Parvin Yazdi, PhD 1994

Thesis Title: Quantitative analysis of immunotoxin action

Immunotoxins are designed to target and specifically kill diseased populations of cells. These agents are produced by coupling potent plant or bacterial toxins to targeting agents such as monoclonal antibodies or ligands. Production of effective immunotoxins requires a quantitative understanding of their mechanism of action on target cells. This thesis describes the development of a framework which allows for the quantitative analysis of the action of immunotoxins.

The protein synthesis inhibition activity of four immunotoxins prepared by conjugating a plant toxin, gelonin, or a mutant form of diphtheria toxin, CRM107, to human transferrin and anti-transferrin receptor monoclonal antibody (MAb) 5E9 was evaluated in two human tumor cell lines. A mathematical model was developed to describe the action of immunotoxins by relating the protein synthesis inhibition activity of immunotoxins to the cellular trafficking of the targeting agents and the translocation and enzymatic properties of the toxins. Using the cellular trafficking of the two targeting agents characterized in this work and the enzymatic activity of the toxins reported in the literature, this model was compared to the experimental data collected on the kinetics of protein synthesis inhibition by the immunotoxins. This comparison indicated a close agreement between the model and experiments.

The comparison between the model and the experimental data provided estimates for the translocation rate constants of the immunotoxins. According to the estimated values, translocation is by far the slowest process in the transport of toxins to the cytosol. The efficiency of gelonin translocation appeared to be independent of the target cell or the targeting agent. That of CRM107, however, was strongly dependent on the properties of the targeting agent. Conjugated to transferrin, CRM107 translocated 100-fold more efficiently than conjugated to MAb 5E9. In general, the translocation rate constants estimated for CRM107 immunotoxins were 40 to 6,000-fold higher than those estimated for gelonin immunotoxins.