Agonists of toll like receptors (TLRs) are promising anticancer vaccine adjuvants because of their ability to induce proinflammatory cytokines necessary to generate a robust immune response. However, currently available TLR agonists suffer from a number of limitations including self-regulatory immunosuppression and unfavorable local pharmacokinetics resulting in poor availability within dendritic cells. Further, current TLR agonist-based anticancer vaccines generate a robust cytotoxic CD8 T cell response but not CD4 Th 1 helper T cell response, which is critical for inducing effective, long-term antitumor immunity. We will address these important challenges through a synergistic combination of drug discovery and drug delivery efforts. Our team has developed a suite of highly substituted imidazoquinolines, which activate TLR7 and/or 8 and induce significantly higher levels of cytokines compared to imiquimod, an FDA approved TLR7 agonist. Our studies show the balance between proinflammatory and immunosuppressive cytokines can be tuned through structural modifications. Encapsulation of these novel agonists in acidic pH responsive nanoparticles (NPs) resulted in robust activation of CD4 and COB T cells as well as natural killer (NK) cells, leading to a stronger anticancer immune response than free agonist or that encapsulated in non-pH responsive NPs. Importantly, intradermal delivery of NP vaccine using a hollow microneedle platform led to an enhanced Th1 immune response, which is essential for effective induction of long-term antitumor immunity. We will build on these exciting findings and further optimize the new agonists for efficient encapsulation in pH responsive NPs, tune the NP properties for improved targeting of dendritic cells following delivery via hollow microneedles, and investigate potentiation of NK cell-mediated antibody-mediated cellular cytotoxicity. The Specific Aims of this revised R01 grant application include: Aim 1: Design and synthesize TLR7/8 agonists that are optimized for NP encapsulation Aim 2: Optimize pH responsive NP formulation for hollow microneedle-assisted ID delivery Aim 3: Determine anticancer efficacy of NP vaccine following hollow microneedle-assisted ID delivery We expect our studies will identify new design principles and delivery strategies for TLR agonists that overcome the limitations of current anticancer vaccines and further advance the field of cancer immunotherapy.