Diffusion of an Anti-Transferrin Receptor Antibody in Cultured Murine Melanoma Cell Layers

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Abstract

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 antibody.

Methods

A mouse melanoma cell line, B16F10, was grown on Matrigel® coated Transwell® inserts. The diffusion of a binding monoclonal antibody which recognizes the murine transferrin receptor (a-TfR) was investigated, and compared to that of an isotype-matched, nonbinding control.

Results

At a cell density of 6.4 ×105 cells/cm2, 37°C and a donor antibody concentration of 10 nM, the permeability of the a-TfR antibody was approximately half that of the control. Blocking experiments, in which the cell layer was pretreated with excess a-TfR, resulted in equal permeabilities for a-TfR and control antibodies, suggesting that the difference in permeabilities observed in the absence of blocking is due to a-TfR binding. Experiments at 4°C and in the presence of metabolic inhibitors also equalized the permeation rates of the two antibodies, indicating that internalization of the antibody/antigen complex is important in retarding the permeation of the binding antibody.

Conclusions

In this system, the diffusion of the a-TfR antibody is retarded by binding and internalization processes. The results have implications for the use of immunoconjugates in cancer chemotherapy, and for the delivery of other binding macromolecules to tissue sites of action.

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