Recombinant BCG overexpressing STING agonist elicits trained immunity and improved antitumor efficacy in non-muscle invasive bladder cancer

Abstract

BCG remains first-line therapy for non-muscle invasive bladder cancer (NIMBC) but its mechanism of action is not fully understood nor is it completely efficacious. We engineered a recombinant BCG (rBCG) that releases increased levels of STING agonist, c-di-AMP and compared effects of rBCG to wild type BCG (WT-BCG) by assessing antitumor efficacy and its ability to elicit trained immunity in NMIBC models. Rat MNU-carcinogen model of NMIBC was used to compare antitumor efficacy of 6 weekly intravesical installations of WT-BCG versus rBCG by pathologic tumor grading of bladder tissue and qRT-PCR for Th1 cytokines. Primary human and murine macrophage and urothelial carcinoma cell lines were treated with WT-BCG and rBCG and cytokine response was compared using ELISAs while macrophage phenotypes were determined by flow cytometry. MB49 cells were injected into flanks of C57Bl/6J mice followed by intratumoral injection of WT-BCG and rBCG to compare infiltrating Tcells and Teffector responses by flow cytometry. An in vitro trained immunity model of primary human monocytes was used to evaluate epigenetic changes (H3K4Me3) on TNF-a and IL-6 gene promoters by ChIP-PCR. Compared to WT-BCG, intravesical installation of rBCG in MNU-rats resulted in significantly lower tumor involvement and a more potent induction of Th1 cytokines. rBCG treated primary macrophages showed increased IFN I, IL-6, TNF‑a MCP-1 and IL-12 levels concomitant with increased M1 polarization shift. More pronounced IL-6, TNF‑a and IL-1b levels were observed in several human and rodent urothelial carcinoma cells in response to rBCG. Intratumoral injection of rBCG caused increased tumor regression and stronger infiltration of IFN‑g producing CD4+ T cells in MB49 tumors. rBCG elicited more potent epigenetic changes (H3K4Me3) on the TNF-a and IL-6 gene promoters following monocyte training. Increased proinflammatory cytokines, potentiated IFN-g+CD4+ T cells, and increased macrophage reprogramming (increased M1 shift and increased epigenetic changes) contributes to enhanced antitumor efficacy of rBCG over WT-BCG in NMIBC tumor models. Recombinant rBCG may be a novel intravesical immunotherapy for patients with NMIBC.