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Bioactive IL7-diphtheria fusion toxin secreted by mammalian cells
1Harvard Skin Disease Research Center, Department of Dermatology, Brigham and Women's Hospital, Boston, MA 02115, USA.
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.
Insights
Researchers developed a novel mammalian cell system for efficiently producing properly folded fusion toxins. This method overcomes challenges in recombinant protein production, enabling rapid evaluation of targeted cytotoxic agents like DAB(389)-IL7.
Area of Science:
- Biotechnology
- Molecular Biology
- Protein Engineering
Background:
- Targeted cytotoxic agents, engineered proteins combining cytotoxic elements with specific ligands, show promise for selectively eliminating pathological cells.
- Current production methods often involve bacterial expression, necessitating denaturation and renaturation, which can be inefficient for certain recombinant proteins.
- Difficulty in achieving proper protein folding has been a significant hurdle in developing these therapeutic agents.
Purpose of the Study:
- To establish a novel system for the rapid and efficient production of properly folded fusion toxins.
- To overcome the limitations of traditional bacterial expression and in vitro refolding methods.
- To enable faster evaluation of novel targeted cytotoxic agents.
Main Methods:
- Engineering mammalian cells to secrete fusion toxins as correctly folded molecules.
- Utilizing cell culture medium for purification of the native, properly folded proteins.
- Production and testing of DAB(389)-IL7, a fusion toxin comprising diphtheria toxin domains and interleukin 7.
Main Results:
- Successfully produced highly active preparations of DAB(389)-IL7 using the engineered mammalian cell system.
- Demonstrated efficient secretion and purification of properly folded fusion toxins from cell culture medium.
- Validated the system's capability to produce functional fusion toxins.
Conclusions:
- The developed mammalian cell-based system provides a rapid and efficient method for producing properly folded fusion toxins.
- This approach overcomes significant technical obstacles associated with traditional recombinant protein production.
- The system is generalizable for producing and evaluating a wide range of fusion toxins with novel ligands.
