Bioactive IL7-diphtheria fusion toxin secreted by mammalian cells

Abstract

A number of targeted cytotoxic agents have been developed that selectively kill malignant or otherwise pathological cells. These engineered proteins consist of a potent cytotoxic element connected to a ligand domain that binds to specific molecules on the surface of the target cell. Several of these agents have shown promise in clinical trials and one is currently administered to patients. A significant technical obstacle that has impeded the development of some of these toxins is the difficulty of preparing certain recombinant proteins in properly folded forms. These fusion proteins have generally been produced in bacteria requiring them to be denatured and renatured in vitro. For some proteins this is an efficient process whereas for others it is not. We describe here a system to produce fusion toxins rapidly and efficiently by engineering mammalian cells to secrete them as properly folded molecules which can be purified in native form from cell culture medium. We have used this system to produce highly active preparations of DAB(389)-IL7, a molecule consisting of the catalytic and transmembrane domains of diphtheria toxin fused to interleukin 7. This system is generalizable and can be used to produce and evaluate rapidly fusion toxins incorporating novel or uncharacterized ligands.

Engineering Toxin-Resistant Therapeutic Stem Cells to Treat Brain Tumors

“…In this study, we proposed that stem cell delivery of PE‐cytotoxins could circumvent current limitations by allowing the continuous release of therapeutic agent. Previous studies have demonstrated that a mutant form of EF‐2 confers resistance to EF‐2‐ADP‐ribosylating toxins , and that mammalian cells can be modified to secrete diphtheria‐fused toxin . We used a similar strategy to engineer a toxin‐resistant stem cell line able to stably secrete PE‐cytotoxins using single‐stranded oligonucleotides encoding mutant EF‐2 to convert endogenous EF‐2 into a toxin‐resistant variant.…”

“…Established human GBM lines U87, LN229, U251, Gli36vIII and primary GBM4, GBM6, GBM23, GBM64, and BT74 cells were grown as described previously . 293DT cells were cultured as previously described . Melanoma and breast cancer cell lines were grown as previously described .…”

Hosts and Heterologous Expression Strategies of Recombinant Toxins for Therapeutic Purposes

“…This line is particularly suitable for transient expression assays because it is readily transfected at high efficiency, and the SV40 T antigen replicates plasmids containing the SV40 origin. In work published in 2005 [ 43 ], this cell line was mutated, and clones that were highly resistant to DT were isolated and used to express DAB389-IL7, a very potent fusion toxin composed of the catalytic and transmembrane domains of diphtheria toxin fused to interleukin 7.…”

Protein engineering: security implications

“…Until recently, major factors limiting the therapeutic use of plant and animal toxins were the difficulty and high cost of bulk extraction or large‐scale chemical synthesis. Now, instead of the labour‐intensive process of purifying toxins directly from their original source, an increasing number can be produced in large quantities through the cloning and expression of toxin genes in bacteria, yeast or cultured cells (Ducancel et al , 1989; Shulga‐Morskoy & Rich, 2005). To facilitate their manufacture, protein toxins can also be truncated to the minimum size that is required for functional activity.…”