DIFFUSION OF AN ANTITRANSFERRIN RECEPTOR ANTIBODY IN CULTURED MURINE MELANOMA CELL-LAYERS

Purpose. The purpose of the study was to develop a cell culture system representing a layer of cells in a solid tumor, and to use the system to study factors affecting the diffusion of a binding monoclonal anti body. Methods. A mouse melanoma cell line, B16F10, was grown on Matrig el(R) coated Transwell(R) inserts. The diffusion of a binding monoclon al antibody which recognizes the murine transferrin receptor (a-TfR) w as investigated, and compared to that of an isotype-matched, nonbindin g control. Results. At a cell density of 6.4 x 10(5) cells/cm(2), 37 d egrees C and a donor antibody concentration of 10 nM, the permeability of the a-TfR antibody was approximately half that of the control. Blo cking experiments, in which the cell layer was pretreated with excess a-TfR, resulted in equal permeabilities for a-TfR and control antibodi es, suggesting that the difference in permeabilities observed in the a bsence of blocking is due to a-TfR binding. Experiments at 4 degrees C and in the presence of metabolic inhibitors also equalized the permea tion rates of the two antibodies, indicating that internalization of t he antibody/antigen complex is important in retarding the permeation o f the binding antibody. Conclusions. In this system, the diffusion of the a-TfR antibody is retarded by binding and internalization processe s. The results have implications for the use of immunoconjugates in ca ncer chemotherapy, and for the delivery of other binding macromolecule s to tissue sites of action.