Mathematical modeling of mutant transferrin-CRM107 molecular conjugates for cancer therapy.

The transferrin (Tf) trafficking pathway is a promising mechanism for use in targeted cancer therapy due to the overexpression of transferrin receptors (TfRs) on cancerous cells. We have previously developed a mathematical model of the Tf/TfR trafficking pathway to improve the efficiency of Tf as a drug carrier. By using diphtheria toxin (DT) as a model toxin, we found that mutating the Tf protein to change its iron release rate improves cellular association and efficacy of the drug. Though this is an improvement upon using wild-type Tf as the targeting ligand, conjugated toxins like DT are unfortunately still highly cytotoxic at off-target sites. In this work, we address this hurdle in cancer research by developing a mathematical model to predict the efficacy and selectivity of Tf conjugates that use an alternative toxin. For this purpose, we have chosen to study a mutant of DT, cross-reacting material 107 (CRM107). First, we developed a mathematical model of the Tf-DT trafficking pathway by extending our Tf/TfR model to include intracellular trafficking via DT and DT receptors. Using this mathematical model, we subsequently investigated the efficacy of several conjugates in cancer cells: DT and CRM107 conjugated to wild-type Tf, as well as to our engineered mutant Tf proteins (K206E/R632A Tf and K206E/R534A Tf). We also investigated the selectivity of mutant Tf-CRM107 against non-neoplastic cells. Through the use of our mathematical model, we predicted that (i) mutant Tf-CRM107 exhibits a greater cytotoxicity than wild-type Tf-CRM107 against cancerous cells, (ii) this improvement was more drastic with CRM107 conjugates than with DT conjugates, and (iii) mutant Tf-CRM107 conjugates were selective against non-neoplastic cells. These predictions were validated with in vitro cytotoxicity experiments, demonstrating that mutant Tf-CRM107 conjugates is indeed a more suitable therapeutic agent. Validation from in vitro experiments also confirmed that such whole-cell kinetic models can be useful in cancer therapeutic design.

Intracellular trafficking considerations in the development of natural ligand-drug molecular conjugates for cancer.

Overexpressed receptors, characteristic of many cancers, have been targeted by various researchers to achieve a more specific treatment for cancer. A common approach is to use the natural ligand for the overexpressed receptor as a cancer-targeting agent which can deliver a chemically or genetically conjugated toxic molecule. However, it has been found that the therapeutic efficacy of such ligand-drug molecular conjugates can be limited, since they naturally follow the intracellular trafficking pathways of the endogenous ligands. Therefore, a thorough understanding of the intracellular trafficking properties of these ligands can lead to novel design criteria for engineering ligands to be more effective drug carriers. This review presents a few commonly used ligand/receptor systems where intracellular trafficking considerations can potentially improve the therapeutic efficacy of the ligand-drug molecular conjugates.

Intratumoral therapy of glioblastoma multiforme using genetically engineered transferrin for drug delivery.

Glioblastoma multiforme (GBM) is the most common and lethal primary brain tumor with median survival of only 12 to 15 months under the current standard of care. To both increase tumor specificity and decrease nonspecific side effects, recent experimental strategies in the treatment of GBM have focused on targeting cell surface receptors, including the transferrin (Tf) receptor, that are overexpressed in many cancers. A major limitation of Tf-based therapeutics is the short association of Tf within the cell to deliver its payload. We previously developed two mutant Tf molecules, K206E/R632A Tf and K206E/K534A Tf, in which iron is locked into each of the two homologous lobes. Relative to wild-type Tf, we showed enhanced delivery of diphtheria toxin (DT) from these mutants to a monolayer culture of HeLa cells. Here, we extend the application of our Tf mutants to the treatment of GBM. In vitro treatment of Tf mutants to a monolayer culture of glioma cells showed enhanced cellular association as well as enhanced delivery of conjugated DT. Treatment of GBM xenografts with mutant Tf-conjugated DT resulted in pronounced regression in vivo, indicating their potential use as drug carriers.

Genetically engineering transferrin to improve its in vitro ability to deliver cytotoxins.

We previously demonstrated that decreasing the iron release rate of transferrin (Tf), by replacing the synergistic anion carbonate with oxalate, increases its in vitro drug carrier efficacy in HeLa cells. In the current work, the utility of this strategy has been further explored by generating two Tf mutants, K206E/R632A Tf and K206E/K534A Tf, exhibiting different degrees of iron release inhibition. The intracellular trafficking behavior of these Tf mutants has been assessed by measuring their association with HeLa cells. Compared to native Tf, the cellular association of K206E/R632A Tf and K206E/K534A Tf increased by 126 and 250%, respectively. Surface plasmon resonance studies clearly indicate that this increase in cellular association is due to a decrease in the iron release rate and not to differences in binding affinity of the mutants to the Tf receptor (TfR). Diphtheria toxin (DT) conjugates of K206E/R632A Tf and K206E/K534A Tf showed significantly increased cytotoxicity against HeLa cells with IC(50) values of 1.00 pM and 0.93 pM, respectively, compared to a value of 1.73 pM for the native Tf conjugate. Besides further validating our strategy of inhibiting iron release, these Tf mutants provide proof-of-principle that site-directed mutagenesis offers an alternative method for improving the drug carrier efficacy of Tf.