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 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.
Corti
A
A
Department of Biological and Technological Research, San Raffaele Scientific Institute, Milan, Italy.
Gasparri
A
A
Sacchi
A
A
Curnis
F
F
Sangregorio
R
R
Colombo
B
B
Siccardi
A G
AG
Magni
F
F
D000818:Animals; D001360:Avidin / Q000378:metabolism*; D001665:Binding Sites; D001710:Biotin / Q000494:pharmacology / Q000627:therapeutic use*; D006801:Humans; D008545:Melanoma / Q000628:therapy; D051379:Mice; D009369:Neoplasms / Q000628:therapy*; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use*
D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D001360:Avidin; D001710:Biotin