Designing probiotic strains to sense and deliver novel treatments for oral cancer - PROJECT SUMMARY Oral squamous cell carcinoma (OSCC) is a significant global health challenge, with over 350,000 new cases annually and a five-year survival rate of less than 50% for advanced stages. Current therapies, including immune checkpoint inhibitors (ICIs), are often ineffective due to therapeutic resistance and immune evasion within the tumor immune microenvironment (TIME). The TIME in OSCC is characterized by chronic inflammation, low pH, and immunosuppressive cytokines, which hinder immune cell infiltration and activation. This project proposes an innovative therapeutic strategy using engineered Lactobacillus fermentum, an oral commensal bacterium with intrinsic antitumor activity, to deliver novel immune-activating components directly to the OSCC TIME. Specifically, we will develop a novel fusion protein, CCL5-NBαPD1, which combines the chemotactic activity of CCL5 with the immune checkpoint modulation of NBαPD1. This approach aims to reprogram the TIME, enhance immune infiltration, and overcome resistance to immunotherapy. Our hypothesis is that engineering L. fermentum to deliver CCL5-NBαPD1 and enhance biofilm formation will reprogram the OSCC TIME and improve antitumor immunity. This hypothesis will be tested through two specific aims: (1) Engineer L. fermentum to deliver CCL5 or CCL5-NBaPD1, a novel approach to reprogram the OSCC TIME and enhance antitumor immunity. We will engineer L. fermentum to secrete either CCL5 to augment existing approaches (e.g., a-PD1 Ab) or a novel, all- in-one agent, CCL5-NBαPD1, a fusion protein we have designed to combine CCL5’s chemotactic activity with a nanobody domain of similar activity to a-PD1 (NBαPD1) to provide immune checkpoint modulation. Using syngeneic HPV- OSCC models, we will evaluate tumor growth inhibition, immune cell infiltration, and therapeutic efficacy compared to a-PD1. (2) Develop and characterize a tumor-responsive circuit in L. fermentum to dynamically regulate therapeutic production. We will engineer L. fermentum to overexpress biofilm- promoting genes, enhancing adherence to the oral mucosa and tumor surface. Additionally, we will incorporate synthetic quorum-sensing circuits with low pH-responsive systems to enable targeted payload release in the acidic OSCC TME (pH <6.5). The impact of this project lies in its potential to transform OSCC treatment by integrating synthetic biology with immunotherapy. By leveraging L. fermentum as a delivery platform, this study addresses the unmet need for effective treatments in resistant OSCC and lays the foundation for microbiota- based therapies that reprogram the TIME and enhance cancer immunotherapy outcomes. The team, combining expertise in bacterial genetics, immunotherapy, syngeneic mouse models, and clinical perspectives, is well- equipped to execute this project and advance treatment for immunotherapy-resistant OSCC.
