In the case of cancer immunotherapy, nanostructures are attractive because they can carry all of the necessary components of a vaccine, including both antigen and adjuvant. Herein, we explore how spherical nucleic acids (SNAs), an emerging class of nanotherapeutic materials, can be used to deliver peptide antigens and nucleic acid adjuvants to raise immune responses that kill cancer cells, reduce (or eliminate) tumor growth, and extend life in three established mouse tumor models. Three SNA structures that are compositionally nearly identical but structurally different markedly vary in their abilities to cross-prime antigen-specific CD8+ T cells and raise subsequent antitumor immune responses. Importantly, the most effective structure is the one that exhibits synchronization of maximum antigen presentation and costimulatory marker expression. In the human papillomavirus-associated TC-1 model, vaccination with this structure improved overall survival, induced the complete elimination of tumors from 30% of the mice, and conferred curative protection from tumor rechallenges, consistent with immunological memory not otherwise achievable. The antitumor effect of SNA vaccination is dependent on the method of antigen incorporation within the SNA structure, underscoring the modularity of this class of nanostructures and the potential for the deliberate design of new vaccines, thereby defining a type of rational cancer vaccinology.

Spherical nucleic acids (SNAs) can be potent sequence-specific stimulators of antigen presenting cells (APCs). When loaded with peptide antigens, they can be used to activate the immune system to train T-cells to specifically kill cancer cells. Herein, the role of peptide chemical conjugation to the DNA, which is used to load SNAs with antigens via hybridization, is explored in the context of APC activation. Importantly, though the antigen chemistry does not impede TLR-9 regulated APC activation, it significantly augments the downstream T-cell response in terms of both activation and proliferation. A comparison of three linker types, (1) noncleavable, (2) cleavable but nontraceless, and (3) traceless, reveals up to an 8-fold improvement in T-cell proliferation when the traceless linker is used. This work underscores the critical importance of the choice of conjugation chemistry in vaccine development.

All metal salts and solvents were obtained from Sigman-Aldrich and used without further purification. 1 H and 13 C NMR spectra were recorded on a Varian 500 Mhz, Varian 400 Mhz or a Varian 300 Mhz spectrometer. High-resolution mass spectra were provided by the California Institute of Technology Mass Spectrometry Facility using JEOL JMS-600H High Resolution Mass Spectrometer. General ProceduresProcedure A for preparative scale (0.5 mmol) oxidation of alkenes (isolation): PdCl 2 (PhCN) 2 (0.05 mmol, 19.2 mg), CuCl 2 •2H 2 O (0.05 mmol, 8.5 mg) and NaNO 2 (0.025 mmol, 1.7 mg) were weighed into a 20 mL vial charged with a stir bar. The vial was sparged for 1 minute with oxygen (1 atm, balloon). Premixed and oxygen saturated tBuOH (7.5 mL) and MeNO 2 (0.5 mL) was added followed by the alkene (0.5 mmol). The solution was saturated with oxygen by an additional 30 seconds of sparging. The reaction was then allowed to stir at room temperature (20-25ºC) for 4 h under 1 atm oxygen (balloon). Next, the reaction was quenched by addition to water (ca. 50mL) and extracted three times with dichloromethane (ca. 25 mL). The combined organic layers were subsequently washed with a saturated solution of NaHCO 3 and dried over Na 2 SO 4 . The solvent was removed under reduced pressure and the desired aldehyde product was purified using flash chromatography (pentane/ether). The selectivity was calculated by 1 H NMR analysis of the unpurified reaction mixture. Long relaxation delays (d1=10) were applied due to the long T 1 of the aldehydic proton signal. Procedure B for analytical scale (0.2 mmol) oxidation of alkenes (NMR analysis):PdCl 2 (PhCN) 2 (0.02 mmol, 7.7 mg), CuCl 2 •2H 2 O (0.02 mmol, 3.6 mg) and NaNO 2 (0.01 mmol, 0.7 mg) were weighed into a 8 mL vial charged with a stir bar. The vial was sparged for 1 minute with oxygen (1 atm, balloon). Premixed and oxygen saturated tBuOH (3 mL) and MeNO 2 (0.2 mL) was added followed by the alkene (0.2 mmol). The solution was saturated with oxygen by an additional 15 seconds of sparging and then sealed under an atmosphere of oxygen. The reaction was then allowed to stir at room temperature (20-25ºC) for 4 h. Next, the reaction was quenched by addition to water (ca. 10mL) and extracted three times with dichloromethane (ca. 5 mL). The combined organic layers were subsequently washed with a saturated solution of NaHCO 3 and dried over Na 2 SO 4 . After volatiles were removed under reduced pressure, nitrobenzene was added as an internal standard. The resulting solution was subsequently subjected to 1 H NMR analysis to determine yield and selectivity.

A novel method for synthesizing and photopatterning colloidal crystals via light‐responsive DNA is developed. These crystals are composed of 10–30 nm gold nanoparticles interconnected with azobenzene‐modified DNA strands. The photoisomerization of the azobenzene molecules leads to reversible assembly and disassembly of the base‐centered cubic (bcc) and face‐centered cubic (fcc) crystalline nanoparticle lattices. In addition, UV light is used as a trigger to selectively remove nanoparticles on centimeter‐scale thin films of colloidal crystals, allowing them to be photopatterned into preconceived shapes. The design of the azobenzene‐modified linking DNA is critical and involves complementary strands, with azobenzene moieties deliberately staggered between the bases that define the complementary code. This results in a tunable wavelength‐dependent melting temperature (Tm) window (4.5–15 °C) and one suitable for affecting the desired transformations. In addition to the isomeric state of the azobenzene groups, the size of the particles can be used to modulate the Tm window over which these structures are light‐responsive.

Although the strategy of therapeutic vaccination for the treatment of prostate cancer has advanced to and is available in the clinic (Sipuleucel-T), the efficacy of such therapy remains limited. Here, we develop Immunostimulatory Spherical Nucleic Acid (IS-SNA) nanostructures comprised of CpG oligonucleotides as adjuvant and prostate cancer peptide antigens, and evaluate their antitumor efficacy in syngeneic mouse models of prostate cancer. IS-SNAs with the specific structural feature of presenting both antigen and adjuvant CpG on the surface (hybridized model (HM) SNAs) induce stronger cytotoxic T lymphocyte (CTL) mediated antigen-specific killing of target cells than that for IS-SNAs with CpG on the surface and antigen encapsulated within the core (encapsulated model (EM) SNAs). Mechanistically, HM SNAs increase the co-delivery of CpG and antigen to dendritic cells over that for EM SNAs or admixtures of linear CpG and peptide, thereby improving cross-priming of antitumor CD8 + T cells. As a result, vaccination with HM SNAs leads to more effective antitumor immune responses in two prostate cancer models. These data demonstrate the importance of the structural positioning of peptide antigens together with adjuvants within IS-SNAs to the efficacy of IS-SNA-based cancer immunotherapy.