PROJECT SUMMARY Our research team is proposing to establish an alternative type of cancer immunotherapy centered on a selective and localized bacterial infection within the tumor mass. The working hypothesis is that directing bacteria selectively to a tumor mass will promote the clearance of the tumor by the patient’s own immune system. Because the bacteria used will not be drug resistant (we can dictate which bacteria are implanted), the bacterial infection can be readily cleared when needed by the application of antibiotics. We propose to control the localization and adhesion of bacteria by grafting tumor-homing epitopes onto bacterial cell surfaces. Once they reach the tumor, the small number of bacteria will grow, multiply, and imbed into the tumor. Aim 1. We will set up a screening platform to efficiently identify bacteria whose surface can be robustly chemically remodeled. Surface remodeling will be performed based on the metabolic incorporation of analogs of precursors to surface bound biomacromolecules. The conserved nature of these biomacromolecules, their exposure to the extracellular space, and established methods of metabolic labeling with unnatural epitopes provides the basis to chemically graft tumor-binding agents. Aim 2. We will graft tumor-targeting moieties onto the surface of bacteria selected from Aim 1 to refine the set of candidate bacteria based on their ability to specifically bind to cancer cells. We will use two modalities of tumor-targeting: cancer biomarkers overexpressed overexpressed on the surface of cancer cells, and the inherently low pH microenvironment of tumors. Aim 3. We will evaluate tumor targeting and colonization by surface reprogrammed bacteria (selected in Aim 2) in xenograft cancer models in mice. Bacterial load levels will be quantified in various tissues and in blood to determine the colonization of bacteria.