PROJECT SUMMARY/ABSTRACT Recent advances in cancer immunotherapy have provided promising treatment options for patients with triple- negative breast cancer (TNBC). Despite overall success in treating these malignancies, immunotherapeutic ap- proaches face a number of unique challenges: (1) dose limitation due to off-target side effects, (2) additive toxicity of combination therapies, (3) and relatively low immunogenicity of breast cancer. To overcome these limitations, this proposal seeks to engineer probiotic strains of bacteria that selectively colonize tumors of breast-cancer origin and locally release therapeutics. The ultimate goal is to create clinic-ready strains that will efficiently local- ize and release high-doses of therapeutics, while maintaining safety for patients. To do so, the accompanying project will focus on engineering genetically encoded encapsulation systems to improve intravenous (IV) delivery of therapeutic bacteria for breast cancer therapy. In previous studies, we have found that a single injection of a probiotic strain E. coli Nissle 1917 (EcN) can colonize multiple primary and metastatic tumors of breast origin, relevant for TNBC patients that have metastatic disease across organs such as the liver, brain, bone, and lung. However, in clinical trials with IV injection of genetically-attenuated bacteria, less than 15% of patients demonstrated efficient colonization of tumors, although safe administration doses were noted. Using synthetic biology approaches, we previously engineered EcN for transient induction of capsule polysaccharides on the bacteria surface (termed iCAP), which increases maximally-tolerated doses in mice by 10-fold. In the first aim, we will genomically integrate this system and combine it with genetic attenuations used in clinical trials, and other safety systems we have built. We will remove antibiotic markers from the probiotic strain for future translation as well. Since humoral antibody responses to our EcN-iCAP will be generated in humans, they will limit the efficacy of the system to generate multiple injections. Thus, we will next construct two other encapsulation systems (polysialic acid, chondroitin), to enable sequential delivery of distinct strains. These approaches will be characterized and tested through multiple in vitro assays and in mouse models. Altogether the approach of using engineered probiotics has several advantages over current therapeutic strat- egies, including: (1) tumor-specific production of therapeutics, (2) bacteria lysis that leads to effective release of novel therapeutics and lipopolysaccharides (LPS) adjuvant, and (3) enhanced efficacy and safety from combi- nations and multiple encapsulation systems. This work seeks to overcome current limitations of immunothera- pies, by providing a targeted vehicle to locally deliver therapeutics that stimulate antitumor immunity while pre- venting systemic toxicity and mitigating immune-related adverse effects.

PROJECT NARRATIVE The engineering of tumor-specific bacteria is a transformative approach to cancer therapy, since bacteria can be genetically programmed to sense and respond to environments and locally release therapeutics. This proposal seeks to engineer genetically encoded encapsulation systems to improve intravenous delivery of therapeutic bacteria for breast cancer therapy. The objective is to create probiotic strains with multiple encapsulation systems, and combine them with previous attenuations tested in clinical trials, to improve colonization efficiency and efficacy while maintaining safety.