Anti-transferrin receptor IgG3 -avidin fusion proteins as universal drug delivery systems and direct tumoricidal agents for the treatment of hematopoietic malignancies

The transferrin receptor (TfR) is the principal cell surface molecule by which most mammalian cells acquire iron, an indispensable element for cell growth and survival. The overexpression of TfR on cancer cells including hematopoietic malignancies, in addition to its high rate of internalization, make the receptor an attractive target for the delivery of therapeutics into these cancer cells. We have developed human IgG3-avidin fusion proteins specific for the rat and human TfRs (anti-rat TfR IgG3-Av, anti-hTfR IgG3-Av), and demonstrated that the fusion proteins are able to deliver biotinylated chemical and proteins into cancer cells through TfR-mediated endocytosis. The internalized proteins remain functional and exhibit activities such as fluorescent light emission and induction of growth inhibition. In addition, we had some success in expressing reporter genes following transfection of rat cancer cell lines using anti-rat TfR IgG3-Av conjugated to biotinylated plasmids.;We also, unexpectedly, discovered an intrinsic proapoptotic activity of the fusion proteins. Anti-rat TfR IgG3-Av exists as a non-covalent dimer and induces apoptosis in two malignant rat hematopoietic cell lines. Further studies using anti-hTfR IgG3-Av in malignant human hematopoietic cell lines suggest that the dimeric structure of the fusion protein increases the cross-linking of cell surface TfR, which is then delivered to the lysosome and rapidly degraded by multiple enzymes including a papain-like cysteine protease. In addition, cells treated with anti-hTfR IgG3-Av exhibit mitochondrial depolarization and activation of caspase 9, 3, and 8. The mitochondrial damage and cell death can be prevented by iron supplement, but cannot be fully blocked by a pan-caspase inhibitor. These results suggest that anti-hTfR IgG3-Av induces a lethal iron deprivation in malignant hematopoietic cells, but the resulting cell death does not necessarily require caspase activity.;In contrast to the established view that anti-TfR antibodies inhibit cell growth by blocking the internalization of transferrin, anti-hTfR IgG3-Av induces cell death by effecting the degradation of the TfR. There is evidence that this mechanism of cytotoxicity is shared by other anti-TfR molecules. This study also describes, for the first time, several important molecular events during cell death induced by an anti-TfR antibody. Additional research in this area will improve our understanding of the cellular response to blockade of TfR-mediated iron uptake. From a medical perspective, further development of anti-hTfR IgG3-Av as a direct therapeutic, or as a drug delivery system may lead to improved treatments for hematopoietic malignancies.