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Production, Purification, and Characterization of scFv TNF Ligand Fusion Proteins

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Book cover Antibody Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 907))

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.

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Authors and Affiliations

  1. Division for Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Würzburg, Germany

    Andrea Fick, Agnes Wyzgol & Harald Wajant

Authors
  1. Andrea Fick
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  2. Agnes Wyzgol
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  3. Harald Wajant
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Corresponding author

Correspondence to Harald Wajant .

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Editors and Affiliations

  1. Antibody Therapeutics & Immunotargeting, INSERM U624, Marseille CEDEX 09, France

    Patrick Chames

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Fick, A., Wyzgol, A., Wajant, H. (2012). Production, Purification, and Characterization of scFv TNF Ligand Fusion Proteins. In: Chames, P. (eds) Antibody Engineering. Methods in Molecular Biology, vol 907. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-974-7_33

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  • DOI: https://doi.org/10.1007/978-1-61779-974-7_33

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-973-0

  • Online ISBN: 978-1-61779-974-7

  • eBook Packages: Springer Protocols

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