Immunotoxin sensitivity of Chinese hamster ovary cells expressing human transferrin receptors with differing internalization rates

Abstract

 Previous studies have shown that immunotoxin action is dependent upon selective binding to the target cell, internalization and then passage into the cytosol. It is important to define precisely how these critical steps are controlled so that the underlying relationship of each to high cytotoxic effectiveness is understood. In order to evaluate the contribution of internalization rate and receptor number on immunotoxin potency, the effects of an anti-(transferrin receptor, TfR)/ricin A chain immunotoxin, 7D3-A, were assessed on a parent Chinese hamster ovary cell line developed in our laboratory with no TfR (TfR^neg) and two lines transfected with either wild-type TfR (Tfr^wt) or an internalization-deficient (TfR^{δ7 – 58del}) mutated human TfR. Potent, receptor-mediated cytotoxicity resulted from the action of 7D3-A on TfR^wt cells (ID₅₀<1 nM) while both TfR^neg cells and TfR^{δ7 – 58del} were only minimally affected (ID₅₀>100 nM). Butyrate up-regulation substantially increased receptor expression on the TfR^wt and TfR^{δ7 – 58del} cells, but no corresponding rise in sensitivity to 7D3-A was observed. In contrast, immunotoxin potency was increased by co-treatment of TfR^wt cells with the carboxylic ionophore monensin and the effect was even more pronounced for TfR^{δ7 – 58del} cells. We conclude that internalization rate or intracellular destination is a much more important determinant of immunotoxin efficacy than receptor number.