Summary This R01 renewal application is focused on triple negative breast cancer (TNBC), which is an aggressive life- threatening disease with poor prognosis and increased likelihood of recurrence and distant metastasis. Advances in cancer immunotherapy have demonstrated that modulation of the patient’s immune system can result in dramatic antitumor activity. The most promising immunotherapy approaches are those that are personalized and take advantage of the unique neoantigens within each patient’s tumor. Toward this goal, we developed a plant virus nanoparticle immunotherapy approach that activates innate immune cells within the tumor microenvironment (TME) to launch adaptive, systemic, and durable antitumor immunity. Specifically, intratumorally injected cowpea mosaic virus (CPMV) demonstrates potent efficacy in multiple mouse models, incl. TNBC. Trials in companion dogs with breast cancer also demonstrate potent antitumor efficacy. During the previous funding cycle, we gained insights into the mechanism of action and demonstrated that CPMV is recognized by pathogen-associated molecular pattern (PAMP) receptors that detect danger signals and activate the innate immune system; specifically, CPMV is recognized by Toll-like receptors (TLR2, 4 and 7). Further, we developed and tested combination and dual-pronged treatment approaches: we demonstrated efficacy of CPMV as solo-treatment as well as in combination with radiation, chemotherapy, immunomodulatory drugs, and checkpoint inhibitors, amongst others. This proposal builds on this strong portfolio of data. Our first goal is to focus on dual-pronged CPMV that combines its immunomodulatory and antitumor immunity properties with checkpoint therapy (Aim 1). Checkpoint blocking antibodies are effective at removing inhibitory signals but as monotherapy have variable and limited efficacy. In situ vaccination with CPMV increases tumor antigen specific effector T cells and our preliminary data indicate that CPMV treatment synergizes with immune checkpoint therapy. Next, we seek to develop targeted approaches that effectively concentrate systemically administered CPMV in tumors and provide further therapy options to treat metastatic disease (Aim 2). S100A9- targeted CPMV will be studied: expression of S100A9 (also known as myeloid-related protein 14 [MRP-14]), is linked to inflammation and carcinogenesis. Higher S100A9 expression in breast cancer correlates with a worse prognosis. S100A9 expression is an early event in tumorigenesis, enhancing tumor aggressiveness and metastasis. In the TME, S100A9 is secreted to the extracellular matrix, making it a highly suitable target for nanomedicine. Recognizing the potential of S100A9 as a pharmacologic target, we developed S100A9-targeted CPMV that efficiently concentrates at sites of metastasis enabling potent efficacy preventing outgrowth of metastases. Here we set out to detail the mechanisms of action and understand the pharmacology of S100A9- targeted CPMV. ...