Malignant gliomas are the most common primary brain tumor in the United States, accounting for 70% of the 21,810 estimated new cases of brain cancer in 2008 (1
). Although the prevalence of malignant gliomas represents only 1.5% of the 1,437,180 new cancers diagnosed in 2008, glial tumors are associated with disproportionately high morbidity and mortality (1
). For instance, grade IV astrocytomas, or glioblastoma multiforme (GBM), represent 60-70% of all malignant gliomas, with median survival of 12-15 months, despite administration of the current standard of care described below.
The first step in the treatment of malignant gliomas typically involves a gross surgical resection of the tumor (2
). Next, to potentially eradicate residual cancer cells not surgically removed, the patient is given 6 weeks of radiation therapy (RT) concomitant with the chemotherapeutic temozolomide (TMZ). This treatment is followed by an additional 6 months of maintenance TMZ (4
). Although this treatment regimen significantly increases the 1-3 month median survival of a patient newly diagnosed with GBM, there is much room for improvement. Possible reasons for the inability to extend survivability beyond ~1 year include the nonspecificity of RT and TMZ in combination with intrinsic cellular resistance to treatment (5
) allowing GBM to recur after a median survival of 32-36 weeks (7
). Consequently, a variety of agents have been investigated to combat GBM, many of which focus on proteins involved in the enhanced proliferative and migratory capacity characteristic of this disease (5
). These approaches target proteins that are selectively expressed or overexpressed in tumors to minimize nonspecific toxicity to normal cells typical of conventional therapies.
Motivated by early clinical successes of GBM therapeutics, we undertook a study to improve upon the current standard of care utilizing the transferrin receptor (TfR) as our therapeutic target (11
). Specifically, we were intrigued by Tf-CRM107, consisting of a human serum transferrin (Tf) molecule chemically conjugated to CRM107, a mutant variant of diphtheria toxin (DT). Though this conjugate drug had shown therapeutic promise in the treatment of progressive and recurrent GBM in both a Phase I and a multicenter Phase II study (5
), a conditional power analysis in Phase III determined that Tf-CRM107 was unlikely to improve overall patient survival compared to the current standard of care (14
). A reasonable hypothesis to account for this poor efficacy is the rapid cycling of Tf through the cell, severely limiting the time in which to deliver its toxin payload (15
The trafficking pathway of Tf, by receptor-mediated endocytosis, serves the crucial physiological role of transporting iron to cells. Each Tf molecule is capable of binding two ferric (Fe3+
) ions, one in each of its two homologous iron binding lobes, the N-terminal lobe (N-lobe) and the C-terminal lobe (C-lobe) (16
). Iron-loaded Tf (holo-Tf) specifically binds to TfR residing on the surface of all actively dividing cells. It is well known that many tumors significantly overexpress TfR relative to non-neoplastic tissue, ascribed to the extra need for iron of their rapidly proliferating cells (17
). However, once Tf delivers its iron to the cell, iron-free Tf (apo-Tf) still bound to TfR is recycled to the cell surface, where it is released from its receptor; Tf cannot reenter cells until it acquires more iron, a rather inefficient process at this post-delivery site (13
). Therefore, each holo-Tf molecule is restricted to a single 4-5 minute passage through the cell, while 30 such successive cycles of trafficking may be required for a Tf conjugated toxin to be delivered into the cytosol (19
To enhance delivery of drugs conjugated to Tf, we developed a mathematical model describing the Tf/TfR trafficking pathway using the principles of mass action kinetics (15
). A sensitivity analysis of the various model parameters identified decreasing the iron release rate in the endosome as a previously unreported design criterion to enhance the cellular association of Tf. By inhibiting the delivery of ferric ions by holo-Tf within the endosomal compartment, we manipulated the cell to recycle holo-Tf, rather than apo-Tf, to the cell surface. Each Tf molecule can now cycle through its trafficking pathway multiple times, since holo-Tf maintains a high affinity for TfR at the cell surface and could reenter the cell, increasing the probability that a Tf conjugated toxin will deliver its cytotoxic payload. Recombinant protein technology was used to develop two mutant variants of Tf exhibiting reduced iron release kinetics (21
). Both mutants contain two point mutations, one within each iron binding lobe specifically targeting amino acids involved in the release of iron within the endosomal compartment. In vitro
studies with HeLa cervical cancer cells displayed significantly higher drug delivery efficacy by both mutant Tf proteins in comparison to the wild-type Tf control (23
Here we utilized these mutant Tf-DT conjugates in the treatment of GBM. In vitro studies with the U87 and U251 glioblastoma cell lines demonstrated increased drug delivery efficacy attributed to the mutant Tf proteins. Moreover, application of mutant Tf-DT conjugates to xenografted U87:EGFRvIII flank tumors demonstrated superior and rapid tumor regression when compared with wild-type Tf-DT controls. These results establish the potential for clinical application of mutant Tf-based therapeutics in the treatment of malignant gliomas.