**1. Introduction**

Cancer is the second leading cause of death in the United States [1, 2]. Unfortunately, current treatments involve invasive surgery followed by nonspecific radiation and chemotherapy that harm both healthy and cancer cells. Accordingly, much research has been dedicated to improving the tumor selectivity of chemotherapy treatments. Since the early 1980s, many receptors were found to have increased expression in cancer cells compared to their normal counterparts. These include transferrin receptor, interleukin‐13 receptor, and growth factor receptors [3‐7]. These receptors have been investigated for several years as cancer‐selective targets for therapeutic purposes.

An understanding of the trafficking pathway of the natural ligand can lead to novel design criteria for engineering the ligand to be a more effective drug carrier [8‐12]. This short review focuses on transferrin ligand‐toxin molecular conjugates with particular emphasis given to the

© 2013 Kamei; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Kamei; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

intracellular trafficking properties of the ligand. Moreover, although transferrin has been conjugated to liposomes [13] and nanoparticles [14] to enhance their targeting to cancer cells, they are not included in this review, since the intracellular trafficking properties of these drug delivery vehicles can vary from that of the ligand alone due to the large differences in size.

**3. Transferrin‐diphtheria toxin conjugates**

the current standard‐of‐care treatments.

conjugated to CRM107.

**4. Concluding remarks**

Diphtheria toxin (DT) is a protein toxin secreted by *Corynebacterium diphtheria*. DT acts by inhibiting protein synthesis through inactivation of elongation factor 2 (EF‐2) [27]. Tf conju‐ gated to a mutant of DT, known as CRM107, has been effective against malignant gliomas [28]. The mutation in CRM107 significantly inhibits the binding of the toxin to its native receptor, a heparin‐binding epidermal growth factor‐like growth factor precursor, thereby reducing the nonspecific toxicity of Tf‐CRM107 [29]. Youle and Oldfield's group performed *in vivo* studies using Tf‐CRM107 on solid human gliomas in the flanks of nude mice, and observed increased cytotoxicity exerted by the conjugate. The success of Tf‐CRM107 eventually led to phase III clinical trials, which were unfortunately canceled in late 2006 following the results of a conditional power analysis suggesting that its efficacy would not significantly improve upon

Transferrin-Toxin Conjugates for Cancer 317

As mentioned above, Tf recycles very rapidly, and this short duration inside the cell can limit the ability of Tf to deliver its cytotoxic payload. Therefore, to identify new approaches to improving the efficacy of Tf‐CRM107, Kamei and coworkers used mass action kinetics to derive a mathematical model of the Tf/TfR trafficking pathway. Analysis of the model helped determine that, by reducing or inhibiting the iron release rate of Tf within the endosome, its drug delivery efficacy would be significantly improved [8]. In this scenario, iron would be retained by Tf upon recycling to the cell surface and the conjugate would be reinternalized to participate in another cycle of trafficking due to the preserved high affinity of holo‐Tf for TfR, increasing its cellular association. The drug delivery efficacy of Tf can therefore be improved, since a single Tf‐drug conjugate would undergo multiple trafficking cycles, thereby increasing the probability of delivering the drug. Kamei and coworkers engineered two Tf variants that satisfied the molecular level design criterion using site‐directed mutagenesis [9]. These Tf mutants were conjugated to DT and were shown to be more effective than wild‐type Tf in delivering DT *in vitro* to U87 and U251 human glioma cell lines [10]. Furthermore, Kamei and coworkers performed *in vivo* experiments, and demonstrated that both mutant Tf‐DT conju‐ gates were more effective than their wild‐type counterpart in shrinking glioma tumors on the flanks of mice [10]. Studies are currently being performed with these mutant Tf molecules

Ligand‐toxin molecular conjugates for cancer have been studied for several years. Though these conjugates have demonstrated some success, not many have obtained FDA approval for the treatment of cancer. The general lack of FDA‐approved ligand‐toxin conjugates may be attributed to the physiological behavior of the targeting ligand. Although ligands can effec‐ tively target cancer cells via their cell‐surface receptors, they will follow the ligand's physio‐ logical pathway once bound to receptors. For instance, the rapid recycling rate of Tf which aids in iron delivery can restrict the ability to deliver a conjugated toxin [8]. Through quanti‐

Human serum transferrin (Tf) has been investigated for several years as a targeting agent due to the overexpression ofits receptor on cancer cells. Tf has a molecular weight of approximately 80 kDa and is responsible for transporting free iron from the circulation to cells. Each Tf molecule has the capability to bind to two ferric (Fe3+) ions, one in the N‐terminal lobe (N‐lobe) and the otherin the C‐terminal lobe (C‐lobe). Each lobe binds to a ferric ion with an equilibrium dissociation constant (KD) of approximately 10‐<sup>22</sup> M [15, 16]. This iron‐bound Tf, or holo‐Tf, then binds to its cell‐surface receptor (TfR). After binding, The Tf/TfR complex is internalized, and holo‐Tf delivers its iron to the cell, promoting cellular growth and proliferation [17]. Since cancer cells require more iron to sustain their rapid proliferation, they have been found to overexpress TfR, and this high expression level of TfR has been exploited to achieve selective targeting of anticancer agents to cancer cells.
