Development of Designer Probiotics for Targeted Delivery of Immunomodulatory Payloads

Drug delivery platforms that target the deposition of therapeutics to sites of disease are needed in order to increase efficacy and decrease off-target effects of current interventions. To address these issues, we have developed a probiotic E. coli capable of secreting therapeutic proteins into its surroundings. Our chosen chassis, E. coli Nissle 1917 (EcN), a probiotic with a well-established safety record in humans, can colonize the intestines as well as solid tumors. To enable EcN to secrete therapeutic payloads, we equipped it with a modified type III secretion system (T3SS), a protein delivery system common to many pathogenic Gram-negative bacteria. Using recombineering-based approaches, we transferred the operons that encode the core Shigella T3SS onto the chromosome of EcN. Next, we developed variants of this strain, referred to as T3EcN, that secrete functional nanobodies, variable domains of heavy chain only antibodies (aka VHH) that were isolated from immunized alpacas and identified to sequester and neutralize the target protein of interest (e.g. pro-inflammatory cytokines or immune checkpoint molecules). This was done by appending the N-termini of the nanobody with a type III secretion sequence. To enable T3EcN to stably colonize and constitutively secrete nanobodies in vivo, the nanobody-coding genes were placed under the control of constitutive synthetic promoters and introduced onto a plasmid that can be maintained in the absence of antibiotic selection. Notably, we observe that nanobody-secreting T3EcN variants stably colonize and maintain secretory activity within the mouse gut as well as tumors. Furthermore, using a bioluminescent reporter for T3SS activity, we have demonstrated that T3EcN’s T3SS is active in the colon and small intestine. Ongoing work in preclinical murine models is focused on developing proof of concept for this novel targeted drug delivery platform.