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Production, purification, and characterization of scFv TNF ligand fusion proteins
Andrea Fick1, Agnes Wyzgol, Harald Wajant
1Division for Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Würzburg, Germany.
Abstract:
Single-chain variable fragments (scFvs) specific for tumor-associated cell surface antigens are the most broadly used reagents to direct therapeutic or diagnostic effector molecules, such as toxins, radioisotopes, and CD3-stimulating scFvs, to tumors. One novel class of effector molecules that can be targeted to tumors by scFvs are ligands of the tumor necrosis factor (TNF) family. Typically, these molecules have apoptosis inducing and/or immune stimulating properties and are therefore highly attractive for cancer treatment. N-terminal fusion of scFvs does not interfere with the receptor binding capabilities of TNF ligands and thus allows the straightforward generation of scFv TNF ligand fusion proteins. We report here a protocol for the purification of eukaryotically produced scFv TNF ligand fusion proteins based on affinity chromatography on anti-Flag agarose and further describe assays for the determination of the targeting index of this type of scFv-targeted proteins.
Insights
This study presents a method for purifying single-chain variable fragment (scFv) fusion proteins targeting tumor necrosis factor (TNF) ligands. These novel cancer therapeutics can be effectively purified and their targeting efficacy assessed.
Area of Science:
- Oncology
- Immunology
- Biotechnology
Background:
- Single-chain variable fragments (scFvs) are crucial for directing therapeutic agents to tumors.
- Tumor necrosis factor (TNF) ligands show promise for cancer treatment due to their apoptosis-inducing and immune-stimulating properties.
Purpose of the Study:
- To develop a protocol for purifying scFv-TNF ligand fusion proteins.
- To establish assays for evaluating the targeting index of these novel fusion proteins.
Main Methods:
- Eukaryotic production of scFv-TNF ligand fusion proteins.
- Affinity chromatography using anti-Flag agarose for purification.
- Development of assays to determine the targeting index.
Main Results:
- A reliable protocol for purifying scFv-TNF ligand fusion proteins was established.
- Assays for assessing the targeting capabilities of these fusion proteins were described.
Conclusions:
- N-terminal fusion of scFvs to TNF ligands preserves ligand function.
- The developed purification and assay methods facilitate the development of targeted cancer therapies using scFv-TNF ligand fusion proteins.
