Transferrin (Tf) conjugates of CRM107, a point mutant of diphtheria toxin (DT), are currently being tested in clinical trials for treatment of malignant gliomas. However, the rapid cellular recycling of Tf limits its efficiency as a drug carrier. We used a mathematical model of the Tf/Tf receptor (TfR) trafficking cycle to identify the Tf iron release rate as a previously unreported factor governing the degree of Tf cellular association. A modified Tf ligand with a significantly lowered iron release rate was generated by altering the iron binding sites of Tf. Trafficking patterns of the modified Tf and native Tf were compared by measuring their cellular association with HeLa cells. The amount of Tf associated with the cells was an average of 51% greater for the modified Tf than for native Tf over a two hour period at Tf concentrations of 0.1 nM and 1 nM. In addition, DT conjugates of modified Tf were significantly more cytotoxic against HeLa cells than conjugates of native Tf. Conjugate IC50 values were determined to be 0.02 nM for the modified Tf conjugate vs. 0.1 nM for the native Tf conjugate. Thus, inhibition of Tf iron release may improve the efficacy of Tf as a drug carrier through increased association with cells expressing TfR.

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