one or more MHC-II antigens are encapsulated in the nanoparticle core and are also associated with the nanoparticle core on the exterior side of the nanoparticle core. In various embodiments, the one or more MHC-I antigens are all the same, while in some embodiments, the one or more MHC-I antigens include any combination of MHC-I antigens described herein. In various embodiments, the one or more MHC-II antigens are all the same, while in some embodiments, the one or more MHC-II antigens include any combination of MHC-II antigens described herein.

[0053] In a further aspect, the present disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides; (c) a first antigen which is a major histocompatibility complex type I (MHC-I) antigen, the first antigen being encapsulated in the nanoparticle core, bound to one or more oligonucleotides in the oligonucleotide shell through a linker, bound to the outer surface of the nanoparticle core through a linker, or a combination thereof; and (d) a second antigen which is a major histocompatibility complex type II (MHC-II) antigen, the second antigen being encapsulated in the nanoparticle core, bound to one or more oligonucleotides in the oligonucleotide shell through a linker, bound to the outer surface of the nanoparticle core through a linker, or a combination thereof.

[0054] In a further aspect, the present disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; (b) a shell of oligonucleotides attached to an exterior surface of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides; (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen, the first antigen being encapsulated in the nanoparticle core, attached to one or more oligonucleotides within the shell of oligonucleotides through a linker, attached to the exterior surface of the nanoparticle core through a linker, or a combination thereof; and (d) a major histocompatibility complex type II (MHC-II) antigen. and (e) a third antigen, which is a major histocompatibility complex type I (MHC-I) antigen, and which is encapsulated in the nanoparticle core, bound to one or more oligonucleotides in the oligonucleotide shell through a linker, bound to the external surface of the nanoparticle core through a linker, or a combination thereof.

[0055] In some aspects, the disclosure provides a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; (b) a shell of oligonucleotides attached to an exterior surface of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides; (c) a first antigen, which is a major histocompatibility complex type I (MHC-I) antigen, which is encapsulated in the nanoparticle core, attached to one or more oligonucleotides in the oligonucleotide shell through a linker, attached to the exterior surface of the nanoparticle core through a linker, or a combination thereof; and (d) a second antigen, which is a major histocompatibility complex type II (MHC-II) antigen, which is encapsulated in the nanoparticle core, attached to one or more oligonucleotides in the oligonucleotide shell through a linker, or attached to the exterior surface of the nanoparticle core through a linker. (e) a third antigen, which is a major histocompatibility complex type I (MHC-I) antigen, and which is either encapsulated in the nanoparticle core, or is attached to one or more oligonucleotides in the oligonucleotide shell through a linker, or is attached to the external surface of the nanoparticle core through a linker, or a combination thereof; and (f) a fourth antigen, which is a major histocompatibility complex type II (MHC-II) antigen, and which is either encapsulated in the nanoparticle core, or is attached to one or more oligonucleotides in the oligonucleotide shell through a linker, or is attached to the external surface of the nanoparticle core through a linker, or a combination thereof.

[0056] In any of the aspects or embodiments of the present disclosure, a spherical nucleic acid (SNA) comprises: (a) a nanoparticle core; (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen which is a major histocompatibility complex type I (MHC-I) antigen and a second antigen which is a major histocompatibility complex type II (MHC-II) antigen, wherein the first antigen is encapsulated in the nanoparticle core and the second antigen is bound through a linker to an oligonucleotide that hybridizes to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core. In any of the aspects or embodiments of the present disclosure, the method described herein comprises administering to a subject a spherical nucleic acid (SNA) comprising: (a) a nanoparticle core; (b) a shell of oligonucleotides bound to the exterior surface of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen which is a major histocompatibility complex type I (MHC-I) antigen and a second antigen which is a major histocompatibility complex type II (MHC-II) antigen, wherein the first antigen is encapsulated in the nanoparticle core and the second antigen is bound through a linker to an oligonucleotide that hybridizes to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core.

[0057] In any of the aspects or embodiments of the present disclosure, a spherical nucleic acid (SNA) comprises: (a) a nanoparticle core; (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen which is a major histocompatibility complex type I (MHC-I) antigen and a second antigen which is a major histocompatibility complex type II (MHC-II) antigen, wherein the second antigen is encapsulated in the nanoparticle core and the first antigen is bound through a linker to an oligonucleotide that hybridizes to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core. In any of the aspects or embodiments of the present disclosure, the methods described herein include administering to a subject a spherical nucleic acid (SNA), comprising: (a) a nanoparticle core; (b) a shell of oligonucleotides bound to the outer surface of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen, wherein the second antigen is encapsulated in the nanoparticle core and the first antigen is bound through a linker to an oligonucleotide that hybridizes to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core.

[0058] Oligonucleotides The present disclosure provides a spherical nucleic acid (SNA) comprising a nanoparticle core and a shell of oligonucleotides bound to the exterior of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides, a first antigen being a major histocompatibility complex type I (MHC-I) antigen, and a second antigen being a major histocompatibility complex type II (MHC-II) antigen. In various embodiments, about or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the oligonucleotides within the shell of oligonucleotides are immunostimulatory oligonucleotides. In various embodiments, the shell of oligonucleotides comprises inhibitory oligonucleotides, targeting oligonucleotides, non-targeting oligonucleotides, or combinations thereof. Oligonucleotides contemplated for use according to the present disclosure include those bound to the nanoparticle core through any means (e.g., covalent or non-covalent). In various embodiments, the oligonucleotides of the present disclosure include DNA oligonucleotides, RNA oligonucleotides, modified forms thereof, or combinations thereof. In any aspect or embodiment described herein, the oligonucleotide is single-stranded, double-stranded, or partially double-stranded. In any aspect or embodiment of the present disclosure, the oligonucleotide comprises a detectable marker.

[0059] Modified forms of oligonucleotides, including those with at least one modified internucleotidic linkage as described herein, are also contemplated by the present disclosure. In some embodiments, the oligonucleotide is all or partly a peptide nucleic acid. Other modified internucleoside linkages include at least one phosphorothioate linkage. Still other modified oligonucleotides include those that include one or more universal bases. A "universal base" refers to a molecule that can replace a linkage to any one of A, C, G, T, and U in a nucleic acid by forming a hydrogen bond without significant structural destabilization. Oligonucleotides incorporating universal base analogs can function, for example, as probes in hybridization. Examples of universal bases include, but are not limited to, 5'-nitroindole-2'-deoxyriboside, 3-nitropyrrole, inosine, and hypoxanthine.

[0060] The term "nucleotide" or its plural forms as used herein are interchangeable with the variants discussed herein and otherwise known in the art. The term "nucleobase" or its plural forms as used herein are interchangeable with the variants discussed herein and otherwise known in the art. Nucleotides or nucleobases include the naturally occurring nucleobases A, G, C, T, and U. Non-naturally occurring nucleobases include, but are not limited to, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosine, N',N'-ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C3-C6)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, and the "non-naturally occurring" nucleobases described in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freier and Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp 4429-4443. The term "nucleobase" includes the known purine and pyrimidine heterocycles, as well as their heterocyclic analogs and tautomers.Additionally, for naturally occurring and non-naturally occurring nucleobases, see U.S. Pat. No. 3,687,808 (Merigan, et al.), Chapter 15 of Antisense Research and Application, Ed. S.T. Crooke and B. Lebleu, CRC Press, 1993, Englisch et al., 1991, Angewandte Chemie, International Edition, 30:613-722 (see especially pages 622 and 623), and the Concise Encyclopedia of Polymer Science and Engineering, J.I. Kroschwitz Ed., John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design 1991, 6, 585-607, each of which is incorporated herein by reference in its entirety). In various embodiments, oligonucleotides also contain one or more "nucleoside bases" or "base units", which are a category of non-naturally occurring nucleotides that contain compounds such as heterocyclic compounds that are not nucleoside bases in the most classical sense, but can function as nucleoside bases, including certain "universal bases". Universal bases include 3-nitropyrrole, optionally substituted indoles (e.g., 5-nitroindoles), and optionally substituted hypoxanthines. Other desirable universal bases include pyrrole, diazole, or triazole derivatives that contain universal bases known in the art.

[0061] Examples of oligonucleotides include those containing modified backbones or non-natural internucleoside linkages. Oligonucleotides with modified backbones include those that have a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are considered to be within the meaning of "oligonucleotide."

[0062] Modified oligonucleotide backbones containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phototriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs thereof, as well as those having reverse polarity in which one or more internucleotide linkages are 3' to 3', 5' to 5', or 2' to 2' linkages. Oligonucleotides having reverse polarity, including a single 3' to 3' linkage at the 3'-most internucleotide linkage, i.e., a single reverse nucleoside residue, which may be abasic (either missing a nucleotide or having a hydroxyl group in its place), are also contemplated. Salts, mixed salts, and free acid forms are also contemplated. Representative United States patents which teach the preparation of the above phosphorus-containing linkages include U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, Nos. 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361, 5,194,599, 5,565,555, 5,527,899, 5,721,218, 5,672,697 and 5,625,050, the disclosures of which are incorporated herein by reference.

[0063] Modified oligonucleotide backbones that do not contain a phosphorus atom therein have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include backbones with morpholino linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and methylenethioformacetyl backbones; riboacetyl backbones; alkene-containing backbones; sulfamic acid backbones; methyleneimino and methylenehydrazino backbones; sulfonic acid and sulfonamide backbones; amide backbones; and other backbones containing a mixture of N, O, S and CH2 constituent moieties. See, for example, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, 5,59 See Nos. 6,086, 5,602,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, 5,792,608, 5,646,269 and 5,677,439, the disclosures of which are incorporated herein by reference.

[0064] In yet further embodiments, there are oligonucleotide mimetics in which one or more sugars and / or one or more internucleotide bonds of nucleotide units are both replaced with "non-naturally occurring" groups. The bases of the oligonucleotide are maintained for hybridization. In some aspects, this embodiment contemplates peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced with an amide-containing backbone. See, for example, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, and Nielsen et al., Science, 1991, 254, 1497-1500, the disclosures of which are incorporated herein by reference.

[0065] In yet further embodiments, oligonucleotides having phosphorothioate backbones and oligonucleosides having heteroatom backbones are provided, including -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2-, -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -ON(CH3)-CH2-CH2- as described in U.S. Patent Nos. 5,489,677 and 5,602,240. Oligonucleotides having morpholino backbone structures as described in U.S. Patent No. 5,034,506 are also contemplated.

[0066] In various forms, the bond between two consecutive monomers in an oligonucleotide can be -CH2-, -O-, -S-, -NR H -, >C=O, >C=NR H , >C=S, -Si(R”)2-, -SO-, -S(O)2-, -P(O)2-, -PO(BH3)-, -P(O,S)-, -P(S)2-, -PO(R”)-, -PO(OCH3)-, and -PO(NHR H ) , wherein R H is hydrogen and C 1-4 -alkyl, R″ is selected from C 1-6Illustrative examples of such bonds are selected from -CH-CH-CH-, -CH-CO-CH-, -CH-CHOH-CH-, -O-CH-O-, -O-CH-CH-, -O-CH-CH=(R 5 including -CH2-CH2-O-, -NR H -CH2-CH2-, -CH2-CH2-NR H -, -CH2-NR H -CH2--, -O-CH2-CH2-NR H -, -NR H -CO-O-, -NR H -CO-NR H -, -NR H -CS-NR H -, -NR H -C(=NR H )-NR H -, -NR H -CO-CH2-NR H -O-CO-O-, -O-CO-CH2-O-, -O-CH2-CO-O-, -CH2-CO-NR H -, -O-CO-NR H -, -NR H -CO-CH2-, -O-CH2-CO-NR H -, -O-CH2-CH2-NR H -, -CH=NO-, -CH2-NR H -O-, -CH2-ON = (R when used as a bond to a subsequent monomer) 5 including -CH2-O-NR H -, -CO-NR H -CH2-, -CH2-NR H -O-, -CH2-NR H -CO-, -O-NR H -CH2-, -O-NR H , -O-CH2-S-, -S-CH2-O-, -CH2-CH2-S-, -O-CH2-CH2-S-, -S-CH2-CH=(when used as a bond to a subsequent monomer R 5including), -S-CH2-CH2-, -S-CH2-CH2--O-, -S-CH2-CH2-S-, -CH2-S-CH2-, -CH2-SO-CH2-, -CH2-SO2-CH2-, -O-SO-O-, -O-S(O)2-O-, -O-S(O)2-CH2-, -O-S(O)2-NR H -, -NR H -S(O)2-CH2-; -O-S(O)2-CH2-, -O-P(O)2-O-, -O-P(O,S)-O-, -O-P(S)2-O-, -S-P(O)2-O-, -S-P(O,S)-O-, -S-P(S)2-O-, -O-P(O)2-S-, -O-P(O,S)-S-, -O-P(S)2-S-, -S-P(O)2-S-, -S-P(O,S)-S-, -S-P(S)2-S-, -O-PO(R”)-O-, -O-PO(OCH3)-O-, -O-PO(O CH2CH3)-O-, -O-PO(O CH2CH2S-R)-O-, -O-PO(BH3)-O-, -O-PO(NHR N )-O-, -O-P(O)2-NR H H-, -NR H -P(O)2-O-, -O-P(O,NR H )-O-,-CH2-P(O)2-O-, -O-P(O)2-CH2-, and -O-Si(R”)2-O-, and among these, -CH2-CO-NR H -, -CH2-NR H -O-, -S-CH2-O-, -O-P(O)2-O-O-P(-O,S)-O-, -O-P(S)2-O-, -NR H P(O)2-O-, -O-P(O,NR H )-O-, -O-PO(R”)-O-, -O-PO(CH3)-O-, and -O-PO(NHR N )-O- (wherein R H is selected from hydrogen and C 1-4 -alkyl, and R” is C 1-6-alkyl and phenyl) are contemplated. Further examples are provided in Mesmaeker et.al., Current Opinion in Structural Biology 1995, 5, 343-355 and Susan M. Freier and Karl-Heinz Altmann, Nucleic Acids Research, 1997, vol 25, pp 4429-4443.

[0067] Still other modified forms of oligonucleotides are described in detail in US Patent Application Publication No. 2004 / 0219565, the disclosure of which is incorporated herein by reference in its entirety.

[0068] Modified oligonucleotides may also contain one or more substituted sugar moieties. In certain embodiments, the oligonucleotide comprises one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl or alkynyl. In another embodiment, O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON [(CH2) n CH3]2, where n and m are from 1 to about 10. Other oligonucleotides include one of the following at the 2' position: C1 to C 10lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, or an RNA cleaving group. In one aspect, the modification includes 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O-CH2CHOCH3) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., an alkoxyalkoxy group. Other modifications include the group 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3), also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2.

[0069] Still other modifications include 2'-methoxy (2'-O-CH), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-allyl (2'-CH2-CH=CH2), 2'-O-allyl (2'-O-CH2-CH=CH2), and 2'-fluoro (2'-F). The 2' modification may be at the arabino (up) or ribo (down) position. In one aspect, the 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, for example, the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and at the 5' position of the 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. See, for example, U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,576,427, 5,591,7 22, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, 5,792,747, and 5,700,920, the disclosures of which are incorporated herein by reference in their entireties.

[0070] In some embodiments, sugar modifications include Locked Nucleic Acids (LNAs), in which a 2'-hydroxyl group is attached to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. In certain embodiments, the linkage is a methylene (-CH2-) bridging the 2' oxygen atom and the 4' carbon atom. n where n is 1 or 2. LNAs and their preparation are described in WO98 / 39352 and WO99 / 14226.

[0071] Modified nucleotides are described in EP 1072679 and WO 97 / 12896, the disclosures of which are incorporated herein by reference. Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (succinimide), ... douracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases can also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, Englisch et al., 1991, Angewandte Chemie, International Edition, 30:613, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993. Some of these bases are useful for increasing binding affinity and include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C, and in certain embodiments are combined with 2'-O-methoxyethyl sugar modifications. See U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, and 5,55 See, for example, Nos. 2,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,830,653, 5,763,588, 6,005,096, 5,750,692, and 5,681,941, the disclosures of which are incorporated herein by reference.

[0072] Methods for making polynucleotides of a given sequence are well known. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press, New York, 1991). Solid-phase synthesis methods are suitable for both polyribonucleotides and polydeoxyribonucleotides (the well-known methods for DNA synthesis are also useful for RNA synthesis). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can also be incorporated into polynucleotides. See, e.g., U.S. Pat. No. 7,223,833; Katz, J. Am. Chem. Soc., 74:2238 (1951); Yamane, et al., J. Am. Chem. Soc., 83:2599 (1961); Kosturko, et al., Biochemistry, 13:3949 (1974); Thomas, J. Am. Chem. Soc., 76:6032 (1954); Zhang, et al., J. Am. Chem. Soc., 127:74-75 (2005); and Zimmermann, et al., J. Am. Chem. Soc., 124:13684-13685 (2002).

[0073] In various embodiments, the oligonucleotides of the present disclosure or modified forms thereof are generally about 5 to about 1000 nucleotides in length. More specifically, the oligonucleotides of the present disclosure may be about 5 to about 1000 nucleotides in length, about 5 to about 900 nucleotides in length, about 5 to about 800 nucleotides in length, about 5 to about 700 nucleotides in length, about 5 to about 600 nucleotides in length, about 5 to about 500 nucleotides in length, about 5 to about 450 nucleotides in length, about 5 to about 400 nucleotides in length, about 5 to about 350 nucleotides in length, about 5 to about 300 nucleotides in length, about 5 to about 250 nucleotides in length, about 5 to about 200 nucleotides in length, about 5 to about 150 nucleotides in length, about 5 to about 100 nucleotides in length, or about 5 to about 150 nucleotides in length. , about 5 to about 90 nucleotides long, about 5 to about 80 nucleotides long, about 5 to about 70 nucleotides long, about 5 to about 60 nucleotides long, about 5 to about 50 nucleotides long, about 5 to about 45 nucleotides long, about 5 to about 40 nucleotides long, about 5 to about 35 nucleotides long, about 5 to about 30 nucleotides long, about 5 to about 25 nucleotides long, about 5 to about 20 nucleotides long, about 5 to about 15 nucleotides long, about 5 to about 10 nucleotides long, about 10 to about 1000 nucleotides long, about 10 to about 900 nucleotides long, about 10 to about 800 nucleotides long nucleotide length, about 10 to about 700 nucleotides, about 10 to about 600 nucleotides, about 10 to about 500 nucleotides, about 10 to about 450 nucleotides, about 10 to about 400 nucleotides, about 10 to about 350 nucleotides, about 10 to about 300 nucleotides, about 10 to about 250 nucleotides, about 10 to about 200 nucleotides, about 10 to about 150 nucleotides, about 10 to about 100 nucleotides, about 10 to about 90 nucleotides, about 10 to about 80 nucleotides, about 10 to about 70 nucleotides , about 10 to about 60 nucleotides in length, about 10 to about 50 nucleotides in length, about 10 to about 45 nucleotides in length, about 10 to about 40 nucleotides in length, about 10 to about 35 nucleotides in length, about 10 to about 30 nucleotides in length, about 10 to about 25 nucleotides in length, about 10 to about 20 nucleotides in length, about 10 to about 15 nucleotides in length, about 18 to about 28 nucleotides in length, about 15 to about 26 nucleotides in length, and all oligonucleotide lengths intermediate to the specifically disclosed sizes such that the oligonucleotide achieves the desired result.In further embodiments, the oligonucleotides of the disclosure are from about 5 to about 100 nucleotides in length, from about 5 to about 90 nucleotides in length, from about 5 to about 80 nucleotides in length, from about 5 to about 70 nucleotides in length, from about 5 to about 60 nucleotides in length, from about 5 to about 50 nucleotides in length, from about 5 to about 40 nucleotides in length, from about 5 to about 30 nucleotides in length, from about 5 to about 20 nucleotides in length, from about 5 to about 10 nucleotides in length, and any oligonucleotide length intermediate the specifically disclosed sizes such that the oligonucleotide achieves the desired result. Thus, in various embodiments, the oligonucleotides of the disclosure comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides in length. In further embodiments, the oligonucleotides of the disclosure are 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 , 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more nucleotides in length.In various embodiments, the shell of oligonucleotides bound to the exterior of the SNA nanoparticle core comprises a plurality of oligonucleotides all having the same length / sequence, and in some embodiments, the plurality of oligonucleotides comprises one or more oligonucleotides having a different length and / or sequence relative to at least one other oligonucleotide in the plurality. For example, and not by way of limitation, in some embodiments, the shell of oligonucleotides comprises a plurality of immunostimulatory oligonucleotides, where one immunostimulatory oligonucleotide has a different sequence from at least one other immunostimulatory oligonucleotide in the plurality.

[0074] In some embodiments, one or more of the oligonucleotides in the shell of oligonucleotides is (GGX) n In some embodiments, the nucleic acid comprises or consists of a nucleotide sequence, in which n is 2 to 20 and X is a nucleobase (A, C, T, G, or U). n The nucleotide sequence is at the 5' end of one or more oligonucleotides. In some embodiments, (GGX) n The nucleotide sequence is at the 3' end of one or more of the oligonucleotides. In some embodiments, one or more of the oligonucleotides within the shell of oligonucleotides has the sequence (GGT) n In some embodiments, the sequence comprises or consists of a nucleotide sequence, where n is between 2 and 20. n The nucleotide sequence is at the 5' end of one or more of the oligonucleotides. In some embodiments, the nucleotide sequence is (GGT) n The nucleotide sequence is at the 3' terminus of one or more oligonucleotides.

[0075] In some embodiments, the oligonucleotides in the shell of oligonucleotides are targeting oligonucleotides, such as aptamers. Thus, all the features and aspects of oligonucleotides described herein (e.g., length, type (DNA, RNA, modified forms thereof), optional presence of spacers) also apply to aptamers. Aptamers are oligonucleotide sequences that can be evolved to bind to various target analytes of interest. Aptamers can be single-stranded, double-stranded, or partially double-stranded.

[0076] Spacer. In some aspects and embodiments, one or more oligonucleotides in the shell of the oligonucleotides that are bound to the nanoparticle core of the SNA comprise a spacer. As used herein, "spacer" refers to a moiety that serves to increase the distance between the nanoparticle core and the oligonucleotide, or to increase the distance between individual oligonucleotides when bound to the nanoparticle core in multiple copies, or to improve the synthesis of the SNA. Thus, it is contemplated that the spacer is located between the oligonucleotide and the nanoparticle core.

[0077] In some aspects, the spacer, when present, is an organic moiety. In some aspects, the spacer is a polymer, including but not limited to a water-soluble polymer, a nucleic acid, a polypeptide, an oligosaccharide, a carbohydrate, a lipid, ethyl glycol, or a combination thereof. In any of the aspects or embodiments of the present disclosure, the spacer is an oligo(ethylene glycol)-based spacer. In various embodiments, the oligonucleotide comprises one, two, three, four, five, or more spacer (e.g., spacer-18 (hexaethylene glycol)) moieties. In further embodiments, the spacer is an alkane-based spacer (e.g., C12). In some embodiments, the spacer is an oligonucleotide spacer (e.g., T5). The oligonucleotide spacer can have any sequence that does not interfere with the ability of the oligonucleotide to become bound to the nanoparticle core or the target. In certain aspects, the bases of the oligonucleotide spacer are all adenylic acids, all thymidylic acids, all cytidylic acids, all guanylic acids, all uridylic acids, or all some other modified base.

[0078] In various embodiments, the length of the spacer is or is equal to at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, 5-10 nucleotides, 10 nucleotides, 10-30 nucleotides, or even more than 30 nucleotides.

[0079] SNA surface density. Generally, at least about 0.5 pmole / cm 2 is suitable to provide a stable SNA. In a further embodiment, an oligonucleotide surface density of at least about 1 pmol / cm 2 , 1.5 pmol / cm 2 , or 2 pmoles / cm 2 is suitable for providing a stable SNA (e.g., LSNA or LNP-SNA). In some embodiments, the surface density of the SNA of the present disclosure is at least 15 pmoles / cm 2In addition, the oligonucleotide is about 0.5 pmol / cm 2 ~about 1000pmol / cm 2 , or about 2 pmol / cm 2 ~about 200pmol / cm 2 , or about 10 pmol / cm 2 ~about 100pmol / cm 2 Also provided are methods in which the SNA is bound to the nanoparticle core at a surface density of about 1.7 pmol / cm. In some embodiments, the surface density is about 1.7 pmol / cm. 2 In some embodiments, the surface density is about 2 pmol / cm 2 In a further embodiment, the surface density is at least about 0.5 pmol / cm 2 , at least about 0.6 pmol / cm 2 , at least about 0.7 pmol / cm 2 , at least about 0.8 pmol / cm 2 , at least about 0.9 pmol / cm 2 , at least about 1 pmol / mc 2 , at least about 1.5 pmol / cm 2 , at least about 2 pmol / cm 2 , at least 3 pmol / cm 2 , at least 4 pmol / cm 2 , at least 5 pmol / cm 2 , at least 6 pmol / cm 2 , at least 7 pmol / cm 2 , at least 8 pmol / cm 2 , at least 9 pmol / cm 2 , at least 10 pmol / cm 2 , at least about 15 pmol / cm 2 , at least about 19 pmol / cm 2 , at least about 20 pmol / cm 2 , at least about 25 pmol / cm 2 , at least about 30 pmol / cm 2 , at least about 35 pmol / cm 2 , at least about 40 pmol / cm 2 , at least about 45 pmol / cm 2, at least about 50 pmol / cm 2 , at least about 55 pmol / cm 2 , at least about 60 pmol / cm 2 , at least about 65 pmol / cm 2 , at least about 70 pmol / cm 2 , at least about 75 pmol / cm 2 , at least about 80 pmol / cm 2 , at least about 85 pmol / cm 2 , at least about 90 pmol / cm 2 , at least about 95 pmol / cm 2 , at least about 100 pmol / cm 2 , at least about 125 pmol / cm 2 , at least about 150 pmol / cm 2 , at least about 175 pmol / cm 2 , at least about 200 pmol / cm 2 , at least about 250 pmol / cm 2 , at least about 300 pmol / cm 2 , at least about 350 pmol / cm 2 , at least about 400 pmol / cm 2 , at least about 450 pmol / cm 2 , at least about 500 pmol / cm 2 , at least about 550 pmol / cm 2 , at least about 600 pmol / cm 2 , at least about 650 pmol / cm 2 , at least about 700 pmol / cm 2 , at least about 750 pmol / cm 2 , at least about 800 pmol / cm 2 , at least about 850 pmol / cm 2 , at least about 900 pmol / cm 2 , at least about 950 pmol / cm 2 , at least about 1000 pmol / cm 2 In a further embodiment, the surface density is about 2 pmol / cm 2 Less than 3 pmol / cm 2Less than 4 pmol / cm 2 Less than 5 pmol / cm 2 Less than 6 pmol / cm 2 Less than 7 pmol / cm 2 Less than 8 pmol / cm 2 Less than 9 pmol / cm 2 Less than 10 pmol / cm 2 Less than 15 pmol / cm 2 Less than 19 pmol / cm 2 Less than 20 pmol / cm 2 Less than about 25 pmol / cm 2 Less than 30 pmol / cm 2 Less than 35 pmol / cm 2 Less than 40 pmol / cm 2 Less than 45 pmol / cm 2 Less than 50 pmol / cm 2 Less than 55 pmol / cm 2 Less than 60 pmol / cm 2 Less than 65 pmol / cm 2 Less than 70 pmol / cm 2 Less than about 75 pmol / cm 2 Less than 80 pmol / cm 2 Less than 85 pmol / cm 2 Less than 90 pmol / cm 2 Less than 95 pmol / cm 2 Less than 100 pmol / cm 2 Less than about 125 pmol / cm 2 Less than 150 pmol / cm 2 Less than about 175 pmol / cm 2 Less than about 200 pmol / cm 2 Less than about 250 pmol / cm 2 Less than 300 pmol / cm 2 Less than about 350 pmol / cm 2 Less than 400 pmol / cm 2 Less than 450 pmol / cm 2 Less than about 500 pmol / cm 2 Less than about 550 pmol / cm 2 Less than 600 pmol / cm 2Less than 650 pmol / cm 2 Less than 700 pmol / cm 2 Less than about 750 pmol / cm 2 Less than 800 pmol / cm 2 Less than about 850 pmol / cm 2 Less than 900 pmol / cm 2 Less than 950 pmol / cm 2 , or about 1000 pmol / cm 2 is less than.

[0080] Alternatively, the density of oligonucleotides bound to the SNA is measured by the number of oligonucleotides bound to the SNA. With respect to the surface density of oligonucleotides bound to the SNA of the present disclosure, it is contemplated that the SNA described herein comprises or consists of about 1 to about 2,500 or about 1 to about 500 oligonucleotides on its surface. In various embodiments, the SNA comprises about 10 to about 500, or about 10 to about 300, or about 10 to about 200, or about 10 to about 190, or about 10 to about 180, or about 10 to about 170, or about 10 to about 160, or about 10 to about 150, or about 10 to about 140, or about 10 to about 130, or about 10 to about 12 ...50, or about 10 to about 160, or about 10 to about 170, or about 10 to about 180, or about 10 to about 190, or about 10 to about 190, or about 10 to about 200, or about 10 to about 200, or about 10 to about 200, or about 10 to about 200, or about 10 to about 200, or about 10 to about 300, or about 10 to about 300, or about 10 to about 400, or about 10 to about 400, or about 10 to about 500, or about 10 to about 500, or about 10 to about 600, or about 10 comprises about 10 to about 110, or about 10 to about 100, or about 10 to about 90, or about 10 to about 80, or about 10 to about 70, or about 10 to about 60, or about 10 to about 50, or about 10 to about 40, or about 10 to about 30, or about 10 to about 20, or about 75 to about 200, or about 75 to about 150, or about 100 to about 200, or about 150 to about 200. In some embodiments, the SNA comprises about 80 to about 140 oligonucleotides within a shell of oligonucleotides bound to the nanoparticle core. In further embodiments, the SNA comprises at least about 5, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 oligonucleotides within the shell of oligonucleotides bound to the nanoparticle core. In further embodiments, the SNA consists of 5, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 oligonucleotides within a shell of oligonucleotides bound to a nanoparticle core.In still further embodiments, the shell of oligonucleotides attached to the nanoparticle core of the SNA comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 60, 70, 75, 80, 90, 100, 150, 160, 170, 175, 180, 190, 200 or more oligonucleotides. In some embodiments, the shell of oligonucleotides attached to the nanoparticle core of the SNA consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 75, 80, 90, 100, 150, 160, 170, 175, 180, 190, or 200 oligonucleotides. In some embodiments, the oligonucleotide shell comprises from about 10 to about 80 oligonucleotides, in some embodiments, the oligonucleotide shell comprises or consists of about 75 oligonucleotides.

[0081] Linkers. The present disclosure provides compositions and methods in which an antigen is associated and / or attached to the surface of an SNA via a linker. In various embodiments, the linker can be a cleavable linker, a non-cleavable linker, a traceless linker, and combinations thereof.

[0082] The linker attaches the antigen to the oligonucleotide within the disclosed SNA or to the surface of the SNA (i.e., antigen-linker-oligonucleotide or antigen-linker). The oligonucleotide can hybridize to another oligonucleotide attached to the SNA or can be attached directly to the SNA (e.g., via a lipid anchor group). Some specifically contemplated linkers include carbamate alkylene, carbamate alkylene aryl disulfide linker, amido alkylene disulfide linker, amido alkylene aryl disulfide linker, thiol linker, and amido alkylene succinimidyl linker. In some cases, the linker is -NH-C(O)-OC 2-5 Alkylene-SSC 2-7Alkylene- or -NH-C(O)-C 2-5 Alkylene-SSC 2-7 The carbon alpha to the -SS- moiety can be branched, e.g., -CHX-SS- or -SS-CHY-, or combinations thereof, where X and Y are independently Me, Et, or iPr. The carbon alpha to the antigen can be branched, e.g., -CHX-C 2-4 alkylene-SS-, where X is Me, Et, or iPr. In some cases, the linker is -NH-C(O)-O-CH2-Ar-SSC 2-7 alkylene- and Ar is a meta or para substituted phenyl. 2-4 Alkylene-N-succinimidyl-SC 2-6 It is alkylene.

[0083] Additional linkers include SH, SM, SE, and SI linkers. The present disclosure contemplates multiple attachment points available for modulating antigen release (e.g., disulfide cleavage, linker cyclization, and dehybridization), allowing control of the kinetics of antigen release at each attachment point. For example, the steric bulk of a disulfide can be increased by S N 2 can slow down the rate of the reaction, increasing the length of the alkyl spacer or the steric bulk attached to the alkyl spacer can affect the rate of ring closure, and mismatched nucleotide sequences can increase the melting temperature (T m ), whereas locked nucleic acids reduce T m Increase the

[0084] Use of SNAs in immune modulation Toll-like receptors (TLRs) are a class of proteins expressed on sentinel cells and play a key role in regulating the innate immune system. The mammalian immune system uses two general strategies to fight infectious diseases. Exposure to pathogens rapidly triggers an innate immune response characterized by the production of immunostimulatory cytokines, chemokines, and polyreactive IgM antibodies. The innate immune system is activated by exposure to pathogen-associated molecular patterns (PAMPs) expressed by a diverse group of infectious microorganisms. Recognition of PAMPs is mediated by members of the Toll-like family of receptors. TLR receptors, such as TLR8 and TLR9, which respond to specific oligonucleotides, are located within specialized intracellular compartments called endosomes. Regulatory mechanisms of TLR8 and TLR9 receptors are based on, for example, but not limited to, DNA-protein interactions.

[0085] As described herein, synthetic immunostimulatory oligonucleotides containing CpG motifs similar to those found in bacterial DNA stimulate similar responses of TLR receptors. Thus, the CpG oligonucleotides of the present disclosure have the ability to function as TLR agonists. Other TLR agonists contemplated by the present disclosure include, but are not limited to, single-stranded RNA and small molecules (e.g., R848 (resiquimod)). Thus, immunomodulatory (e.g., immunostimulatory) oligonucleotides have a variety of promising therapeutic applications, including the treatment of diseases (e.g., cancer).

[0086] Thus, in some embodiments, a method is disclosed that utilizes the SNAs described herein to modulate toll-like receptors. The method upregulates toll-like receptor activity through the use of TLR agonists. The method includes contacting a cell having a toll-like receptor with the SNA of the present disclosure, thereby modulating the activity and / or expression of the toll-like receptor. The toll-like receptors that are modulated include one or more of toll-like receptor 1, toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 11, toll-like receptor 12, and / or toll-like receptor 13.

[0087] Methods for inducing an immune response The present disclosure also includes methods for eliciting an immune response in a subject in need thereof, comprising administering to the subject an effective amount of a SNA of the present disclosure (e.g., formulated as an antigenic composition). In various embodiments, administering to the subject a SNA of the present disclosure (e.g., formulated as a composition, pharmaceutical formulation, or antigenic composition) results in an increase in the amount of neutralizing antibodies against the antigen(s) produced in the subject compared to the amount of neutralizing antibodies against the antigen(s) produced in a subject who was not administered the SNA. In further embodiments, the increase is a 2-fold increase, a 5-fold increase, a 10-fold increase, a 50-fold increase, a 100-fold increase, a 200-fold increase, a 500-fold increase, a 700-fold increase, or a 1000-fold increase.

[0088] In further embodiments, the SNAs of the disclosure activate human peripheral blood mononuclear cells to generate an antibody response against one or more antigens described herein. In some embodiments, the antibody response is a total antigen-specific antibody response. In further embodiments, administering a SNA of the disclosure (e.g., formulated as a composition, pharmaceutical preparation, or antigenic composition) to a subject results in an increase in the amount of total antigen-specific antibodies against the antigen(s) produced in the subject compared to the amount of total antigen-specific antibodies against the antigen(s) produced in a subject that was not administered the SNA. In further embodiments, the increase is a 2-fold increase, a 5-fold increase, a 10-fold increase, a 50-fold increase, a 100-fold increase, a 200-fold increase, a 500-fold increase, a 700-fold increase, or a 1000-fold increase. A "total antigen-specific antibody response" is a measure of all of the antibodies (including neutralizing and non-neutralizing antibodies) that bind to a particular antigen.

[0089] The immune response elicited by the methods of the present disclosure generally includes antibody responses, preferably neutralizing antibody responses, T and B cell maturation and memory, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and CD4 + , CD8 +The immune response generated by the SNAs disclosed herein generates an immune response and preferably treats a disease (e.g., cancer) described herein. Methods for evaluating antibody responses following administration (immunization or vaccination) of an antigenic composition are known in the art and / or described herein. In some embodiments, the immune response includes a T cell mediated response (e.g., a peptide-specific response such as a proliferative response or a cytokine response). In any of the aspects or embodiments of the present disclosure, the immune response includes both a B cell response and a T cell response. The antigenic composition may be administered in a number of suitable ways, such as intramuscular injection, subcutaneous injection, intradermal administration, and mucosal administration, e.g., orally or intranasally. Additional modes of administration include, but are not limited to, intravenous, intraperitoneal, intranasal, intravaginal, intrarectal, and oral administration. Combinations of different routes of administration in the subject to be immunized, e.g., simultaneous intramuscular and intranasal administration, are also contemplated by the present disclosure.

[0090] Administration may include a single dose or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and / or a booster immunization schedule. In a multiple dose schedule, various doses may be given by the same or different routes, for example, parenteral prime and mucosal boost, mucosal prime and parenteral boost, or subcutaneous prime and subcutaneous boost. Administration of more than one dose (typically two doses) is particularly useful in immunologically naive subjects or subjects in low-responder populations, such as diabetic patients, or subjects with chronic kidney disease (e.g., dialysis patients). In various embodiments, the second dose is administered about or at least about two weeks after the first dose. Multiple doses are typically administered at least one week apart (e.g., about two weeks, about three weeks, about four weeks, about six weeks, about eight weeks, about ten weeks, about twelve weeks, or about sixteen weeks). In some embodiments, multiple doses are administered 1, 2, 3, 4, or 5 months apart. The antigenic compositions of the present disclosure may be administered to a patient substantially simultaneously with other vaccines (eg, during the same medical consultation or visit to a health care professional).

[0091] Use of LNP-SNA to treat disorders In some aspects, the SNAs of the present disclosure are used to treat disorders. Thus, in some aspects, the present disclosure provides a method of treating a disorder, comprising administering an effective amount of the SNAs of the present disclosure to a subject (e.g., a human subject) in need thereof, thereby treating the disorder. In some aspects, the present disclosure provides a method of treating cancer, comprising administering to a subject an effective amount of the SNAs of the present disclosure, thereby treating the cancer in the subject. In various embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, glioblastoma, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, lymphoma, non-Hodgkin's lymphoma, osteocarcinoma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and human papilloma virus-induced cancer, or a combination thereof.

[0092] Additional drugs In any of the aspects or embodiments of the present disclosure, the additional agent is administered separately from the SNA of the present disclosure. Thus, in various embodiments, the therapeutic agent is administered before, after, or simultaneously with the SNA of the present disclosure to treat a disorder (e.g., cancer).

[0093] In some aspects, the SNAs provided herein optionally further comprise an additional agent or agents. The additional agent, in various embodiments, is simply associated with an oligonucleotide in the shell of the oligonucleotide bound to the exterior of the nanoparticle core of the SNA, and / or the additional agent is associated with the nanoparticle core of the SNA, and / or the additional agent is encapsulated in the nanoparticle core of the SNA. In some embodiments, the additional agent is associated with the terminus of the oligonucleotide in the shell of the oligonucleotide that is not bound to the nanoparticle core (e.g., if the oligonucleotide is bound to the nanoparticle core through its 3' terminus, the additional agent is associated with the 5' terminus of the oligonucleotide). Alternatively, in some embodiments, the additional agent is associated with the terminus of the oligonucleotide in the shell of the oligonucleotide that is bound to the nanoparticle core (e.g., if the oligonucleotide is bound to the nanoparticle core through its 3' terminus, the additional agent is associated with the 3' terminus of the oligonucleotide). In some embodiments, the additional agent is covalently associated with an oligonucleotide in the shell of the oligonucleotide that is bound to the exterior of the nanoparticle core of the SNA. In some embodiments, the additional agent is non-covalently associated with an oligonucleotide in the shell of the oligonucleotide that is bound to the exterior of the nanoparticle core of the SNA. However, it should be understood that the present disclosure provides SNAs in which one or more additional agents are both covalently and non-covalently associated with oligonucleotides within a shell of oligonucleotides bound to the exterior of the lipid nanoparticle core of the SNA, and it will also be understood that non-covalent association includes hybridization, protein binding, and / or hydrophobic interactions.

[0094] Additional agents contemplated by the present disclosure include, but are not limited to, a protein (e.g., a therapeutic protein), a growth factor, a hormone, an interferon, an interleukin, an antibody or antibody fragment, a small molecule, a peptide, an antibiotic, an antifungal agent, an antiviral agent, a chemotherapeutic agent, or a combination thereof. In some embodiments, the additional agent is an anti-programmed cell death protein 1 (PD-1) antibody.

[0095] The term "small molecule" as used herein refers to a chemical compound or drug, or any other low molecular weight organic compound, either natural or synthetic. By "low molecular weight" we mean a compound having a molecular weight of less than 1500 Daltons, typically between 100 and 700 Daltons.

[0096] Use of SNAs in gene regulation In some aspects of the disclosure, oligonucleotides associated with the SNAs of the disclosure (e.g., LNP-SNAs, LSNAs) inhibit gene expression. Thus, in some embodiments, the SNAs perform both a vaccine function and a gene-inhibiting function. In such aspects, the shell of oligonucleotides bound to the exterior surface of the nanoparticle core comprises one or more immunostimulatory oligonucleotides and one or more inhibitory oligonucleotides designed to inhibit target gene expression.

[0097] Methods of inhibiting expression of a gene product include those in which expression of the target gene product is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% compared to expression of the gene product in the absence of SNA. In other words, the methods provided encompass essentially any degree of inhibition of expression of the target gene product.

[0098] The degree of inhibition is determined in vivo from bodily fluid or biopsy samples, or by imaging techniques well known in the art, or, alternatively, in cell culture assays as a predictable measure of the degree of inhibition that can generally be expected in vivo resulting from the use of a particular type of SNA and a particular oligonucleotide.

[0099] In various embodiments, the methods involve the use of inhibitory oligonucleotides that are 100% complementary, i.e., a perfect match, to the target oligonucleotide, while in other embodiments the oligonucleotide is at least (meaning greater than or equal to) about 95% complementary to the polynucleotide over the length of the oligonucleotide, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20% complementary to the polynucleotide over the length of the oligonucleotide to the extent that the oligonucleotide is capable of achieving the desired degree of inhibition of the target gene product.

[0100] Percent complementarity is determined over the length of the oligonucleotide. For example, consider an antisense compound in which 18 of the 20 nucleotides of an inhibitory oligonucleotide are complementary to a 20 nucleotide region of a target polyoligonucleotide with a total length of 100 nucleotides, and the oligonucleotide will be 90 percent complementary. In this example, the remaining non-complementary nucleotides can be clustered or interspersed with complementary nucleotides, and do not have to be adjacent to each other or to complementary nucleobases. Percent complementarity of an inhibitory oligonucleotide with a region of a target nucleic acid can be routinely determined using BLAST (basic local alignment search tool) and PowerBLAST programs (Altschul et al., J.Mol.Biol., 1990,215,403-410; Zhang and Madden, Genome Res., 1997,7,649-656) known in the art.

[0101] The oligonucleotides utilized in such methods are either RNA or DNA. The RNA may be an inhibitory oligonucleotide, such as an inhibitory RNA (RNAi) that performs a regulatory function, and in various embodiments is selected from the group consisting of small inhibitory RNA (siRNA), single-stranded RNA (ssRNA), and ribozymes. Alternatively, the RNA is a microRNA that performs a regulatory function. The DNA, in some embodiments, is antisense-DNA. In some embodiments, the RNA is a piwi-interacting RNA (piRNA).

[0102] The following examples are provided merely to illustrate the present disclosure and not to limit its scope. EXAMPLES

[0103] Example 1 Methods for synthesizing SNA of the present disclosure. In some embodiments, multi-antigen targeted SNA vaccines are synthesized using liposome cores. Lipid films containing DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) are hydrated with PBS containing dissolved peptide antigens. They are then extruded and dialyzed overnight to remove excess unencapsulated peptides and characterized using DLS, Pierce assay (for peptide quantification), and PC assay (for lipid quantification). Separately, peptide-conjugated complementary DNA (to adjuvant) is synthesized. This process can take many chemical reactions, but involves disulfide interactions between the peptide (method 1-cysteine, or method 2-linker conjugation) and complementary DNA (through thiol-terminated treatment). In the case of cysteine ​​(method 1), the peptide is first activated with aldrithiol at room temperature for at least 1 hour. This product is then mixed overnight with thiol-terminated complementary DNA at a molar ratio of 5:1. In method 2, the N-terminus of the peptide on the resin is reacted with an NHS ester-terminated linker overnight at room temperature. The product is cleaved from the resin, deprotected, and purified prior to conjugation with thiol-terminated complementary DNA at a molar ratio of 10:1. In both methods, the final product is purified through polyacrylamide gel electrophoresis and analyzed using ESI. The purified product is mixed 1:1 with cholesterol-terminated DNA adjuvant and lyophilized. It is resuspended in duplex buffer and slowly cooled to duplex (70°C for 10 min, 25°C for 1.5 h, 4°C overnight). The duplex is then mixed with the liposomally encapsulated peptide in an equimolar ratio and the liposomes are reloaded (up to 75 strands per liposome) to form the SNA.

[0104] Example 2 Cancer vaccines must activate multiple immune cell types to be effective against aggressive tumors, but design considerations for these multi-targeted vaccines have not been fully validated. Herein, we demonstrate the impact of structural presentation of multiple antigenic peptides on immune responses at the transcriptomic, cellular, and organismal levels. Using spherical nucleic acid (SNA) nanoconstructs, we investigated differences in antigen processing, cytokine production, and memory derived from the spatial distribution and nanoscale arrangement of two antigen classes to activate two T cell types. Compared with two single antigen SNAs, a single double antigen SNA (DA-SNA) induced a 30% and 2-fold increase in antigen-specific T cell activation and proliferation, respectively. Antigen positioning within the DA-SNA altered immunological gene expression and tumor growth. Encapsulated helper T cell antigen and externally conjugated cytotoxic T cell antigen (referred to as DA-SNA2) elevated antitumor gene pathways and stalled tumors in lymphoma mice. When combined with an anti-PD-1 checkpoint inhibitor in clinically relevant melanoma, DA-SNA2 suppressed tumors and increased circulating T cell memory. This example demonstrates the importance of implementing the structural control afforded by modular nanoscale architectures to synthesize multi-antigen vaccines with improved efficacy.

[0105] Vaccination is an attractive strategy for cancers that express targetable tumor-associated antigens and neo-antigens. For melanoma, there are increasing efforts to develop vaccines targeting identified tumor-associated proteins (e.g., gp100, MAGE-A3, MART-1, NY-ESO-1). 1-4 However, although these vaccines induce some benefits (i.e., an increase in activated melanoma-specific T cells), most are designed to activate only one immune cell type. Tumors have a significant heterogeneity and high genetic mutation burden that allows for easy escape from immune surveillance. 5、6 may have the following structure: 7Therefore, vaccines that activate primarily one immune cell type are insufficient, and vaccines containing antigens that target multiple cell types are needed to induce complete tumor remission.

[0106] Common approaches to eliciting multifaceted immune responses are 1) administration of "long peptides" whose sequences cover multiple epitopes to activate both cytotoxic and helper T cells, or 2) administration of multiple "minimal" peptide antigens, each unique to a T cell subclass. 8-11 However, many of these ongoing efforts involve pools of peptides delivered in saline as a mixture, with or without adjuvants.

[0107] In this example, we explored the vaccine design space with multiple cell-targeting antigens. By employing structural changes in antigen positioning, we uncovered the impact of the resulting immune response, helping to boost the success of translational efforts. The antigens used activate cytotoxic (CD8+) T cells to effectively kill tumors, and helper (CD4+) T cells to synergize the interaction for long-term tumor rejection. 17、18 CD4+ T cells maintain tumor-tropic CD8+ functionality by recruiting CD8+ cells to the tumor site and enhancing their proliferation and effector function. 19-23 A cancer vaccine pathway with both cytotoxic and helper T cell antigens can activate different classes of T cells through the presentation of cytotoxic antigens on MHC-I receptors and helper antigens on MHC-II receptors. (See Figures 40-43). See also Hoos, A. Nat. Rev. Drug Discov. 2016, 15(4), 235-247. Thus, the vaccines described herein take into account the precise structural positioning of both major histocompatibility complex (MHC)-I and major histocompatibility complex (MHC)-II restricted antigen targets (CD8+ and CD4+ activating, respectively) to most effectively prime the immune system.

[0108] Herein, we utilize the spherical nucleic acid (SNA) platform to elucidate the effect of nanoscale architectures on multi-antigen immunological processes. SNAs consist of a nanoparticle core (e.g., liposomes) with a high density, radially arranged surface of oligonucleotides. SNAs are valuable due to their biocompatibility, 24 the ability to rapidly enter cells in large quantities; 25、26 Strong immune activation when employing toll-like receptor 9 (TLR9) agonist DNA as a shell, 27 And because of their modularity, allowing defined nanoscale positioning of components using well-known chemistry, they are powerful tools for examining these complex relationships. 28-30 This example demonstrates how the structure of an SNA vaccine carrying multiple immune cell targeting antigens significantly impacts immune activation. Changing the location of antigen types within the SNA upregulates immune cell pathways at the transcriptome level, enhances the production and secretion of cytokines and memory markers at the cellular level, and slows tumor growth at the organismal level. Taken together, these changes define the efficacy of the vaccine against an aggressive B16-F10 melanoma tumor model and, importantly, elucidate design insights for multi-antigen positioning that can be linked to other therapies.

[0109] Results and Discussion In the experiments described in this example, we sought to determine optimal antigen processing conditions for a multi-antigen SNA vaccine to generate robust cytotoxic and helper T cell responses. Specifically, we investigated how delivery of two antigen classes (MHC-I and -II restricted) to dendritic cells (DCs) alters their processing in vitro. DCs are the key professional antigen-presenting cells that induce signaling for effective T cell priming. Previous literature has demonstrated the possibility of enhancing DC activation through the simultaneous delivery of both cytotoxic and helper antigens. 31、32 However, we do not have a platform that can help us understand the best way to present such antigens. 14It was hypothesized that co-delivery of both antigen classes on the same nanoparticle, in contrast to delivery of both antigen classes on different nanoparticles, would enhance activation of both T cell types, and that the structural location of the antigen would have a pronounced impact on vaccine performance.

[0110] Processing of multiple antigens in vitro based on antigen distribution on SNA. To test this theory, dual antigen SNA vaccines (DA-SNAs) containing both MHC-I and -II restricted antigens at different nanoscale locations (designated DA-SNA1 and DA-SNA2 based on the placement of each antigen (see, e.g., Figures 1, 4, and 26A) were designed and synthesized. Due to SNA modularity, there are many different locations within the SNA construct where antigens can be placed. For this study, encapsulation and hybridization placements for antigen positioning were selected and compared to each other. To evaluate how the distribution of antigens and delivery on different nanoparticles affects immune activation, a formulation containing two individual SNAs, each presenting only one antigen class in the same position as the DA-SNA vaccine (Figure 27) was synthesized. The formulations were designated "separate" for the individual SNAs delivering a single antigen and "combined" for the dual antigen-containing DA-SNAs.

[0111] To synthesize DA-SNAs, peptides from one antigen class were encapsulated into 50 nm 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes during their formation (Figures 28 and 29). In parallel, peptides from the other antigen class were conjugated to strands complementary to the CpG motif adjuvant DNA shell ("CpG complement") of the SNA using disulfide bond formation (Figure 30). The DNA and peptide sequences used in this example can be found in Tables 1 and 2). Hybridized duplexes were formed by slow cooling of the CpG complement with the antigen appended to the complementary 3'-cholesterol-terminated CpG strand. The cholesterol anchors the duplex to the surface of the liposome. This hybridized product was added to liposomes to obtain equimolar amounts of each antigen. The liposome surface was reloaded with non-targeting DNA that did not contain antigen to obtain 1.6 pmol / cm 2 We obtain a total of 75 DNA strands per liposome, corresponding to a density of 100 μg / ml, and observe the properties associated with SNAs that make them useful in biology (see Figures 2, 3, and 10). 16 SNAs containing either a single encapsulated antigen or a single hybridized antigen ("separate" formulations) were synthesised according to previous protocols. 16 The formation of SNA was confirmed using dynamic light scattering (Figure 31). [Table 1] [Table 2]

[0112] Once these different structures were synthesized, their activation in DCs was assessed using the MHC-I and -II restricted model antigens of ovalbumin (OVA), known as OVA1 and OVA2, respectively. The activation of DC cues and their ability to prime T cells were characterized using mouse bone marrow derived DCs and antigens delivered as two separate SNA structures for a combined DA-SNA vaccine. Specifically, CD11c DCs expressing the innate costimulatory markers CD86 and CD80 were also expressed.+ The proportion of DCs did not vary significantly depending on the structural configuration (Figure 26B). This is likely due to the fact that adjuvant delivery is equivalent in the combined and separate formulations, indicating that hybridization to CpG does not interfere with TLR9 activation. Furthermore, presentation of antigen to naive splenic T cells to elicit antigen-specific T cells, an indicator of the ability of the immune system to recognize tumor antigens, was not observed. 33 was not affected by the way the antigen was placed. Naive T cells could be clonally expanded into OVA1-specific and OVA2-specific T cells (Figure 26C). Gating strategy in Figure 32. Indeed, about 0.6-0.7% of the viable CD19 population expressed OVA1-H-2kb dimers and CD8 + A similar trend for OVA2-specific T cell differentiation was observed, with approximately 0.9–1.1% expressing OVA2-H-2-Iad tetramers and CD4 + The DA-SNA2 construct contained dual positive markers for the antibodies. Only the DA-SNA construct, not the separate formulations, was able to significantly elevate the amount of antigen-specific T cells above the amount of T cell control baseline. This suggested that the combined delivery of both antigens to DCs led to stronger T cell differentiation. This was more evident when evaluating the expression of the early activation marker CD69 within either antigen-specific T cell population. When the antigens were delivered in combination as one DA-SNA, there was an increased amount of CD69 signal (measured by median fluorescence intensity, MFI), with the DA-SNA2 construct outperforming all test groups (Figure 26D). Furthermore, regardless of antigen positioning, delivery of the antigen in the DA-SNA construct was linked to a two-fold increase in T cell proliferation, a key step in the anti-tumor response, using OT1 spleen cells specific for the OVA1 antigen (Figure 26E, Figure 13). Based on these findings, DA-SNA was used in all subsequent experiments instead of separate SNA formulations.

[0113] In vivo activation via DA-SNA. Based on these promising in vitro findings using DA-SNA, C57BL / 6 mice were immunized in vivo to depict how differences in the positioning of MHC-I and -II restricted antigens within the DA-SNA vaccine affect immune activity. Mice were given a total of three injections (6 nmol with DNA and each peptide, FIG. 33A). On day 35, spleen cells were harvested to evaluate the specific immune response elicited towards both peptide antigens. After a 5 week period, CD8 + Levels were significantly elevated with DA-SNA2 immunization (referred to as "admixture", FIG. 33B). CD4 + Although levels did not vary significantly between treatment groups, an approximately 8% reduction in helper T cells was observed as a result of DA-SNA2 immunization (Figure 33B). DA-SNA2 was the only vaccine that was able to significantly increase production of the key proinflammatory cytokine IFN-γ and the degranulation marker CD107a upon restimulation with OVA1 peptide ex vivo. DA-SNA2 immunization also induced a greater proportion of polyfunctional splenic CD8 + T cells (approximately 17%, FIG. 33C, FIG. 5, FIG. 20). Moreover, this generated effector memory CD8 + T cells (CD44 + The increase in the percentage of CD4+ / CD62L- cells was approximately 48% (Figure 33D, Figure 6, Figure 17). +Ex vivo stimulation of T cells showed an overall increase in these same parameters for both DA-SNAs compared to the admixture treatment, further demonstrating the importance of the combined delivery of both antigen and adjuvant to immune cells. No significant differences were observed between the two DA-SNA constructs (Figures 33C-D, 5, 6). When IFN-γ secretion was assessed through enzyme-linked immunosorbent spot (ELISpot) assays, an increase in spot-forming cells (SFCs) was observed for both DA-SNA constructs compared to the admixture, with the greatest enhancement seen for DA-SNA2 (Figures 33E, 18, and 19). Furthermore, when stimulated ex vivo with either OVA1 or OVA2, spleen cells elicited by DA-SNA2 immunization led to at least two-fold more SFCs than DA-SNA1 immunization, demonstrating the efficacy of utilizing this array of antigens on DA-SNAs to respond ex vivo to MHC-I or -II restricted antigen cues. Overall, these results suggest that the positioning of MHC-I restricted antigens in the hybridized architecture is important for CD8 + Optimizing the presentation of DC processing to potently activate T cell responses while preserving cytotoxic function by encapsulating MHC-II restricted antigens within the core, we have developed a novel antibody that can inhibit CD4 + It was emphasized that this induces a modest increase in activity.

[0114] Mechanistic understanding of DA-SNA-induced immune activation and proliferation via RNA sequencing. To understand how different DA-SNA and admixture treatments induced such diverse immunological responses in vivo, splenic CD8 + and CD4 + T cells were collected and isolated after immunization and bulk RNA sequencing (RNAseq) was performed. Principal component analysis (PCA) showed that CD8 T cells were significantly higher in mice immunized with the admixture formulation than in mice immunized with the admixture formulation. + and CD4 +Collectively, we found that T cell gene expression profiles were most similar to those from naive mice, suggesting that this was the cause of the low overall activation (Figure 34A). Mice immunized with DA-SNA2 differed most from naive mice in their CD8+ transcriptome, whereas both DA-SNA1 and DA-SNA2 similarly upregulated the CD4 + The gene expression profiles of CD8 T cells in DA-SNA2-induced T cells were different from those in naive mice (Figure 14). + Although T cell function was significantly increased, CD4 + Evidence suggests that T cell function levels are comparable. Furthermore, differentially regulated genes in DA-SNA2 immunized mice showed greater absolute log fold changes (LFC) in both T cell types compared to other treatments, with at least double the number of differentially regulated genes as a result of DA-SNA2 immunization compared to DA-SNA1 (Figure 34B). Differentially regulated genes were enriched in pathways involving inflammatory responses and upregulation of proinflammatory cytokines, chemotaxis, and migration of key immune cell populations (Figure 34C, Figure 14, Figure 15, and Figure 16). Although some of the enriched pathways from DA-SNA2 treatment were shared with admixture treatment and others with DA-SNA1 treatment, overall, the widespread activation induced at the transcriptome level for DA-SNA2 architecture correlated with enhanced immunological output.

[0115] Associated gene signatures were identified for adaptive and innate immune activation and function across all treatments, including, for example, CXCR3, TNFSF9, and GZMK (Figure 34D). These genes have particular relevance to T cell effector function and trafficking, antigen presentation and generation of cytotoxic T cells, and cytolytic function of helper T cells, respectively. Specific comparison of DA-SNA2 to DA-SNA1 demonstrates unique nanoscale-induced gene differences induced by simply changing antigen class positioning (Figure 34E). CD8 + and CD4+ A total of 452 and 229 overlapping significant genes were detected between both DA-SNAs in T cells, respectively. Specifically, DA-SNA2 significantly increased the expression of CD8 + IL2RA, CD44, XCL1, and CD4 in T cells + It induced higher expression of LAG3, CCR7, and CCL9 in T cells. Finally, comparing gene signatures across all immunization treatments highlighted the profound impact of vaccine architecture, particularly nanoscale antigen positioning, on genomic and expression patterns. These results corroborate the immunological measurements detected, provide mechanistic evidence highlighting pathways leading to T cell activation and sustained responses, and detail a framework for vaccine design using fit-for-purpose structural considerations.

[0116] DA-SNA Structure-Driven Tumor Inhibition and Immune Activation To evaluate the therapeutic efficacy and immunological impact of DA-SNA, we employed a murine E.G7-OVA lymphoma cancer model due to its stable expression of OVA protein, which expresses both the OVA1 and OVA2 epitopes used above. 34Briefly, C57BL / 6 mice were subcutaneously inoculated with E.G7-OVA cells and immunized weekly with either DA-SNA or admixture formulations (6 nmol each of OVA1 and OVA2 antigens, 6 nmol adjuvant DNA) (Figure 35A). Tumor-bearing mice immunized with DA-SNA2 demonstrated approximately 3-fold tumor growth reduction compared to both control (saline treatment) and admixture groups as early as 5 days after the second immunization (day 15), and demonstrated a greater than 16-fold difference in tumor growth 22 days after tumor inoculation compared to saline-treated mice (Figure 35B, Figure 9, and Figure 36). Importantly, DA-SNA1 treatment, unlike DA-SNA2 treatment, did not effectively halt tumor growth compared to the admixture. Compared to the admixture and DA-SNA1, DA-SNA2 caused an approximately 7-fold reduction in tumor growth at day 24, highlighting the striking effect of antigen positioning, ultimately leading to a significant extension of animal survival (median survival in days: PBS=27, admixture=24, DA-SNA1=28, DA-SNA2=35) (Figure 35C, Figure 11). To further explore the physical effects of treatment on tumor growth, tumors were excised from mice on day 15 after the same treatment regimen and then weighed (Figure 35D and Figure 37). Interestingly, at this point in the tumor growth curve, both SNA groups showed a significant reduction in tumor weight compared to PBS-treated mice, suggesting that DA-SNA1 was able to elicit an anti-tumor immune response, but that it was not as sustained as that elicited from DA-SNA2.

[0117] Spleens were harvested and CD8 + and CD4 + T cell changes were assessed to elucidate differences as a result of treatment that may have contributed to tumor regression (Figure 35E, Figure 12). Spleens from DA-SNA2-treated mice showed significantly higher CD8 + Generated a significantly higher percentage of T cells and CD8 + CD4 +They also displayed an overall high ratio to T cells. To assess immunological differences that contributed to tumor suppression, tumor-bearing C57BL / 6 mice were assessed for circulating peripheral blood mononuclear cells (PBMCs) on day 15, when differences in tumor growth were first observed, and when the impact of DA-SNA2 treatment began to halt tumor growth while other treatments had negligible effects. Of note, DA-SNA2-treated mice showed significantly higher levels of circulating antigen-specific CD8 + The highest levels of CD8 T cells were observed (Figure 35F, Figure 7, Figure 21). + This subset of lymphocytes was further evaluated for their memory phenotype, in which DA-SNA2 treatment induced an effector memory phenotype in OVA1-specific circulating CD8 + The antigen-specific CD4 + T cells were also significantly elevated in mice treated with DA-SNA (Fig. 35H, Fig. 7, Fig. 21). As expected, CD4 + There were minimal differences between the two DA-SNA groups for the transcriptomic profiles and immunological parameters previously validated herein for T cells. There was sufficient OVA2-specific CD4 T cell expression to accurately represent the memory phenotype within this subpopulation. + There were no T cells, but CD4 + When treated with DA-SNA1, approximately 10% of T cells matured compared to treatment with DA-SNA2 (CD4 + Approximately 30% of T cells are CD4 + Overall, T cells demonstrated an enhanced effector memory state (Figure 35I, Figure 8).

[0118] Structural impact of antigen positioning in a clinically relevant melanoma tumor model. What has been learned so far about DA-SNA structure in a model system was utilized to evaluate the ability of dual antigen positioning to affect the growth of B16-F10 melanoma tumors. This model is established as highly invasive with enhanced immunosuppressive properties. 35 A recently reported mutation in the MART-1 protein that is present only in tumors 36M27 and M30 neoepitopes were selected as MHC-I and -II antigens, respectively. Initially, C57BL / 6 mice inoculated with B16-F10 tumor cells, which received weekly vaccinations with either DA-SNA1 or DA-SNA2, showed substantial inhibition of tumor growth at day 17 (68% and 48%, respectively) compared to saline-treated mice (Figures 38A-B, Figure 23). Indeed, there was a significant increase in circulating effector memory antigen-specific CD8+ cells in mice treated with either DA-SNA compared to saline treatment. + We observed a statistically significant 4-fold increase in T cells (Figure 38C). Thus, an antigen-specific immune response was generated, but this had only a minimal effect on tumor growth, indicating that the highly immunosuppressive tumor environment may have inhibited the therapeutic potential of these T cells.

[0119] Due to these observations and the inherent aggressiveness of this tumor model, in an effort to overcome tumor immunosuppression, DA-SNA therapy was combined with the immune checkpoint inhibitor anti-PD-1, an FDA-approved treatment for advanced melanoma. 37、38 Of note, when anti-PD-1 was co-administered with DA-SNA, a significant reduction in tumor growth was observed as early as 17 days after tumor inoculation for animals treated with the DA-SNA2+anti-PD-1 combination, whereas no substantial reduction in tumor growth was observed for mice in other treatment groups (Figure 38D). See also Figures 24, 25, and 39. This translated into a 40% increase in median survival for these mice compared to saline or anti-PD-1 monotherapy treatment groups (Figure 38E). Furthermore, comparison between DA-SNA combination treatment groups revealed a substantial improvement in overall median survival for the combined DA-SNA2+anti-PD-1 animals (p=0.0507). Evaluation of immune cells isolated from peripheral blood further highlights this dramatic conformation-induced difference where DA-SNA2 appears to act synergistically with checkpoint inhibitors. Circulating CD8 for animals receiving combination DA-SNA2 + anti-PD-1 treatment compared to all other groups +A significant increase in T cells was observed (Figure 38F). Interestingly, among these circulating PBMCs, total CD8 + When evaluating effector memory T cell populations, we detected a significant increase for both combination DA-SNA + anti-PD-1 groups, and DA-SNA2 + anti-PD-1 increased circulating CD8 + DA-SNA2+anti-PD-1 treatment generated an effector memory phenotype in approximately 60% of T cells (Figure 38G). Furthermore, only this combination DA-SNA2+anti-PD-1 treatment generated robust antigen-specific CD8 + It was able to induce a T cell response (Figure 38H). + Observation of circulating T cells revealed a similar high increase as a result of combination DA-SNA2 + anti-PD-1 therapy (Figure 38I), but both combination DA-SNA + anti-PD-1 groups maintained an effector memory phenotype in CD4 + The combination DA-SNA2+PD-1 treatment significantly increased antigen-specific CD4 T cell counts to approximately 28% of the T cell population (Figure 38J). Compared to DA-SNA1+anti-PD-1 treatment, the combination DA-SNA2+PD-1 treatment, along with anti-PD-1 monotherapy, significantly increased antigen-specific CD4 T cell counts to approximately 28% of the T cell population (Figure 38J). + increased levels of T cell production, but no significant differences were observed between groups (Figure 38K).

[0120] conclusion Extensive research has generally examined the importance of adjuvants and antigens in generating potent new immunotherapies, but thus far there has been no body of work examining the importance of structural presentation of multiple antigens within a specific construct and its role in eliciting a strong and desired immune response. Taken together, the data provided herein indicates that antigen positioning is as important as antigen selection in vaccine efficacy. Indeed, in certain embodiments, altering the positioning of MHC-I and -II restricted antigens in two compositionally nearly identical vaccines dramatically altered the therapeutic benefit against tumors, with one vaccine being potent and the other being totally ineffective. The origin of these differences may be due to antigen positioning, which affects the pathways of processing it undergoes in immune cells, and its residence time in different cellular compartments. By altering the processing pathways and the kinetics of these signaling, this impacts the resulting immune response at the genetic, cellular, and organismal levels. Encapsulated MHC-II and hybridized MHC-I restricted antigens upregulate genes specific to key immune cell inflammatory responses, chemotaxis, and migration, which together affect immune cell activity. These structurally defined genetic differences are coupled to immunological behavior upon repeated in vivo immunization and ultimately define tumor growth profiles in both model E.G7-OVA lymphoma and clinically relevant B16-F10 melanoma murine tumor systems, a key demonstration of the impact of vaccine antigen positioning across multiple cellular processes.

[0121] The present examples demonstrate that the ability to optimize antigen presentation to match a desired signaling profile is critical to generating a potent vaccine, with small vaccine changes in antigen positioning significantly increasing cell-cell communication, crosstalk, and cellular synergy.

[0122] Materials and Methods Materials and Animals: Unless otherwise stated, all reagents were purchased commercially and used as received. Oligonucleotides were synthesized as described below. Peptides were purchased from Genscript or Northwestern's peptide synthesis core. Chemicals were purchased from the sources listed in brackets. 8-12 week old C57BL / 6 mice and C57BL / 6-Tg(TcraTcrb)1100Mjb / J(OT-1,003831) female mice were purchased from Jackson Laboratory. Mice were used in accordance with all national and local guidelines and regulations, and the protocols carried out were approved by the Laboratory Animal Use Committee of Northwestern University (IUCAC). E.G7-OVA and B16-F10 cells were purchased from ATCC. Antibodies were purchased and clones are provided in Table 3. [Table 3]

[0123] Synthesis and purification of oligonucleotides: Oligonucleotides were synthesized on an ABI394 synthesizer using standard phosphoramidite chemistries with phosphate or phosphorothioate backbones as indicated (Table 1). Following synthesis, strands were deprotected using a 1:1 solution of 37% ammonium hydroxide / 40% methylamine at 55°C for 35 min unless the strand contained a dye, in which case the strand was deprotected overnight at room temperature (RT) using 37% ammonium hydroxide. Strands were then purified using a C18 or C4 (for strands containing dyes or cholesterol) column on a reversed-phase HPLC, with peaks collected as fractions. Dimethoxytrityl (DMT) groups were removed from product strands by incubation in 20% aqueous acetic acid at room temperature for 1 h, followed by three washes with ethyl acetate to remove DMT. The final products were lyophilized and resuspended in deionized water (diH2O). Concentrations were measured using UV-vis absorbance at 260 nm with extinction coefficients calculated through the IDT OligoAnalyzer online tool (listed in Table 1).

[0124] Synthesis and purification of oligonucleotide-peptide conjugates: Thiol-functionalized oligonucleotides in diH2O were reduced to generate free thiols for future reactions. Reduction was performed using dithiothreitol (100 mM, DTT, Sigma) dissolved in phosphate-buffered saline (PBS) pH 8.5 at a final concentration of 100 mM for 45 min at room temperature. The solution was washed at least three times with diH2O in a 3 kDa molecular weight cut-off (MWCO) spin filter (Amicon). For OVA-peptide conjugates, the peptide on resin was purchased and washed three times each with dimethylformamide (DMF) and acetone, after which 5 μmol was reacted overnight at room temperature with a solution of succinimidyl 2-(2-pyridyldithio)ethyl carbonate (SDEC, prepared using a previous protocol39) dissolved in DMF (10 equivalents relative to the initial peptide loading on the solid support) and N,N-diisopropylethylamine (5 equivalents). The beads were then washed three times each with DMF and acetone, dried in air, and then deprotected with 95% trifluoroacetic acid (2.5% triisopropylsilane, 2.5% diH2O) at room temperature for 1 h. The TFA was blown off using nitrogen, and the beads were redissolved in DMF and filtered through glass wool. The peptide product was precipitated by adding approximately 5-6 volumes of diethyl ether and left at -20 °C for 1-2 h to further precipitate. The solution was centrifuged (2,000 × g, 3 min) to pellet the peptide, which was collected, dried, and dissolved in DMF. The reduced DNA (0.5 μmol) was reacted with the dissolved peptide (5 μmol) in 70-75% DMF in water overnight at room temperature to bring the total volume of the reaction to approximately 1.5 mL. For the MART-1 peptide conjugate, the peptide was activated using 2,2'-dithiodipyridine (150 μmol) dissolved in 10 equivalents of DMF under gentle agitation at room temperature for 30 min. The activated peptide was then washed three times in diethyl ether, pelleted by centrifugation (2,000×g, 3 min) and dried. The reduced DNA (0.3 μmol) was reacted overnight at room temperature with the peptide (5 μmol) dissolved in approximately 70% DMF in water to a total reaction volume of approximately 1.5 mL.

[0125] Following peptide conjugation, the solution was centrifuged at 17,000 x g for 2 min to pellet any ground peptide, and the supernatant was transferred to a 3 kDa MWCO spin filter and washed 3-4 times with diH2O. The volume was concentrated to <500 μL, and the solution was purified by preparative-scale denaturing (8 M urea) 15% PAGE gel (no more than 0.5 μmol of DNA loaded onto a single gel). The gel was run at 175 V for 30 min, then at 350 V for approximately 3 h, after which it was imaged using a UV lamp to excise the desired bands. The excised gel bands were ground and the products were collected by washing three times with 1x Tris / borate / EDTA (TBE) buffer approximately every 4 h. Product mass was confirmed by electrospray ionization mass spectrometry (ESI-MS), and concentrations were measured by UV-vis at 260 nm, assuming an attenuation coefficient for DNA.

[0126] SNA synthesis: SNA was synthesized as previously reported with minor modifications. 16、40Briefly, a dried lipid film of 50 mg 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, Avanti Polar Lipids) was hydrated with 3-4 mL of PBS or PBS containing peptide for encapsulated liposomes. (Note: Peptide-containing solutions included: OVA1: 1 mg / mL dissolved in PBS containing approximately 100 μL of 1 M NaOH; OVA2: 1 mg / mL dissolved in PBS containing approximately 60 μL of 1 M NaOH; M27: 2.25 mg / mL dissolved in PBS containing approximately 60 μL of 1 M NaOH; M30: 1 mg / mL dissolved in PBS containing approximately 100 μL of 1 M NaOH.) The solutions were subjected to 20 freeze-thaw cycles in liquid nitrogen followed by sonication at 37-40 °C. Liposomes were extruded using sequential high-pressure extrusion (Northern Lipids Inc.) using polycarbonate filters with pore sizes of 200, 100, 80, and 50 nm, with liposomes passed three times through each pore size. Following extrusion, liposomes were concentrated to approximately 2-3 mL using a 100 kDa MWCO spin filter and dialyzed overnight against 3.5 L of PBS to remove unencapsulated peptide. Liposome concentration was determined using a phosphatidylcholine (PC) assay kit (Sigma, MAK049-1KT), assuming that 50 nm liposomes contain 18,140 lipids per liposome. 24 If the liposomes encapsulated peptides, the peptide concentration was measured using the Pierce™ Fluorescence Assay Kit (Thermofisher, 23290) by adding 1% sodium dodecyl sulfate (SDS) to disrupt the liposomes and release the peptide for quantification, using peptide dissolved in 1% SDS as a standard curve. The peptide loading per liposome was calculated by dividing the peptide concentration by the liposome concentration. The amount of each antigen per particle ranged from approximately 25 to 40, depending on the encapsulation rate of each batch, which was adjusted using different starting concentrations.

[0127] The purified oligonucleotide-peptide conjugates were mixed with complementary 3'-cholesterol terminated CpG DNA in a 1:1 molar ratio and centrifuged overnight. The next day, approximately 20-40 μL of duplex buffer (IDT) was added and the solution was slowly cooled to duplex the strands according to the program: 70 °C for 10 min, 23 °C for 1.5 h, 4 °C for ≥ 2 h. The duplexes were added to a solution of liposomes synthesized in equimolar amounts to the peptide encapsulated within the liposomes. The remaining space was filled with 3' cholesterol terminated T20 DNA to obtain up to 75 strands per liposome. The mixture was incubated overnight at 37 °C and then stored at 4 °C.

[0128] Cell culture: All cells were maintained at 37°C in a 5% CO2 incubator. E.G7-OVA cells and DCs were cultured in RPMI 1640 medium (Gibco, 11875093) containing 10% heat-inactivated fetal bovine serum (HI-FBS) and 1% penicillin-streptomycin, referred to herein as RPMI+ / +. B16-F10 were handled using DMEM medium (Gibco, 11965092) containing 10% HI-FBS and 1% penicillin-streptomycin.

[0129] Collection of bone marrow derived dendritic cells (BMDCs): Bone marrow cells were collected from mice according to a previous protocol. Briefly, red blood cells were lysed with 2-3 mL of ACK lysis buffer (Gibco, A1049201) for approximately 4 min, and then cultured in 10 cm aliquots of BMDCs with 40 ng / mL GM-CSF (BioLegend, 576304) for 5-7 days prior to use to differentiate DCs from the population. 2 Plated onto cell culture dishes.

[0130] Activation and cross-priming of BMDCs with T cells in vitro: 2 The cells were collected from cell culture dishes and DCs were isolated from the mixture using a magnetic biotin positive selection kit (Stemcell Technologies, 17665). +DCs were selected using a biotin-labeled antibody (BioLegend), and after isolation, purified DCs were counted using a Vi-CELL BLU cell viability analyzer. For DC activation, 6 × 10 4 DCs were cultured in a final volume of 200 μL SNA treatment. After 22 h in the incubator, cells were washed with PBS to terminate the treatment, stained with 0.5 μL of each antibody (L / D, CD11c, CD86, and CD80) per tube for 15 min at 4°C, washed with PBS, and fixed with 100 μL fixation buffer (BioLegend, 420801). To assess T cell specificity and activation, 1.6 × 10 5 Purified DCs were pulsed with SNA treatment for 30 min in an incubator with a final volume of 200 μL. After 30 min pulsing, cells were washed twice with RPMI+ / + to remove any residual SNA from the cell solution, and the cells were resuspended in 500 μL of RPMI+ / +. At the same time, splenocytes were isolated from naive mice. After dissociation of the spleen and lysis of red blood cells, cells were counted and aliquoted at 3 × 10 in warmed RPMI+ / +. 6 The cells were resuspended to a concentration of 1000 cells / mL, and 100 μL of this cell solution was transferred to each well of a 96-well round-bottom plate. Each well was inoculated with treated DCs (3.3 × 10 4 100 μL of 100x10 cells were added. Cells were co-cultured in an incubator for 3 days, after which cells were washed with PBS and stained according to the manufacturer's instructions for either DimerX Mouse H-2Kb:Ig fusion protein (BD, 552944) or OVA2 tetramer (ProImmune). Staining antibodies in addition to peptide-specific TCR markers included L / D, either CD8 or CD4, CD19, as well as CD69. After staining, cells were fixed with 100 μL of fixation buffer. To assess T cell proliferation, 2.6 × 10 cells were added to 100x10 cells. 5Purified DCs were pulsed with SNA treatment for 30 min in an incubator with a final volume of 200 μL. After 30 min pulsing, cells were washed twice with RPMI+ / + to remove any residual SNA from the cell solution, and cells were resuspended in 266.6 μL of RPMI+ / +. At the same time, spleen cells were isolated from C57BL / 6-Tg(TcraTcrb)1100Mjb / J(OT-1) mice (Jackson, 003831). After dissociation of spleens and lysis of red blood cells, 4 × 10 cells were collected in PBS for cell counting and staining with cell proliferation dye eFluor™ 450 (eBioscience, 65-0842-85). 7 After staining, cells were washed, counted, and resuspended at a concentration of 3 × 10 cells / mL in RPMI+ / +. 6 The cells were resuspended to a concentration of 1000 cells / mL, and 100 μL of this cell solution was transferred to each well of a 96-well round-bottom plate. Each well was added with treated DCs (3.3 × 10 4 33.3 μL of CD40 (0.5 μL cells) was added and each well was brought up to a final volume of 200 μL with media. Cells were co-cultured in an incubator for 3 days after which the cells were washed with PBS and stained for CD8 (0.5 μL antibody per tube), washed and immediately analyzed by flow cytometry using dilutions of eFluor™ 450 as a measure of T cell proliferation. All samples were analyzed using a BD A3 Symphony flow cytometer with data analyzed in FlowJo.

[0131] In vivo immunization to measure cumulative immune responses over 5 weeks: Female C57BL / 6 mice were subcutaneously immunized 3 times over 2 weeks with different treatments. Treatments included a simple mixture (mixture, 6 nmol of each peptide and 6 nmol of CpG 1826 DNA) or either DA-SNA (6 nmol of each OVA peptide and CpG1826). The volume of the injected treatments was kept at 100 μL. One week after the final immunization, the mice were sacrificed and the spleens were harvested for subsequent immune evaluation.

[0132] Harvesting procedure: Removed spleens were collected and temporarily held in 3-5 mL RPMI+ / + until all spleens were collected, at which point they were passed through a 70 μm cell strainer with a constant flow of PBS. Cells were centrifuged at 1,200 rpm for 5 min, after which the supernatant was removed and cells were resuspended in 2-3 mL ACK lysis buffer (Gibco, A1049201) for 4 min. To dilute the lysis buffer, PBS was then added to a final volume of 30 mL, and cells were counted prior to centrifugation to obtain a total cell count of 1 × 10 8 cells mL -1 The cells were resuspended in RPMI+ / + medium at a concentration of 100 mM.

[0133] IFN-γ cytokine production: T cells were restimulated ex vivo to assess antigen-specific intracellular IFN-γ production. 4×10 6 Spleen cells were cultured for 4 hours at 37°C in a 5% CO2 incubator with 450 μL of RPMI+ / + medium containing either OVA1 or OVA2 peptide (10 μg / mL), monensin (2 μM), brefeldin A (5 μg / mL), and CD107a antibody (0.5 μL). After 4 hours of incubation, cells were centrifuged at 1,200 rpm for 5 minutes, aspirated, washed with 600 μL of PBS, and then stained with surface antibodies (0.5 μL per sample for each of L / D, CD8, and CD4) for 15 minutes at 4°C. Cells were washed with 600 μL of PBS, centrifuged at 1,200 rpm for 5 minutes, aspirated, and resuspended in 100 μL of Cytofix fixation and permeabilization solution (BD, 554722) for 20 minutes at 4°C. Cells were then washed with 600 μL Perm / Wash buffer (BD, 554723), centrifuged at 1,200 rpm for 5 min, aspirated, and resuspended in 100 μL Perm / Wash buffer containing intracellular antibody IFN-γ (0.5 μL per sample). Samples were stored at 4° C. prior to flow cytometry analysis.

[0134] T cell memory phenotype: T cells were assessed for effector memory phenotype. 3×10 6Spleen cells were washed with 600 μL PBS and stained with surface antibodies (0.5 μL per sample for each of L / D, CD8, CD4, CD44, and CD62L) for 15 min. Cells were washed with 600 μL PBS, centrifuged at 1,200 rpm for 5 min, aspirated, resuspended in 100 μL fixation buffer (BioLegend, 420801), and stored at 4° C. prior to flow cytometry analysis.

[0135] ELISpot assay: ELISpot analysis was performed using a commercially available Mouse INF-γ ELISpot Set (BD, 551083) according to the manufacturer's instructions. Briefly, the provided clean plates were coated with capture antibody overnight at 4°C. Afterwards, the plates were washed with RPMI+ / + medium and then blocked with 200 μL of RPMI+ / + medium at room temperature for 2 hours. The blocking buffer was removed by pipetting, taking care not to let the wells dry, and 2 × 10 IgG in 100 μL of RPMI+ / + was added. 5 The wells were rapidly replaced with 100 spleen cells. To each well, an additional 100 μL of either antigen, non-specific peptide, medium (negative control), or positive control solution was added (antigen and non-specific peptide were added to a final concentration of 5 μg / mL, and the positive control was prepared as a mixture of anti-CD3 and anti-CD28 antibodies at a final concentration of 2 μg / mL each). The solutions were left in an incubator at 37° C. in 5% CO2 for 48 hours. After this incubation, the plates were washed and detection antibodies, enzyme conjugates, and chromogenic substrates were added according to the manufacturer's instructions. The dried plates were imaged and analyzed using a CTL immunospot imager.

[0136] Bulk RNA sequencing: CD4 RNA was isolated from total spleen cells from individual treatment groups using magnetic positive selection kits. + and CD8 +T cells were isolated (Stemcell Technologies, 18952 and 18953). From these isolated cell populations, RNA extraction was performed using RNeasy® Plus Mini Kit (Qiagen) in combination with QIAshredder (Qiagen) according to the manufacturer's specifications. RNA concentration was quantified using NanoDrop 8000 (Thermo Scientific) and RNA samples were stored at -80°C until further use. Sequencing was performed at the Northwestern University NUSeq Core Facility. Briefly, total RNA examples were checked for quality using RNA integrity numbers (RIN) generated from an Agilent Bioanalyzer 2100. RNA quantity was confirmed with a Qubit fluorimeter. Sequencing libraries were prepared from 125ng of high-quality RNA samples (RIN>7) using the Illumina TruSeq Stranded mRNA Library Preparation Kit. Kit procedures, including mRNA purification and fragmentation, cDNA synthesis, 3'-end adenylation, Illumina adapter ligation, library PCR amplification and validation, were performed without modification. Libraries were sequenced using an Illumina HiSeq 4000 sequencer to generate 50 bp single reads at a depth of 20-25 million reads per sample. Read quality in FASTQ format was assessed using FastQC. Reads were trimmed to remove Illumina adapters from the 3'-end using cutadapt. 41 The adjusted reads were aligned to the Mus musculus genome (mm10) using STAR. 42 Read counts for each gene were calculated using htseq-count with the mm10 gene annotation file obtained from Ensembl (http: / / useast.ensembl.org / index.html). 43 Normalization and differential expression were calculated using DESeq2 employing the Wald test. 44The cutoff for determining significantly differentially expressed genes was an FDR-adjusted p-value of less than 0.05 using the Benjamini-Hochberg method.

[0137] Gene Set Enrichment Analysis (GSEA): To understand whether differentially expressed genes are connected to differentially enriched pathways, GSEA45 was performed. Genes detected by RNA sequencing were compared with the log 1s obtained from DeSeq2 analysis. 10 They were ranked based on transformed nominal P values ​​and compared to naive T cells. Pathway enrichment analysis was performed using GSEA software (v4.0.3) following the protocol of Reimand et al. 46 Gene sets were obtained from the Molecular Signatures Database, including Reactome, KEGG. The ranked list was remapped using the CHIP platform from the Molecular Signatures Database, which was remapped to Human Orthologs (v7.1) using mouse gene symbols. Terms were defined as differentially enriched if they had an FDR<0.05. A subset of strongly enriched pathways was selected for visualization in R using the pheatmap package (v1.0.12). This selection included all pathways with an FDR<0.05, including CD8 + and CD4 + It was related to immune responses in T cells.

[0138] Gene Expression Profiles: Genes whose expression was significantly altered in both SNA treatment groups, as defined by an FDRp value <0.05, were selected for visualization as a heatmap. Gene expression scores in FPKM were converted to z-scores across treatment groups and gene expression values ​​were clustered using K-means clustering. Naïve CD4 + or CD8 +Pairwise combinations were performed between the two conditions of interest, with T cells set as control. Genes up- or downregulated between groups, as defined by FDRp values ​​<0.05 and log2 fold change >0.5 (upregulated) or log2 fold change <0.05 (downregulated), were visualized using a Volcano plot. In vivo efficacy studies: Female C57BL / 6 mice aged 8-12 weeks were obtained from The Jackson Laboratory. 5 × 10 5 E.G7-OVA or 10 5 Tumor inoculation was performed by subcutaneous (sc) injection of B16-F10 cells into animals. Immunizers were administered at doses of either 6 nmol (OVA1 / 2) or 9 nmol (M27 / 30) of each antigen and CpG by sc injection into the abdomen. Immunizers were administered as listed in the treatment schedule provided in the respective figures by sc injection into the abdomen. For combination therapy with immune checkpoint inhibitor anti-PD-1, mice were administered 100 μg of anti-mouse PD-1 (clone RMP1-14, BioXCell) via intraperitoneal injection. Tumor growth was measured on the given days and the volume was calculated using the following formula: tumor volume = length x width. 2 ×0.5. Tumor volume is 2,000 mm 3 (E.G7-OVA) or 1,500mm 3 (B16-F10) or when the animals became moribund, they were euthanized.

[0139] Immune activation of PBMC: For collection of peripheral blood mononuclear cells (PBMC), animals were inoculated with cancer cells as described above. Treatment was performed according to the same schedule and animals were euthanized on day 15 (E.G7-OVA) or day 17 (B16-F10). Blood was collected via cardiac puncture into EDTA-lined collection tubes (BD) and mixed briefly by inversion. Red blood cells were lysed using ACK lysis buffer (Gibco) and washed, after which the remaining cells were stained with antibodies against L / D, CD4, CD8, CD19, CD44, and CD62L using the methods described above.

[0140] Assessment of overall organ immunity: Tumor weight and spleen cell assessments were performed on C57BL / 6 mice bearing E.G7-OVA tumors on the right flank. The first immunization was administered 3 days after tumor inoculation, followed by a booster dose 7 days later (day 10). Tumors and spleens were excised from the animals on day 15 and subsequently analyzed. To generate a single cell splenocyte solution, the spleen was mechanically pressed through a 70 μm cell strainer while maintaining hydration in PBS solution. The cells were then centrifuged at 1200 rpm for 5 minutes. The pellet was then resuspended in ACK lysis buffer (Thermo) for 4 minutes to lyse red blood cells, and then neutralized in PBS prior to centrifugation. Following centrifugation, the cells were labeled using the following antibodies: CD4, CD8, and CD19.

[0141] Statistical analysis: Statistics were calculated using GraphPad Prism 8 software, and the specific statistical analysis used is highlighted in the caption of each figure. Due to the lack of assumptions that could be made based on large differences in standard deviations between groups, comparisons between multiple groups were analyzed using one-way ANOVA with Sidak or Tukey's multiple comparison test, or Welch ANOVA with Dunnett's multiple comparison test. Statistics for animal survival were calculated using the log-rank test. Outliers for Figures 35E-F and Figures 38H,J were identified using the ROUT method with Q set to 10% or 1%, respectively. In all cases, p values ​​were indicated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns indicates significance was not determined, and nd indicates no value was detected. Allocation of animals to each group, immunizations administered, and measurements for tumor studies were performed blinded. Values ​​in the graphs are presented as means±standard error, which is indicated in the captions of each figure, as are sample sizes.

[0142] Example 3 This example describes experiments using SNA constructs according to the present disclosure in a colon cancer model.

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Claims

1. Spherical nucleic acid (SNA), (a) a nanoparticle core; (b) a shell of oligonucleotides attached to an exterior surface of the nanoparticle core, the shell of oligonucleotides comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen, which is either a major histocompatibility complex type I (MHC-I) antigen or a major histocompatibility complex type II (MHC-II) antigen; and (d) a second antigen that is different from the first antigen and is either a major histocompatibility complex type I (MHC-I) antigen or a major histocompatibility complex type II (MHC-II) antigen.

2. The SNA described in claim 1, wherein the first antigen is a major histocompatibility complex type I (MHC-I) antigen and the second antigen is a major histocompatibility complex type II (MHC-II) antigen.

3. The SNA according to claim 1 or 2, wherein the first antigen is encapsulated in the nanoparticle core.

4. SNA according to any one of claims 1 to 3, wherein said second antigen is attached to one or more oligonucleotides within the shell of said oligonucleotides through a linker.

5. The SNA of claim 4, wherein the second antigen is bound through the linker to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core, or the second antigen is bound through the linker to an oligonucleotide that hybridizes to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core.

6. The SNA according to any one of claims 1 to 3, wherein the second antigen is attached to the outer surface of the nanoparticle core through a linker.

7. The SNA according to any one of claims 1 to 3, wherein the second antigen is encapsulated in the nanoparticle core.

8. A SNA according to any one of claims 1, 2 or 4 to 7, wherein the first antigen is attached to one or more oligonucleotides within the shell of the oligonucleotide through a linker.

9. 9. The SNA of claim 8, wherein the first antigen is bound through the linker to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core, or the first antigen is bound through the linker to an oligonucleotide that hybridizes to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core.

10. A SNA according to any one of claims 1 or 3 to 7, wherein the first antigen is attached to the outer surface of the nanoparticle core through a linker.

11. A SNA according to any one of claims 1 to 10, which comprises a third antigen which is a major histocompatibility complex type I (MHC-I) antigen.

12. A SNA according to any one of claims 1 to 11, which comprises a fourth antigen which is a major histocompatibility complex type II (MHC-II) antigen.

13. 13. The SNA of claim 11 or 12, wherein the third antigen is encapsulated in the nanoparticle core.

14. 14. An SNA according to claim 12 or 13, wherein the fourth antigen is attached to one or more oligonucleotides within the shell of the oligonucleotide through a linker.

15. The SNA of claim 14, wherein the fourth antigen is bound through the linker to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core, or the fourth antigen is bound through the linker to an oligonucleotide that hybridizes to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core.

16. 14. The SNA of claim 12 or 13, wherein the fourth antigen is attached to the outer surface of the nanoparticle core through a linker.

17. 17. SNA according to any one of claims 11, 12 or 14 to 16, wherein said third antigen is attached to one or more oligonucleotides within the shell of said oligonucleotides through a linker.

18. 18. The SNA of claim 17, wherein the third antigen is bound through the linker to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core, or the third antigen is bound through the linker to an oligonucleotide that hybridizes to an oligonucleotide in the shell of the oligonucleotide bound to the nanoparticle core.

19. A SNA according to any one of claims 11, 12 or 14 to 16, wherein the third antigen is attached to the outer surface of the nanoparticle core through a linker.

20. A SNA according to any one of claims 12, 13 or 17 to 19, wherein said fourth antigen is encapsulated in said nanoparticle core.

21. 21. The SNA according to any one of claims 11 to 20, wherein the first antigen and the third antigen are the same, the first antigen and the third antigen are different, the second antigen and the fourth antigen are the same, and / or the second antigen and the fourth antigen are different.

22. The MHC-I antigen is OVA ２５７－２６４ (OVA1) (SEQ ID NO: 7), GP100 (25-33) (SEQ ID NO: 11), TC-1 E6 (49-58) (SEQ ID NO: 12), TC-1 E7 (49-57) (SEQ ID NO: 13), PSMA (634-642) (SEQ ID NO: 14), SPAS-1 (SNC9-H8) (SEQ ID NO: 15), SIMS2 (237-245) (SEQ ID NO: 16), PAP (115-123) (SEQ ID NO: 17), B16 22. The SNA according to any one of claims 1 to 21, which is MART-1(M27) (SEQ ID NO: 9), TRP-1(252-260) (SEQ ID NO: 18), TRP-1(252V260M) (SEQ ID NO: 19), TRP-1(455-463) (SEQ ID NO: 20), TRP-1(455A463M) (SEQ ID NO: 21), TRP-2(180-188) (SEQ ID NO: 22), Melan-A / MART-(127-135), Tyrokinase(1-9), Tyrokinase(369-377D), MC38 Adpgk (SEQ ID NO: 23), Irgq-minimal (SEQ ID NO: 24), Irgq-long peptide (SEQ ID NO: 25), or a combination thereof.

23. The MHC-II antigen is OVA ３２３－３３９ (OVA2) (SEQ ID NO: 8), GP100: (46-58) (SEQ ID NO: 26), TC-1 E6 (43-57) (SEQ ID NO: 27), SIMS2 (240-254) (SEQ ID NO: 28), PAP (114-128) (SEQ ID NO: 29), B16 23. The SNA according to any one of claims 1 to 22, which is MART-1(M30) (SEQ ID NO: 30), TRP-1(113-127) (SEQ ID NO: 31), TRP-1(106-130) (SEQ ID NO: 32), Li-Key(77-92) (SEQ ID NO: 33), tyrosinase(56-70), GP100(44-59), GP100(167-189), Melan-A / MART-1(102-111) (SEQ ID NO: 34), Melan-A / MART-1(27-40) (SEQ ID NO: 35), Melan-A / MART-1(51-70) (SEQ ID NO: 36), Melan-A / MART-1(51-73) (SEQ ID NO: 37), Melan-A / MART-1(43-57) (SEQ ID NO: 38), or a combination thereof.

24. The SNA according to any one of claims 1 to 23, wherein at least one of said one or more immunostimulatory oligonucleotides is a toll-like receptor (TLR) agonist.

25. The SNA according to any one of claims 1 to 24, wherein each of said one or more immunostimulatory oligonucleotides is a toll-like receptor (TLR) agonist.

26. 26. The SNA according to claim 24 or 25, wherein the TLR is selected from the group consisting of toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 5 (TLR5), toll-like receptor 6 (TLR6), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), toll-like receptor 9 (TLR9), toll-like receptor 10 (TLR10), toll-like receptor 11 (TLR11), toll-like receptor 12 (TLR12), and toll-like receptor 13 (TLR13).

27. The SNA according to any one of claims 24 to 26, wherein said TLR is TLR9.

28. The SNA according to any one of claims 1 to 27, wherein said immunostimulatory oligonucleotide comprises a CpG nucleotide sequence.

29. 29. A SNA according to any one of claims 1 to 28, wherein one or more of the oligonucleotides within the shell of oligonucleotides comprises or consists of one or more of the sequences 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 39), 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 40), 5'-TCCATGACGTTCCTGACGTT(spacer-18(hexaethylene glycol)) 2 cholesterol-3' (SEQ ID NO: 41) and 5'-TCGTCGTTTTGTCGTTTTGTCGTT(spacer-18(hexaethylene glycol)) 2 cholesterol-3' (SEQ ID NO: 6).

30. 30. The SNA of any one of claims 1 to 29, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the oligonucleotides within the oligonucleotide shell are immunostimulatory oligonucleotides.

31. 31. The SNA of any one of claims 1 to 30, wherein the linker is a carbamate alkylene disulfide linker, a thiol linker, a disulfide linker, an amido alkylene disulfide linker, an amido alkylene thio-succinimidyl linker, or a combination thereof.

32. The SNA according to any one of claims 1 to 31, wherein the nanoparticle core is a micelle, a liposome, a polymer, a lipid nanoparticle (LNP), or a combination thereof.

33. 33. The SNA of claim 32, wherein the polymer is a polylactide, a polylactide-polyglycolide copolymer, polycaprolactone, polyacrylate, alginate, albumin, silica, polypyrrole, polythiophene, polyaniline, polyethyleneimine, poly(methyl methacrylate), or chitosan.

34. The SNA of claim 33, wherein the polymer is poly(lactic-co-glycolic acid) (PLGA).

35. The SNA according to any one of claims 1 to 34, wherein the nanoparticle core is a liposome.

36. The liposome may be selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPC ... PG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), and cholesterol.

37. A SNA according to any one of claims 1 to 36, wherein one or more of the oligonucleotides within the shell of oligonucleotides are attached to the outer surface of the nanoparticle core via a lipid anchor group.

38. 38. The SNA of claim 37, wherein said lipid anchor group is attached to the 5' or 3' end of said one or more oligonucleotides.

39. 39. The SNA of claim 37 or 38, wherein the lipid anchor group is tocopherol or cholesterol.

40. A SNA according to any one of claims 1 to 39, wherein one or more of the oligonucleotides within the shell of said oligonucleotides are modified at their 5'-end and / or 3'-end with dibenzocyclooctyl (DBCO) and / or thiol.

41. A SNA according to any one of claims 1 to 40, wherein the shell of oligonucleotides comprises DNA oligonucleotides, RNA oligonucleotides or a combination thereof.

42. A SNA according to any one of claims 1 to 41, wherein said shell of oligonucleotides comprises DNA oligonucleotides and RNA oligonucleotides.

43. 43. The SNA according to any one of claims 1 to 42, wherein the shell of oligonucleotides comprises single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA or a combination thereof.

44. A SNA according to any one of claims 1 to 43, wherein one or more of the oligonucleotides within the shell of said oligonucleotides is a modified oligonucleotide.

45. A SNA according to any of claims 1 to 44, wherein the shell of oligonucleotides comprises between 2 and 200 oligonucleotides, between 2 and 100 oligonucleotides, between 150 oligonucleotides, between 200 oligonucleotides, between 10 and 80 oligonucleotides, or between 75 oligonucleotides.

46. 46. ​​A SNA according to any one of claims 1 to 45, wherein each oligonucleotide within said shell of oligonucleotides is between 5 and 1000 nucleotides in length, between 10 and 50 nucleotides in length, or between 20 and 30 nucleotides in length.

47. 47. The SNA of any one of claims 1 to 46, wherein the diameter of the SNA is between 1 nanometer (nm) and 500 nm, up to 80 nanometers, or up to 50 nanometers.

48. 48. The SNA according to any one of claims 1 to 47, wherein the shell of oligonucleotides comprises targeting oligonucleotides, inhibitory oligonucleotides, non-targeting oligonucleotides, or a combination thereof, wherein the inhibitory oligonucleotides are antisense oligonucleotides, small interfering RNA (siRNA), aptamers, short hairpin RNA (shRNA), DNAzymes, or aptazymes.

49. A composition comprising a plurality of SNAs according to any one of claims 1 to 48.

50. 50. The composition of claim 49, wherein at least two SNAs in the plurality comprise different nanoparticle cores.

51. A pharmaceutical formulation comprising a plurality of SNAs according to any one of claims 1 to 48 or a composition according to claim 49 or 50, and a pharma- ceutically acceptable carrier or diluent.

52. A vaccine comprising a SNA according to any one of claims 1 to 48, a composition according to claim 49 or 50, or a pharmaceutical formulation according to claim 51.

53. 53. The vaccine of claim 52, comprising an adjuvant.

54. 52. An antigenic composition comprising an SNA according to any one of claims 1 to 48, or a pharmaceutical formulation according to claim 51, in a pharma- ceutically acceptable carrier, diluent, stabilizer, or preservative, which is capable of generating an immune response in a subject, including antibody generation, activation of cytotoxic T cells, activation of helper T cells, or a protective immune response.

55. 55. The antigenic composition of claim 54, wherein the immune response comprises an antibody response.

56. 56. The antigenic composition of claim 55, wherein the antibody response is a neutralizing antibody response or a protective antibody response.

57. A method for inhibiting expression of a gene product, comprising hybridizing a polynucleotide encoding the gene product to an inhibitory oligonucleotide described in claim 48, wherein hybridization between the polynucleotide and the inhibitory oligonucleotide occurs over a length of the polynucleotide that has a sufficient degree of complementarity to inhibit expression of the gene product.

58. 58. The method of claim 57, wherein expression of the gene product is inhibited in vivo or in vitro.

59. A SNA as described in any one of claims 1 to 48, a composition as described in claim 49 or 50, a pharmaceutical formulation as described in claim 51, a vaccine as described in claim 52 or 53, or an antigenic composition as described in any one of claims 54 to 56 for generating an immune response in a subject.

60. 60. The SNA, composition, pharmaceutical formulation, vaccine, or antigenic composition of claim 59, wherein said immune response comprises an antibody response.

61. 61. The SNA, composition, pharmaceutical formulation, vaccine, or antigenic composition of claim 60, wherein said antibody response is a pan-antigen-specific antibody response.

62. 61. The SNA, composition, pharmaceutical formulation, vaccine, or antigenic composition of claim 60, wherein said antibody response is a neutralizing antibody response or a protective antibody response.

63. A SNA according to any one of claims 1 to 48, a composition according to claim 49 or 50, a pharmaceutical formulation according to claim 51, a vaccine according to claim 52 or 53, or an antigenic composition according to any one of claims 54 to 56 for immunizing a subject against one or more antigens.

64. 64. The composition or vaccine of claim 63, wherein the composition or vaccine is a cancer vaccine.

65. A SNA according to any one of claims 1 to 48, a composition according to claim 49 or 50, a pharmaceutical formulation according to claim 51, a vaccine according to claim 52 or 53, or an antigenic composition according to any one of claims 54 to 56 for the treatment of cancer.

66. 66. The SNA, composition, pharmaceutical formulation, vaccine, or antigenic composition of claim 64 or 65, wherein the cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, glioblastoma, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, lymphoma, non-Hodgkin's lymphoma, osteocarcinoma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and human papillomavirus-induced cancer, or a combination thereof.

67. 67. The SNA, composition, pharmaceutical formulation, vaccine, or antigenic composition of claim 65 or 66, wherein the cancer is melanoma, colon cancer, or lymphoma.

68. The SNA, composition, pharmaceutical formulation, vaccine, or antigenic composition of any one of claims 59 to 67, comprising: The SNA, composition, pharmaceutical formulation, vaccine, or antigenic composition is administered to a subject together with an additional agent; The SNA, composition, pharmaceutical formulation, vaccine, or antigenic composition, wherein the additional agent is an anti-programmed cell death protein 1 (PD-1) antibody, an anti-programmed death-ligand 1 (PD-L1) antibody, a cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody, a T-cell immunoglobulin and ITIM domain (TIGIT) antibody, or a combination thereof.

69. 59. The method of claim 57 or 58, wherein the SNA is an SNA according to claim 5 or an SNA according to claim 9.

70. The SNA, composition, pharmaceutical formulation, vaccine or antigenic composition of any one of claims 58 to 68, wherein the SNA is an SNA of claim 5 or an SNA of claim 9.

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