Abstract More than 80,000 Americans are diagnosed with bladder cancer each year, and more than 17,000 die from the disease. Approximately 75% of new bladder cancer patients present with non-muscle invasive bladder cancer (NMIBC). Not only is NMIBC associated with high recurrence rates (>50%) and risk of progression, but 30% of patients are unresponsive to the standard of care treatment: transurethral resection with intravesical Bacillus Calmette-Guérin (BCG) instillation, the only approved microbial therapeutic for cancer. These individuals are left with limited therapeutic options. While there have been efforts to generate improved recombinant BCG (rBCG) strains, such efforts have not yet yielded demonstrable improvement over traditional BCG. With very few advances in treatment over the past two decades for early stage disease and a limited pipeline of therapeutics in development, there is a major unmet need for improved treatments for NMIBC. To address this need, OncoSTING is developing OS-101, a breakthrough rBCG immunotherapy that overexpresses a potent Stimulator of IFN Genes (STING) agonist. STING agonists potentiate anti-tumor responses through the innate immune STING-IRF3-NF-κB pathway. While other companies are developing small molecule STING agonists as novel anti-cancer immunotherapies, OncoSTING is the only company developing STING agonist delivery by a live bacterial vaccine—BCG—that itself is a well-known immunotherapy already in use for the treatment of bladder cancer, thus offering the benefits of both. Because it is a live bacteria, OS-101 allows for continuous and prolonged delivery of the STING agonist to the tumor microenvironment. Compared with wild type BCG, OS-101 demonstrates superior antitumor efficacy in models of NMIBC; more potent pro-inflammatory cytokine responses; greater myeloid cell reprogramming, producing an M1 shift with enhanced phagocytosis/autophagy; more pronounced epigenetic changes in key cytokine promoter regions; and metabolomic changes favoring antitumor immunity. In Phase I of this Fast Track project, OncoSTING will create a next-generation, antibiotic resistance-free version of 0S-101 using a novel, patent-pending method. The current rBCG prototype, OS-101, relies on a bacterial plasmid that is maintained using an antibiotic resistance cassette. However, Phase 3 studies will require the removal of antibiotic resistance genes. The efficacy of the new construct, called OS-151, will then be confirmed in four relevant bioassays. In Phase II, OS-151 will be compared to ADU-S100 (Aduro's small molecule STING agonist which is in current clinical trials) and wild type BCG in relevant models of bladder cancer. Pre-IND, pharmacokinetic and safety studies of OS-151 will also be conducted, and optimization work will ensure production of OS-151 at scale. Finally, tumor repetitive dosing studies and rechallenge studies will be carried out in mouse syngeneic models. Once approved, OS-151 will first be used to treat...