Introduction:

Nadofaragene firadenovec is a novel recombinant adenoviral vector-based intravesical gene therapeutic that delivers IFNA2 to urothelial cells, and was approved for the treatment of BCG-unresponsive NMIBC in 2022. Posttreatment biopsy of patients enrolled on the trial revealed relative PD1 and PD-L1 overexpression in tumor-infiltrating lymphocytes of nonresponders. In vivo orthotopic models replicated these findings, demonstrating PD1 overexpression in tumor-infiltrating T lymphocytes of mice following intravesical adenoviral-IFN treatment. Hence, we sought to examine the treatment efficacy of combination therapy with lentiviral (LV)-IFNα and PD-1 blockade in a murine bladder cancer model. We then examined the IFN-signaling and immune mechanisms responsible for the therapeutic effect of the combination therapy.

Methods:

GFP/luciferase-labeled MB49 cells were administered intravesically into C57BL/6 female mice, and tumor growth was confirmed using bioluminescence imaging. Mice were randomized into one of six groups, including four treatment groups (LV-IFNα alone, anti-PD-1 alone, LV-Ctrl + anti-PD-1 and LV-IFNα + anti-PD-1), and two control groups (no treatment and LV-Ctrl). LV was delivered intravesically and anti-PD-1 mAB was administered via an intraperitoneal injection twice weekly. Mice were sacrificed when moribund or at the end of the 9-week study period. Bladder weights were recorded ex vivo and morphologic examination of specimens was performed to assess presence or absence of tumor. Overall survival (OS) was determined using Kaplan-Meier analysis. To assess changes induced by the combination therapy, we compared the gene signatures of 3 groups using RNAseq of tumors from the same murine model 72 hours post-treatment: 1) PD1 vs Ctrl, 2) LV-IFN vs LV-Ctrl, and 3) LV-IFN/PD1 vs LV-Ctrl/PD1.

Results:

Mice treated with combination therapy (LV-IFNα + anti-PD-1) had the longest survival (log-rank p<0.0001). Bioluminescence decreased amongst all treatment groups compared to controls at week 2; however, durable treatment effect was noted only in the combination mice (week 3 and beyond). At necropsy, bladder tumors were evident on gross morphologic examination in all control mice, whereas no tumors were identified in 14%, 71%, and 86% of mice in the LV-IFNα, anti-PD-1 and LV-IFNα + anti-PD-1 groups, respectively (p<0.0001). IFN pathway and immune-related pathways were significantly upregulated in the combination group compared to either treatment alone. IFN pathways included cytokine storm, IL-1, and IL-6 signaling, among others. Immunological gene sets activated by combination treatment included those from both innate and adaptive immune pathways. Importantly, crosstalk between different immune compartments (such as dendritic cells and natural killer cells or lymphoid and non-lymphoid cells) was upregulated following treatment.

Conclusion:

Combination therapy with LV-IFNα and anti-PD-1 antibody effectively eradicated bladder tumors in immune-competent mice and improved OS. Additionally, combination therapy resulted in upregulation of IFN and immune-related pathways. These findings suggest a therapeutic role for immune checkpoint inhibitors in BCG-unresponsive NMIBC patients treated with nadofaragene treatment. 

Funding: University of Texas MD Anderson Cancer Center SPORE in Genitourinary Cancer, FKD Therapies Oy, AI Virtanen Institute for Molecular Sciences