Abstract 3608: Nonpathogenic E. coli engineered to surface display human cytokines for enhanced immunotherapy

Abstract Background: Poor trafficking, exhaustion and low persistence of adoptively transferred T and NK cell-based therapies in solid tumors remain major barriers in the field. Leveraging safety, tumor tropism and ease of genetic manipulation in bacteria present potentially a novel approach to overcome these barriers. Prior approaches to bacteria-based biologics delivery encounter issues of low concentration and fast clearance. In this study, we hypothesized that displaying immune-activating cytokines on outer membrane of Escherichia coli (E. coli) could modulate immunosuppressive tumor microenvironment (TME) enhancing migration, activation, and tumor control mediated by T and natural killer (NK) cells. Methods: To investigate our hypothesis, we expressed human and murine cytokines: IL-15, decoy resistant IL-18 (DR-18), and IL-21 on outer membrane of a non-pathogenic facultative bacteria E. coli K-12. We first evaluated their ability to activate human mesothelin (MSLN) NK cell CARs in vitro and performed gene expression analysis. For the in vivo experiments we used C57BL/6 mouse model with syngeneic tumor cell lines MC38 and B16F10. We also assessed the ability of human cytokine-displaying bacteria with human MSLN-CAR NK cells in NSG mice carrying human mesothelioma H226. Results: OmpA displaying murine DR-18 on bacterial surface was most effective in tumor control. After optimization, intra-tumoral injection of the bacteria resulted in 50% of cured mice bearing MC38 and 30% of cured mice bearing B16F10. Rechallenging these mice with respective cells showed no tumor growth, indicating induction of tumor-specific immune memory. Tumor tissue analysis showed upregulation of CD8 T and NK cells and depletion experiments demonstrated the contribution of these cells to tumor control. When systemically injected, the bacteria inhibited tumor growth with no side effects. Bio-distribution analysis demonstrated bacterial enrichment in TME. Through the utilization of in vitro killing assays by NK cells, YiaT232 scaffold with human DR-18 emerged as the optimal candidate. RNA-sequencing demonstrated that bacteria with DR-18 were capable of upregulating IL-12 and oxidative phosphorylation pathway in NK cells. Furthermore, administration of bacteria displaying DR-18 and systemic (IV) MSLN-CAR-NK cells led to better tumor control (vs soluble DR-18 or bacteria alone) in NSG mice carrying mesothelin-high H226. Conclusions: Here we develop a novel platform for immunotherapy involving surface display of cytokines in non-pathogenic E. coli. The bacteria were safe and demonstrated tumor tropism and CD8 T and NK cells were identified as the major effector cells mediating tumor control in immune-competent mice. Subsequently, we could display human DR-18 on these bacteria which resulted in superior tumor control in NSG mice when combined with MSLN-CAR-NK cells, paving the way for clinical translation in the near future. Citation Format: Shaobo Yang, Michal Sheffer, Isabel Kaplan, Zongqi Wang, Mubin Tarannum, Roman Shapiro, Yasmin Abdulhamid, Rebecca Porter, David Barbie, Robert Soiffer, Jessica Little, Jerome Ritz, Mengdi Yang, Valeria Márquez-Pellegrin, Jiahe Li, Rizwan Romee. Nonpathogenic E. coli engineered to surface display human cytokines for enhanced immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3608.