DDDR-45. Probiotics Engineered for Specificity and Reduced Immunogenicity Improve Survival in a Glioma Model

Abstract Glioblastoma multiforme (GBM) is an aggressive cancer with a dismal prognosis despite standard care—maximal safe resection, chemotherapy, and radiation. GBM’s highly immunosuppressive environment has limited immunotherapy approaches. Bacteria with intrinsic tumor-homing behavior and programmability as living therapeutics may overcome these challenges by accessing hypoxic, necrotic tumor cores inaccessible to standard treatments. While bacteria are safe and effective as therapeutics in non-CNS cancer models, their infectiousness and immunogenicity have impeded their use in the brain. Here, we engineered the probiotic E. coli Nissle 1917 (EcN) with a biocontainment circuit dependent on tumor-specific cues (hypoxia and elevated lactate) to restrict bacterial survival and growth to the tumor microenvironment (“iBio” strain). We further attenuated this strain via knockout of a membrane protein and lipopolysaccharide to reduce recognition by toll-like receptors 2 and 4, respectively, minimizing inflammation and intracranial toxicity (“BLL” strain). Healthy and orthotopic GBM-bearing mice were injected intracranially with bacteria; survival, body weight, and end-point bacterial load were measured. The iBio and BLL strains exhibited drastically reduced toxicity in healthy mice, with BLL safety comparable to saline controls. The engineered safety strains showed significantly better survival curves and clinical signs in mice than wild-type EcN. Relative to wild-type EcN, BLL increased the maximum tolerable dose of injected bacteria by 1,000-fold in the brain. In an orthotopic mouse model of GBM (GL261), the BLL strain was as safe as saline and more than doubled median survival from 4 to 9 days (p < 0.0001). These findings indicate that engineered EcN can be safely injected into the brain and impart a survival benefit in a GBM model, supporting further development of bacterial therapeutics for GBM. These results lay the foundation for clinical translation of bacterial-based therapies in GBM. Future studies will evaluate BLL-induced immune activation, inflammation, and tumor cell death.