Project Abstract The most important unmet need in oncology is to overcome the lack of effective therapy for advanced solid tumors in adults and children. Despite major advances in immunotherapy, radiation therapy, precision medicine, and nuclear medicine, the vast majority of advanced solid tumors presenting clinically today are still incurable. Radiotheranostic therapies offer the tremendous advantages of precision medicine and patient selection over other cancer treatment modalities but lead to objective responses in only 30-60% of patients. Innovations in radiopharmaceutical therapy (RPT) to address the major barriers to consistent tumor control are sorely needed: suboptimal drug delivery and lack of retention of radionuclides at the target site. For cancer, RPT is administered as an unconjugated or chelated radionuclide or in combination with a delivery vehicle, such as a peptide or antibody (radioimmunotherapy). This project aims to develop a highly versatile RPT platform that harnesses engineered bacteria to concentrate therapeutic radionuclides in tumors. We hypothesize that an engineered bacterial fusion protein can serve as an in vivo artificial receptor for a small radionuclide carrier (as anti-2,2′,2”,2”'- (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA)-radiohapten). This construct, DOTA- binding Salmonella, can specifically colonize tumors following intravenous administration and is cleared via the liver and spleen. Notably, bacteria do not colonize in the radiosensitive red bone marrow or kidneys, which are typically organs-at-risk during RPT. Based on the known pharmacokinetics, biodistributions, and clearance properties of engineered bacteria, the DOTA-radiohapten can be injected precisely at the time of peak bacterial tumor-to-normal tissue accumulation ratios (eg, after 48 hours, tumor-to-spleen ratios of 104 are typical). The intratumoral bacteria will capture the DOTA-radiohapten and plasma DOTA-radiohapten will be rapidly and efficiently excreted from the body via the renal route. This project has two Aims. In Aim 1 we will genetically engineer Salmonella to express surface-anchored DPB characterize its functionality in vitro. In Aim 2, we will demonstrate the efficacy of our proposed radio-theranostic treatment paradigm based on Salmonella-DPB + 86/90Y-DOTA in mouse tumor models. This strategy has several specific advantages over other radioimmune approaches. The number of radiohapten (DOTA) binding sites per gram of tumor has the potential to be orders of magnitude greater than the number of the surface-marker sites on cancer cells. The strategy will produce unprecedented therapeutic indices for critical organs (tumor vs. kidneys and bone marrow) because Salmonella are cleared from the blood hours after injection, and accumulation in the kidneys is minimal. Salmonella, engineering with its high tumor specificity, deep tissue penetration, and plasticity, make it a highly promising for RPT.Engineered ...