Poster CC9 - Using engineered bacteria to activate the STING pathway for anti-tumor immune response in immunotherapy

Introduction: Cancer has emerged as a major health concern, contributing to a myriad of complications and standing as one of the leading causes of death globally in recent years. Traditional cancer therapies, such as surgery, chemotherapy, and radiation, aim to eliminate cancer cells but often come with considerable drawbacks. Immunotherapy, particularly checkpoint inhibitors, has gained prominence for its ability to treat certain cancers more effectively, sometimes leading to complete remission. Nonetheless, these therapies are not universally effective, addressing the limitations and enhancing the effectiveness of immunotherapy has become a key focus in cancer research. Advancements in synthetic biology and genetic engineering have opened new pathways for innovation in cancer treatment. Researchers have successfully engineered bacteria to deliver various therapeutic agents, including immune modulators and toxins. The stimulator of interferon genes (STING) is a pivotal component in the immune response, connecting DNA sensing through cyclic GMP-AMP synthase (cGAS) to the production of interferons (IFNs). STING agonists have shown considerable promise, demonstrating significant efficacy and safety in both human and animal studies by enhancing humoral, cellular, and mucosal immunity1-2. Bacillus subtilis (BS) probiotics have historically been used to improve gut health. Building on this, we employed bioengineering techniques to transform BS to express dacA, allowing it to function as a STING agonist. This innovative approach seeks to assess whether these modified probiotics can enhance the efficacy of immunotherapy, offering a new strategy to overcome the existing challenges in cancer treatment.

Materials and

Methods: The integration fragment used to create ΔamyE:cat_sfGFP was assembled from various plasmids and Bacillus subtilis chromosomal DNA, serving as a template for subsequent dacA constructs. After verifying that the engineered BS strains could express GFP and dacA, bacterial cultures were plated on selective 2*Schaeffer's medium-glucose plates and incubated for one week at 37 °C to allow for spore formation. Spores were collected from these plates into centrifuge tubes, then subjected to washing and sonication three times a day for three consecutive days. To test the biological effects of these spore preparations, five female C57BL/6 mice were assigned to each treatment group, housed in individual cages per experimental condition. Mice were gavaged daily with either 100 µL of Phosphate-Buffered Saline (PBS) or 100 µL of spore suspension (109 CFU). After three days of gavage, each mouse received a subcutaneous injection of 0.5 million MC38 cells in the right flank. When tumors reached an approximate volume of 100 mm3, mice were given either PBS or 200 mg of anti-PD-L1 intraperitoneally (i.p.) every three days, for a total of three doses. Tumor size was measured every three days, and mice were euthanized at the experimental endpoint.

Results, Conclusions, and Discussions: The experimental setup included six groups based on different oral gavage treatments and corresponding i.p. administrations. Among all groups, the combination of dacA gavage and PD-L1 therapy demonstrated the most effective tumor control (Figure 2A). Both the GFP group and the dacA group showed some level of tumor growth inhibition, with dacA proving more effective. However, these effects were generally mild compared to the PBS group (Figure 2B). In the PD-L1 treatment group, mice that received dacA gavage exhibited more pronounced tumor control compared to controls, suggesting that engineered BS may work synergistically with PD-L1 therapy (Figure 2C). This is one of the key reasons for focusing on BS—it not only meets the Food and Drug Administration's generally recognized as safe criteria but also can form spores, allowing it to survive in extreme environments like acidic pH and harsh gastric conditions3.
Engineered BS strain have been developed to express dacA, with studies indicating that oral administration of these modified bacteria can exert a measurable degree of tumor control. Furthermore, when combined with immunotherapy, the treatment demonstrates a significant therapeutic effect against tumors. For further modification to overexpress dacA, potentially enhancing its activity as a STING agonist. This approach offers a promising avenue for combination therapy in the context of immunotherapy. Notably, the inherent stability of BS, owing to its ability to form spores, underscores its potential for commercial applications, suggesting a strong prospect for translational development and broader clinical impact.

Acknowledgements (Optional):

References:
1. Gao P, Ascano M, Wu Y, Barchet W, Gaffney BL, Zillinger T, Serganov AA, Liu Y, Jones RA, Hartmann G, Tuschl T, Patel DJ. Cyclic [G(2',5')pA(3',5')p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell. 2013 May 23;153(5):1094-107. doi: 10.1016/j.cell.2013.04.046.
2. Zhang X, Shi H, Wu J, Zhang X, Sun L, Chen C, Chen ZJ. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol Cell. 2013 Jul 25;51(2):226-35. doi: 10.1016/j.molcel.2013.05.022.