Murine IL-2 secreting recombinant Bacillus Calmette-Guerin augments macrophage-mediated cytotoxicity against murine bladder cancer MBT-2

Purpose: This study was to establish a more effective anti-cancer immunomod ulating agent by constructing recombinant (r) Bacillus Calmette-Guerin (BCG ) secreting alpha-antigen (cu-Ag) fused murine (m) interleukin (IL)-2, and to study its biological activity on cell-mediated cytotoxicity against muri ne bladder cancer cell, MBT-2, in vitro. Materials and Methods: pSO246 plasmid vector ligated with mIL-2 gene was in troduced into BCG by electroporation. Thioglycollate-elicited murine perito neal exudate cells (PEC) were stimulated in vitro with parental BCG or rBCG and their cytotoxic activity and the cytokine production was studied. Cyto kines were assayed by an enzyme-linked immunosorbent assay (ELISA) and L929 bioassay. Cytotoxicity was measured by Cr-51 releasing assay. Results: rBCG (alpha-Ag-IL-2) secreted functional IL-2 and augmented more e fficient cytotoxicity to MBT-2 and cytokines such as IL-12, tumor necrosis factor and interferon (IFN)-gamma in PEC than parental BCG did. rBCG (alpha -Ag) had the same activity as BCG. Anti-IL-2 antibody reduced rBCG (alpha-A g-IL-2)-mediated cytotoxicity and IFN-gamma production. Exogenous IL-2 also enhanced BCG-mediated cytotoxicity, but 100 times more IL-2 was required t o express the same activity as rBCG (alpha-Ag-IL-2). Anti-IL-12 neutralizin g antibody and the depletion of T cells and NK cells reduced IFN-gamma prod uction by PEC stimulated with rBCG (alpha-Ag-IL-2), suggesting that T cells , NK cells and IL-12 participate in the enhancement of IFN-gamma production . Conclusions: rBCG secreting IL-2 showed significant antitumor activity and cytokine production and this will be a promising agent for bladder cancer p atient to reduce both clinical dose and side effects of BCG for immunothera py.