Michael Chan, PhD 1998

Thesis Title: Immunotoxin cytotoxicity against cancer cells: Intracellular targeting and utility of protein synthesis inhibition model

Immunotoxins, which are constructed by linking a plant or bacterial toxin to a tumor-specific monoclonal antibody, could potentially provide better cancer therapy than currently available treatments. In practice, non-specific toxicity limits the effectiveness of immunotoxins. Developing more potent immunotoxins, which are effective at lower concentrations, could alleviate nonspecific toxicity, and make immunotoxins more viable as a cancer therapy.

A model relating the toxicity of gelonin immunotoxin to the cellular trafficking of transferrin receptor targeting agents was proposed by Yazdi and Murphy in 1994. Their analysis indicated that immunotoxin effectiveness was most sensitive to the translocation rate of the toxin into the cytosol and the intracellular concentration of the immunotoxin.

To determine the general applicability of the model, we have applied this model to the immunotoxin, 9.2.27-gelonin, which targets a melanoma-associated antigen. Despite the very different cellular trafficking of 9.2.27 and transferrin receptor targeting agents, the model proposed by Yazdi and Murphy fits the 9.2.27-gelonin protein synthesis inhibition data well, indicating the model may be broadly applicable.

Recently, researchers have added the endoplasmic reticulum retention signal, Lys-Asp-Glu-Leu (KDEL, single letter amino acid nomenclature), to the C-terminus of plant and bacterial toxins, and observed that the modified toxins exhibited increased toxicity compared with native toxins. It has been hypothesized that the increase in toxicity may be due to intracellular targeting of the toxins to the endoplasmic reticulum, which may provide a favorable environment for translocation of the toxin into the cytosol. This hypothesis has been difficult to test directly because a single toxin molecule entering the cytosol is sufficient to kill the cell.

Application of the model to immunotoxins constructed with and without the KDEL retention sequence indicates that increases in toxicity from intracellular targeting with KDEL are attributable to increases in intracellular concentration and not the translocation rate constant of the immunotoxin. At this time, significantly increasing immunotoxin activity by the inclusion of a translocation domain seems more feasible than by intracellular targeting.