Engineering of Bacterial Species in Cancer Immunotherapy
Authors:   , Waanders Lisette 
Published: October 2024

The human immune system is crucial in the protection against microbes. The innate immune system is the first to respond to the threat upon breaching the epithelial barrier. Although the immune system is a sophisticated defense mechanism, some pathogenic species have co-evolved to persist and thrive in the human body. M. tuberculosis is a pathogenic bacterium transmitted through the air and is causative of the lung disease tuberculosis. To understand how mycobacteria interact with the immune system, we conducted a literature study presenting different studies on how intracellular pattern recognition receptors can recognize mycobacterial species. The type of mycobacterial species and the infection model dramatically influence the immune response and bacterial outcome.
The ESX-I secretion system in mycobacteria has an essential role in mycobacterial virulence and cytokine response. Frame-shift mutants were generated to identify the contribution of several ESX-I genes and the associated espACD operon on the immune response. For this, the pathogenic M. marinum was used as a model. Unlike the other frameshift mutants, the EspD M. marinum frameshift mutant was investigated further as it showed high IFN-β expression upon infection. IFN-β in the context of vaccination is interesting as it aids the adaptive immune response. Notably, the EspD frameshift mutant showed a similar infection profile in an embryonic zebrafish model. Therefore, we hypothesize that inhibition of IFN-β signaling is beneficial for the pathogen to prevent clearance by the immune system and could be mediated by EspD or a protein that is stabilized/secreted with the help of EspD.
In addition to optimizing bacteria for vaccination purposes, the natural properties of bacteria also hold great potential in cancer treatment. A hallmark of cancer is the local suppression of the immune system. Recognition of bacteria by the immune cells could provide a strong signal that might overcome this suppression. To further improve Ty21a as a bladder cancer therapeutic, we hypothesized that enhanced bladder binding of the bacterium stimulates the anti-tumor immune response. We have used the SpyCatcher-SpyTag technology to label Ty21a with adhesin FimH. We show that bacteria can be efficiently labeled with FimH utilizing this technique. In addition, FimH-coated Ty21a showed enhanced binding to mouse bladders in vivo. However, FimH-mediated enhanced bladder binding did not improve survival in a bladder cancer mouse model. We have also used SpyCatcher-SpyTag technology to label bacteria with Death receptor 5 nanobodies. Multimerization of death receptors triggers apoptotic cell death in susceptible cancer cell lines. We show that labeling bacteria with DR5nb triggers apoptosis in the epithelial cell line HeLa and the melanoma cell line Mel Juso.
In addition to bacterial surface labeling with SpyCatcher-SpyTag technology, we have also investigated enhancing the immunostimulatory properties of bacteria in immunotherapy. For this, we have investigated improving IFN-β induction via stimulation of the STimulator of INterferon Genes (STING) pathway. We have engineered. S. typhimurium to express murine cGAS and show that these engineered strains produce cGAMPs. Co-culture of the vaccine strain of S. typhimurium with ectopic murine cGAS expression also induced IFN-β in human donor DCs. In addition, in vitro activation of human CD8+ T cells with this engineered strain shows enhanced IFN-y expression. This indicates that our engineered strain can promote the adaptive immune response.
In conclusion, we have investigated the effect of modified intracellular bacteria M. marinum and S. typhimurium on the host immune system. We took different approaches: frame-shift mutations, protein-labeling with SpyCatcher-SpyTag, and ectopic protein expression. The opportunities are endless in the search for the most optimal bacterial vaccine. This thesis shows bacterial engineering is feasible and has many advantages for vaccine optimization and immunotherapy.