MURINE IL-2 SECRETING RECOMBINANT BACILLUS CALMETTE-GUÉRIN AUGMENTS MACROPHAGE-MEDIATED CYTOTOXICITY AGAINST MURINE BLADDER CANCER MBT-2

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Abstract
Purpose: This study was to establish a more effective anti-cancer immunomodulating agent by constructing recombinant (r) Bacillus Calmette-Guérin (BCG) secreting α-antigen (α-Ag) fused murine (m) interleukin (IL)-2, and to study its biological activity on cell-mediated cytotoxicity against murine bladder cancer cell, MBT-2, in vitro. Materials and Methods: pSO246 plasmid vector ligated with mIL-2 gene was introduced into BCG by electroporation. Thioglycollate-elicited murine peritoneal exudate cells (PEC) were stimulated in vitro with parental BCG or rBCG and their cytotoxic activity and the cytokine production was studied. Cytokines were assayed by an enzyme-linked immunosorbent assay (ELISA) and L929 bioassay. Cytotoxicity was measured by 51Cr releasing assay. Results: rBCG (α-Ag-IL-2) secreted functional IL-2 and augmented more efficient cytotoxicity to MBT-2 and cytokines such as IL-12, tumor necrosis factor and interferon (IFN)-γ in PEC than parental BCG did. rBCG (α-Ag) had the same activity as BCG. Anti-IL-2 antibody reduced rBCG (α-Ag-IL-2)-mediated cytotoxicity and IFN-γ production. Exogenous IL-2 also enhanced BCG-mediated cytotoxicity, but 100 times more IL-2 was required to express the same activity as rBCG (α-Ag-IL-2). Anti-IL-12 neutralizing antibody and the depletion of T cells and NK cells reduced IFN-γ production by PEC stimulated with rBCG (α-Ag-IL-2), suggesting that T cells, NK cells and IL-12 participate in the enhancement of IFN-γ production. Conclusions: rBCG secreting IL-2 showed significant antitumor activity and cytokine production and this will be a promising agent for bladder cancer patient to reduce both clinical dose and side effects of BCG for immunotherapy.

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