Engineering a novel cellular trafficking pathway for transferrin to improve its drug carrier efficacy

An effective cytotoxic agent for cancer therapy should demonstrate reduced toxicity by having an increased specificity towards cancer cells. To address this challenge, several research groups have investigated conjugating various cytotoxins to the iron transport protein transferrin (Tf), since its receptor (TfR) is overexpressed on the surfaces of cancer cells. Tf undergoes receptor-mediated endocytosis into a cell following TfR binding, where it can then deliver its drug to the cell. However, Tf is recycled rapidly to the cell surface following internalization (∼5 min), limiting the timeframe in which it can deliver its drug. We engineered Tf to improve its therapeutic properties by applying a mathematical model to identify molecular parameters which could be altered to manipulate its trafficking pathway so as to increase its cellular association. Slow iron release from Tf was predicted to increase cellular association of Tf with cancer cells. We generated a version of Tf with a slower iron release rate by replacing its synergistic carbonate anion with oxalate. This Tf variant associated with HeLa cells to a greater extent when compared to wild-type Tf. In addition, this increase in cellular association translated into an increase in drug carrier efficacy, as diphtheria toxin (DT) conjugates of the Tf variant were significantly more cytotoxic than DT conjugates of wild-type Tf in an in vitro cell-killing assay. Currently, the utility of this strategy for engineering Tf variants is further being explored by generating Tf mutants with site-directed mutagenesis.