Presentation Authors: Alok Singh*, Monali Praharaj, Gregory Joice, Takahiro Yoshida, Max Kates, David McConkey, William Bishai, Trinity Bivalacqua, Baltimore, MD
Introduction: INTRODUCTION: Stimulator of interferon genes (STING), a cytosolic sensor of cyclic dinucleotide (CDN; c-di-AMP and c-di-GMP) activates type I interferons (IFN I) and pro-inflammatory cytokines. Recombinant Bacillus-Calmette Guerin (BCG) overexpressing CDN (STING-agonist) remains unexplored in non-muscle invasive bladder cancer (NMIBC). We hypothesize that intravesical recombinant BCG is more efficacious than BCG for NMIBC. We constructed a novel STING-agonist-like recombinant BCG Pasteur and Tice strains (rBCG-disA-OE) carrying plasmids that harbors di-adenylate cyclase (disA) gene to test this hypothesis.
Methods: METHODS: WT and rBCG-disA-OE were tested for immunotherapeutic potential in WT and STING-deficient murine macrophages, to ascertain STING-dependent IFN I and pro-inflammatory cytokines activation. Similar experiments were conducted in RT4, J5637, and NBT2 cell lines. Fisher 344 rats intravesically received 4 doses of 1.5 mg/kg N methyl N nitrosurea (MNU) to induce NMIBC. Intravesical instillation of WT- and rBCG-disA-OE was administered weekly x 6. Histologic tumor grade, tumor involvement index, gene expression (q-PCR) as well as systemic immune responses (ELISA) were assessed. Growth and virulence of rBCG-disA-OE in BALB/c and SCID mice were tested using aerosol infection protocol.
Results: RESULTS: rBCG-disA-OE Pasteur and TICE strains overexpressing STING-agonist were potent inducers of IFN I in STING-dependent manner in murine macrophages. IFN I induction by rBCG-disA-OE increased TNF-Î± (P=0.01), IL-6 (P=0.04) and IL-1Î² (P=0.004) in bladder cancer cells and murine macrophages. Intravesical instillation of rBCG-disA-OE in MNU-rats resulted into significantly (P=0.004) lower tumor involvement index accompanied by a potent induction of IFN I signaling, M1 macrophage associated cytokines (Nos2, P=0.05 & IL-6 P=0.05) and chemokines (CCL2, P=0.048 & MCP-1, P=0.05). rBCG-disA-OE prevented invasive cancer when compared WT-BCG. BALB/c mice infected with rBCG-disA-OE showed significantly less lung bacillary burden (P=0.005) suggesting strain attenuation. SCID-mice infected with rBCG-disA-OE had prolonged survival.
Conclusions: CONCLUSION: We demonstrate STING-agonist like rBCG-disA-OE is superior to WT-BCG for induction of macrophage cytotoxicity and activation of IFN I signaling. rBCG-disA-OE demonstrated enhanced antitumor activity and cytokine production in a pre-clinical model of NMIBC suggesting an alternative intravesical agent in BCG unresponsive population.
Source of Funding: Source of Funding: Maryland Tedco, Willowcroft Foundation, and Cigarette Restitution Fund