Imaging and modulation of immunophenotype Most importantly, we have created TLR delivery nanotherapeutics and find that TLR NPs combined with aCD40, aPD-1 and aCTL4 (abbreviated as CP4 as in emerging pancreatic cancer studies) efficiently regressed implanted multisite invasive murine pancreatic tumors. We have developed multiple strategies and are particularly focused on 18 nm biodegradable, multi-functional particles that combine immune-modulating peptides, targeting peptides and toll-like receptor (TLR) agonists. We specifically included the immune modulating peptide PADRE (T helper modulation) and mannose (to enhance macrophage uptake) in addition to the TLR7/8 agonist (resiquimod) in preliminary work. To maximize payload, we built upon biocompatible unimicellar nanoparticles via the combination of highly efficient esterification and metal-free click reactions and find that the particle metabolites clear through the kidneys. In our preliminary studies, TLR7/8-nanoparticle treatment combined with CP4 enhanced response in a highly metastatic, multi-site implanted pancreatic cancer model (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre model: abbreviated as KPC). New preliminary data indicate that TLR7/8 agonists and aCD40 each have direct efficacy against pancreatic tumor cells. RNAseq results demonstrate that TLR7/8 agonists and CD40 enhance complementary pathways (C-lectin for CD40 (among others) and TLR/interferon for TLR agonists). We find that the combination enhances anti-tumor leukocytes, regresses KPC tumors and for responders, 100% do not grow tumor on re-challenge. By monitoring OX40 expression (a marker of T cell activation), we demonstrated that unlike other immune modulating approaches involving aCD40, T cells were activated. We have simultaneously developed the ability to monitor OX40 expression using positron emission tomography in a noninvasive fashion. Our primary goal in the proposed work is to move the nanotherapy strategy forward to human translation. As a result, we will evaluate efficacy in models of pancreatic cancer in rodents and safety in a larger animal model. Further, we will evaluate samples from patients undergoing biopsy for pancreatic cancer to better characterize the immune environment. We have 2 major goals: 1) the development of an effective strategy for systemically-administered T cell modulation and 2) combining this with positron emission tomographic imaging and RNA sequencing to optimize multi-component protocols. Within Aim 1, we will determine the optimal carrier properties to maximize T cell modulation by 1a) modulating nanoparticle characteristics and evaluating resulting efficacy, 1b) using positron emission tomography (PET) imaging to quantify accumulation of the systemically-injected NP agonists, and 1c) assessing toxicity through dose escalation and a large animal study, leading to IND filing. Within Aim 2, develop an imaging and in vitro assessment strategy for T cell activation by utilizing 2a) OX40 PET imaging and 2b) flow cytometry and RNAsequencing.