Background EG-70 (detalimogene voraplasmid) is a novel, investigational, non-integrating, non-viral gene therapy specifically engineered to elicit local stimulation of anti-tumor immune response in the bladder while mitigating the risk of systemic toxicities from immune stimulation. EG-70 is administered by intravesical instillation (IVI) to eligible patients with non-muscle-invasive bladder cancer (NMIBC) to drive the expression of innate (retinoic acid-inducible gene I [RIG-I] agonists) and adaptive (interleukin-12 [IL-12]) immune regulators and remodel the local tumor microenvironment, while mitigating the risk of systemic toxicities from immune stimulation. The ongoing Phase 1/2 LEGEND study (ClinicalTrials.gov: NCT04752722) continues to investigate the safety and efficacy of EG-70 in patients with BCG-unresponsive NMIBC. Here we present preclinical data defining the immunomodulatory mechanism of action of EG-70, involving immune cell recruitment, tumor microenvironment remodeling and, ultimately, immune training on neoantigens and tumor clearance.

Methods Preclinical evaluation of EG-70 efficacy was conducted in an orthotopic syngeneic mouse model of bladder cancer to recapitulate a physiological tumor microenvironment in immunocompetent C57BL/6 mice. Luciferase-expressing MB49 cells were instilled in the bladder on study Day 1; following confirmation of tumor engraftment by in vivo imaging on Day 9, mice received two weekly IVIs of mEG-70 (a murine surrogate of EG-70) on Days 10 and 17. Animal experimentation was approved by the Institutional Animal Care Committee (IACC) and conducted in accordance with the guidelines of the Canadian Council on Animal Care (CCAC).

Results Immune profiling by flow cytometry, immunoassay, and immunohistochemistry revealed a profound remodeling of the tumor microenvironment from an immunosuppressive phenotype to a pro-inflammatory milieu supportive of tumor clearance. Accordingly, administration of mEG-70 was associated with marked reduction in tumor burden and significant improvement of survival. As demonstrated by either bladder or flank implantation rechallenge, the anti-tumor immune response had resulted in durable protection against subsequent tumor re-challenge, demonstrating systemic immune memory.

Conclusions The preclinical findings demonstrate that EG-70 delivers genetically encoded immunostimulatory payloads locally to the bladder. The mechanism of action described preclinically has been translated into the clinic in the Phase 1 portion of the LEGEND study, where treatment of patients with BCG-unresponsive NMIBC with carcinoma in situ was well tolerated with an overall complete response rate of 73%.