Synergistic Immunostimulation through the Dual Activation of Toll-like Receptor 3/9 with Spherical Nucleic Acids

Toll-like receptors (TLRs) are a family of proteins that modulate the innate immune system and control the initiation of downstream immune responses. Spherical nucleic acids (SNAs) designed to stimulate single members of the TLR family (e.g., TLR7 or TLR9) have shown utility in cancer immunotherapy. We hypothesized that SNAs synthesized with multiple TLR agonists would enable the simultaneous activation of multiple TLR pathways for maximally synergistic immune activation. Here, we describe the synthesis of SNAs that incorporate both a TLR3 agonist (polyinosinic:polycytidylic acid, poly(I:C)) and TLR9 agonist (CpG oligonucleotide) on the same liposomal scaffold. In this design, CpG comprises the SNA oligonucleotide shell, and poly(I:C) is encapsulated in the liposome core. These dual-TLR activating SNAs efficiently codeliver high quantities of both agonists to the same target cell, yielding enhanced immunostimulation in various murine and human antigen-presenting cells (APCs). Moreover, codelivery of TLR agonists using the SNA both synchronizes and prolongs the duration of costimulatory molecule and major histocompatibility complex class II expression in APCs, which has been shown to be important for efficient downstream immune responses. Taken together, this SNA design provides a strategy for potently activating immune cells and increasing the efficiency of their activation, which likely will inform the preparation of nanomaterials for highly potent immunotherapies.