Radiolytic Escherichia coli Nissle: A Novel Radiosensitizer Delivery Platform Using a Live Bacterial Therapeutic

Abstract

<h3>Purpose/Objective(s)</h3> There is a critical need to develop novel radiosensitizing strategies targeting the tumor microenvironment (TME). <i>Escherichia coli Nissle</i> (EcN) is a facultative anaerobic bacterium amenable to modification for recombinant protein synthesis. Preclinically, it grows within the hypoxic TME after intratumoral injection. It has been used orally and intratumorally in clinical trials. We have engineered a novel EcN strain, Radiolytic EcN (RLE), which releases therapeutic payloads within the TME following radiation (RT) induced lysis. <h3>Materials/Methods</h3> RLE includes the RT-inducible promoter gene, RecA coupled to a lytic gene obtained from bacteriophages. A second RecA (LexA) promoter with an ablated LexA repressor site enables constitutive expression of encoded therapeutic payloads. Activation of the recA promoter by RT drives the expression of the lytic gene leading to bacterial lysis and release of payload. A luciferase reporter was incorporated for bioluminescence imaging. Based on previous RT synergy, a PD-L1 inhibitor nanobody was used as payload for proof-of-concept in vitro and in vivo studies. All RT experiments were performed with an Xstrahl animal irradiator. <h3>Results</h3> Following construct design, RLE and non-radiolytic EcN were treated in vitro using 0.5, 2, 4, 8, and 25 Gy single fraction RT. Significant cell lysis for RLE was observed in all groups exposed to RT without a dose response. No lysis was observed in the unirradiated RLE group or non-radiolytic EcN group after RT. MC38/C57BL6 subcutaneous tumor models were developed for in vivo testing of lysis. RLE was delivered intratumorally (1 × 10⁷ CFU) and intramuscularly in the flank followed by 4 Gy RT. Following RT, a significant reduction in bioluminescence was observed in the irradiated group compared to untreated controls confirming in vivo lysis. In fractionated experiments using 4 Gy x 2 over 3 days, bioluminescence revealed RLE lysis following RT with repopulation between fractions and return to baseline at day 5. Increased bioluminescence without lysis was observed in untreated RLE and non-radiolytic EcN+ RT over 5 days. Western blot of supernatant and cell fractions in RLE and non-radiolytic EcN clones producing PD-L1 demonstrate selective release of PD-L1 after RT in RLE. Efficacy was examined in flank MC38/C57BL6 models for RLE producing PD-L1+RT and 6 controls (RLE-PD-L1-no RT, EcN-PD-L1+RT, EcN-PD-L1 no RT, RLE+RT, RT alone, untreated). Among all groups, RLE-PD-L1 with RT showed a >2-fold increase in median survival (∼30 days) compared to the control groups with a median survival of 11-18 days. <h3>Conclusion</h3> RLE is a novel platform for delivery of radiosensitizing and immune modulating payloads within the TME. Preclinical data demonstrates in vitro/vivo RT induced payload release, preliminary efficacy, and repopulation between fractions allowing for metronomic payload delivery. Future work will examine combinations of immune modulating payloads and sensitizers with toxicity prohibitive for systemic use.