2025 Theses Doctoral

Engineering Probiotics as Versatile Vehicles for Precision and Broadly Applicable Immunotherapy in Solid Tumors

Im, Jongwon

Advances in synthetic biology have revolutionized the field of cell-based cancer therapeutics, enabling comprehensive genetic programming of bacteria for targeted therapy. Engineered probiotics offer a unique advantage in cancer immunotherapy due to their selective tumor-colonizing capabilities and ability to be programmed for controlled on-site therapeutic production and release.

My dissertation aims to engineer bacteria as a versatile therapeutic platform for solid tumor immunotherapy, integrating synthetic biology tools to advance both precision and broadly applicable bacterial cancer therapeutics.

Bacteria serve as natural immune adjuvants due to their intrinsic immunostimulatory properties. Leveraging this feature, we engineered Escherichia coli Nissle 1917 as a probiotic-based cancer vaccine platform for precision immunotherapy. The bacteria were designed to express tumor-specific neoantigens and immunotoxin listeriolysin O, enhancing immune activation. To further improve antigen availability, we deleted two key proteases responsible for intracellular and extracellular protein degradation, which led to increased neoantigen accumulation for delivery. These modifications also improved the safety and specificity of the bacteria by increasing their susceptibility to immune-mediated clearance, including phagocytosis by antigen-presenting cells, which in turn facilitated robust antigen presentation and subsequent activation of CD4+ and CD8+ T cells. This approach effectively remodeled the tumor microenvironment and demonstrated efficacy across multiple cancer models, underscoring its potential as a versatile and potent precision cancer immunotherapy platform.

To broaden the applicability of bacterial therapeutics beyond precision therapy, we engineered a strain with reduced immunogenicity, tumor specificity, and adaptability for diverse therapeutic payload in situ production and extracellular release. To achieve this, we first screened multiple gene knockouts to attenuate lipopolysaccharide-mediated immunogenicity, significantly improving the strain’s safety profile. To further confine bacterial growth to the tumor microenvironment, we constructed a synthetic AND-gated promoter that integrates hypoxia and high lactate as dual input signals to drive essential gene expression, ensuring bacterial survival only within tumors.

Additionally, we modified the bacterial membrane to increase permeability, enabling the passive secretion of a broad range of therapeutic payloads within a defined size limit. This three-component engineered strain demonstrated a 100-fold increase in the maximum tolerable dose compared to the unmodified strain in animal tumor models, leading to significantly improved therapeutic efficacy. To validate its versatility in payload delivery, we programmed the strain to produce four distinct cytokines that activate lymphocytes and confirmed its ability to enhance anti-tumor immunity in vivo.

Together, these findings highlight the potential of engineered probiotics as a next-generation living medicine, capable of both precision and broadly applicable cancer immunotherapies, establishing a foundation for clinically translatable and highly tunable bacterial therapeutics.