Efficient in vivo anti-tumoral effect of the anti-CEA, granulysin-based immunotoxin MFE23GRNLY in a lung adenocarcinoma model. Combination with a Bcl-xL-selective BH3 mimetic

MFE23GRNLY is an immunotoxin formed by the recombinant fusion of granulysin and the single chain fraction variable (scFv) of the anti-CEA antibody MFE23. Granulysin is a protein stored in the granules of human cytotoxic T lymphocytes (CTL) and NK cells with antimicrobial and antitumoral activity. Previous work from our group demonstrated that recombinant MFE23GRNLY preserves the cytotoxic activity of GRNLY and the specific binding to CEA, targeting efficiently the antitumoral activity of GRNLY towards CEA-expressing tumors after systemic injection in vivo . In the present work we observed that the lung adenocarcinoma A549 expressed CEA and was sensitive to the immunotoxin in vitro . We have then demonstrated the efficacy of MFE23GRNLY against this tumor in vivo, broadening the application of the immunotoxin to the treatment of lung tumors. Furthermore, we attempted tu unveil MFE23GRNLY mechanism of action compared to GRNLY on HeLa-CEA and on A549 cells. We demonstrated that MFE23GRNLY treated human tumor cells showed signs of ER stress and autophagosome accumulation. Remarkably, the general caspase inhibitor Z-VAD-fmk prevented MFE23GRNLY-induced death of HeLa-CEA cells but not of A549 cells, a feature that correlated with high levels of expression of the anti-apoptotic protein Bcl-x _L in A549 cells. Accordingly, the combination of the BH3 mimetic A1155463, inhibitimg the activity of the anti-apoptotic member of the Bcl-2 family Bcl-x _L , with MFE23GRNLY, showed a potent in vitro synergistic effect with MFE23GRNLY in A549 cells. However, when we combined both agents in vivo in athymic mice xenotransplanted with A549 cells, the synergistic effect was not observed, although the anti-tumoral potential of the systemic injection of the immunotoxin was again confirmed.