Targeted local activation of Fas in pancreatic beta cells for treatment of insulinomas and disorders of pathological autonomic insulin secretion

The prognosis of patients with insulinomas is poor when multiple or malignant tumors are present. Also, pancreatectomy is frequently necessary as life-saving procedure to rescue patients from hypoglycaemic episodes associated with persistent hyperinsulinemic hypoglycaemia of infancy (PHHI, nesidioblastosis). Therapeutic compounds that selectively destroy autonomically insulin producing cells may cure both multifocal or malignant insulinomas and nesidioblastosis. The aim of this study is to target the apoptosis-inducing actions of Fas-ligand (FasL) specifically to autonomic pancreatic beta cells. We demonstrate that artificial immobilisation of the extracellular inactive domain of FasL on the cell surface by fusion to an appropriate immobilisation domain is sufficient to convert these fusion proteins to bioactive compounds mimicking the apoptosis inducing actions of membrane-bound forms of FasL. To target this principle to pancreatic beta cells, fusion proteins of FasL with a peptide domain of the glucagon-like peptide 1 (GLP-1) analog exendin 4 (FasL-Ex4) were generated. Receptors for the incretin hormone GLP-1 (GLP-1R) are specifically expressed on the surface of insulin producing cells and exendin 4 (Ex4) binds to GLP-1R with high affinity. Expression of GLP-1R was verified by RT-PCR, fluorescence immunocytochemistry and FACS phenotyping in pancreatic beta cell lines INS-1, betaTC3 and BHK cells stably overexpressing rat GLP-1R and in primary human pancreatic beta cells. GLP-1R negative BHK and Cos-7 cells served as controls. Surface immobilisation of FasL-Ex4 fusion proteins specifically in GLP-1R positive cells was demonstrated by fluorescence immunocytochemistry and FACS. FasL dependent induction of apoptosis and activation of apoptotic signal transduction pathways are currently examined. These results demonstrate that FasL-Ex4 fusion proteins hold the potential as therapeutic compounds to target FasL apoptotic activity specifically to GLP-1R expressing insulin producing cells for non surgical treatment of insulinomas and nesidioblastosis in vivo.