We have recently described a new strategy for targeting biotinylated
tumor necrosis factor-alpha (
TNF-alpha) to
tumors, based on pretargeting with biotinylated
antibodies and
avidin. Here, we have analyzed the structure-activity relationships of several
biotin-
TNF-alpha conjugates and studied the mechanism of their interaction with
avidin and
TNF-alpha receptors on
tumor cells. The study has been carried out using an in vitro model based on human
melanoma Colo 38 cells and
monoclonal antibody 225, an antibody against the
high molecular weight melanoma-associated antigen. Immunochemical and cytotoxicity studies showed that
biotin-
TNF-alpha but not
TNF-alpha persists for several hours on the surface of cells pretargeted with
biotin-
monoclonal antibody 225 and
avidin and triggers cytolytic effects. Studies on the mechanism of action showed that
biotin-
TNF-alpha trimers can slowly dissociate from targeted cells in a bioactive form, through trimer-monomer-trimer transitions. Structure-activity relationship studies showed that nonbiotinylated subunits must be present in the
biotin-
TNF-alpha trimers for efficient release of bioactive
TNF-alpha. Colo 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-alpha receptors expressed by bystander cells. In conclusion, the targeting complex works as a system that slowly releases bioactive
TNF-alpha in the microenvironment of the targeted cell. This opens up the possibility that cells other than those reached by the targeting antibody (e.g., endothelial cells and local cells of the immune system) can be affected in vivo.