PD12-07: Addition of Anti-PD-L1 Enhances Antitumor Efficacies of BCG and BCG-STING by CD8+ T Cell expansion in a Murine Syngeneic MB49 Model of Urothelial Carcinoma

Kara Lombardo*, Monali Praharaj, Russell Becker, Kelly Harris, William Bishai, Andres Matoso, Baltimore, MD, Trinity Bivalacqua, Philadelphia, PA, Alok Singh, Baltimore, MD

Introduction: BCG induces PD-L1 in bladder carcinoma cells which decreases antitumor T cell responses in the tumor microenvironment (TME). In the present study aimed to compare efficacy of a PD-L1 checkpoint inhibitor combined with both wild-type BCG (BCG-WT) and our previously reported recombinant BCG overexpressing STING agonist, c-di-AMP (BCG-STING) in a murine MB49 flank model of urothelial carcinoma.

Methods: Syngeneic mouse MB49 flank tumors in C57BL/6J female mice were randomized into treatment groups: 1. Isotype (250 mg/animal 4x every 3rd day), 2. intratumorally (IT) treated BCG-WT (Tice, 5 x 106 CFU, 4x every 3rd day), 3. IT treated BCG-STING (Tice, 5 x 106 CFU, 4x every 3rd day), 4. Intraperitoneally (IP) treated with anti-PD-L1 Ab (250 mg/animal, 4x every 3rd day), 5. combination therapy of IT WT-BCG plus IP andi-PD-L1 or 6. combination of IT BCG-STING and IP anti-PD-L1. Endpoint measurement included tumor volume, histopathological analyses for percent necrosis and flow cytometry analyses of tumor infiltrating lymphocytes. Bulk RNA sequencing on MB49 tumor cells following IT administration of WT-BCG and BCG-STING was performed. Data analysis was performed in R studio, FlowJo and Graphpad with ANOVA tests for significance.

Results: Addition of anti-PDL1 improved antitumor efficacies of both BCG-WT and BCG-STING, but anti-PD-L1 alone was less effective than either BCG strains alone. BCG-STING plus anti-PD-L1 combination showed greatest antitumor response with lowest tumor volume and highest percentage of necrosis (P <0.0001). BCG-STING caused increased recruitment of infiltrating CD4+ and CD8+ T cells in MB49 tumors . However, anti-PD-L1 alone or in combination with BCG-WT or BCG-STING caused significantly higher recruitment of effector (IFN-g+ or IFN-g+PD-1+) CD8+ T cells over BCG-WT or BCG-STING. Bulk RNAseq unveiled significant reprogramming of TME as BCG-STING induced a stronger upregulation of hallmark gene sets from antitumor type I IFN (IFN-a), type II INF (IFN-g) and TNF-a pathways over BCG-WT.

Conclusions: Therapeutic combination of anti-PD-L1 improves antitumor efficacies of BCG-WT or BCG-STING by inducing stronger recruitment of effector CD8+ T cells at tumor sites, while BCG-STING alone more strongly activates antitumor inflammatory pathways than BCG-WT. These findings highlight the importance of detailed investigations of BCG-resistant mechanisms in preclinical models to address suboptimal efficacies of BCG and develop improved or novel therapies for urothelial carcinoma.

Source of Funding: None