TUMOR TARGETING WITH BIOTINYLATED TUMOR-NECROSIS-FACTOR-ALPHA - STRUCTURE-ACTIVITY-RELATIONSHIPS AND MECHANISM OF ACTION ON AVIDIN PRETARGETED TUMOR-CELLS

We have recently described a new strategy for targeting biotinylated t umor necrosis factor-alpha (TNF-alpha) to tumors, based on pretargetin g with biotinylated antibodies and avidin. Here, we have analyzed the structure-activity relationships of several biotin-TNF-alpha conjugate s and studied the mechanism of their interaction with avidin and TNF-a lpha receptors on tumor cells. The study has been carried out using an in vitro model based on human melanoma Cole 38 cells and monoclonal a ntibody 225, an antibody against the high molecular weight melanoma-as sociated antigen. Immunochemical and cytotoxicity studies showed that biotin-TNF-alpha but not TNF-alpha persists for several hours on the s urface of cells pretargeted with biotin-monoclonal antibody 225 and av idin and triggers cytolytic effects. Studies on the mechanism of actio n showed that biotin-TNF-alpha trimers can slowly dissociate from targ eted cells in a bioactive form, through trimer-monomer-trimer transiti ons. Structure-activity relationship studies showed that nonbiotinylat ed subunits must be present in the biotin-TNF-alpha trimers for effici ent release of bioactive TNF-alpha. Cole 38 cells targeted with biotin -TNF-alpha were able to kill mouse L-M cells in coculture experiments, indicating that the released TNF-alpha can interact also with TNF-alp ha receptors expressed by bystander cells. In conclusion, the targetin g complex works as a system that slowly releases bioactive TNF-alpha i n the microenvironment of the targe ted cell. This opens up the possib ility that cells other than those reached by the targeting antibody (e .g., endothelial cells and local cells of the immune system) can be af fected in vivo.