Summary This R01, submitted to the Toward Translation of Nanotechnology Cancer Interventions (TTNCI) program, is a continuation of our successful U01 CA218292 (09/2017-07/2022), funded through the NCI Alliance for Nanotechnology in Cancer program. During the U01 project period, we demonstrated that our nanoparticles derived from the plant virus cowpea mosaic virus (CPMV) stimulate a potent antitumor immune response against multiple tumor mouse models. Additionally, trials in companion dogs with melanoma, sarcoma, and breast cancer demonstrate potent antitumor efficacy with our unique immunotherapy approach. CPMV is an intratumoral injected in situ vaccine (ISV) technology, with an ability to induce durable immune-mediated antitumor efficacy against treated and untreated tumors as well as immunological memory against recurrence. Mechanistically, CPMV activates the innate immune system to improve processing of tumor-associated and neoantigens, thereby resulting in a more functional adaptive antitumor immunity against antigens expressed by the tumor. This results in remissions of treated and untreated malignant tumors, and protection from recurrence via the adaptive arm and immune memory. Building on this strong foundation of data as well as intellectual property, this project will focus on optimizing GMP manufacturing and pharmacology in murine tumor models and canine patients to provide the foundational basis of future human oncology trials. The multi-PI leadership team Steinmetz (UC San Diego), Fiering (Dartmouth) and Ranjan (OSU) brings complimentary expertise in plant virus-based CPMV nanotechnology, cancer immunology, and veterinary oncology/immunotherapy trials. Our team is also supported by a group of consultants with expertise in regulatory (Garnick, Mosaic IE Inc.), pharmacology/toxicology (Luksic, Intrinsik), clinical (Garovoy, Mosaic IE Inc.) affairs, as well as experience in vertical farming (Eisenberg, OPO). We believe that our strong team of collaborators can help establish the future manufacture of the plant-derived biologic. We will fulfil the following Specific Aims: (1) Chemistry, Manufacturing, and Control (CMC) method development for scalable manufacture and QAC of the CPMV- ISV, (2) Pharmacology: Determine biodistribution, dosing, and toxicology of the CPMV-ISV drug candidate in orthotopic and metastatic mouse models of melanoma; and (3) Canine trials: establish dosing and safety in canine oncology trials in varying tumor types amenable for in situ vaccination (melanoma, sarcoma, and mast- cell tumor). Through parallel trials in murine tumor models and dog patients, we will identify biopsy or blood biomarkers for longitudinal monitoring of treatment response. Successful completion would position us for good manufacturing practice (GMP) production of this plant virus-based biologic nanotechnology and to continue the developmental path, e.g. through the NCI Experimental Therapeutics (NExT) program, thereby translating the CPMV-ba...