Basic StudiesSmall Cleavable Adapters Enhance the Specific Cytotoxicity of a Humanized Immunotoxin Directed Against CD64-positive CellsHetzel, Christian*; Bachran, Christopher‡; Fischer, Rainer* †; Fuchs, Hendrik‡; Barth, Stefan* §; Stöcker, Michael† Author Information *Fraunhofer IME, Department of Pharmaceutical Product Development †Institute for Molecular Biotechnology, RWTH-Aachen §Department of Experimental Medicine and Immunotherapy, Helmholtz-Institute for Applied Medical Engineering, AACHEN ‡Zentralinstitut für Laboratoriumsmedizin und Pathobiochemie, Charite, Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm, Berlin, Germany Financial Disclosure: All authors have declared there are no financial conflicts of interest in regard to this work. This work was partially funded by grants 106521 from the German cancer foundation, and IMHOTEP of the Fraunhofer-Gesellschaft, both to Stefan Barth. Reprints: Christian Hetzel, Fraunhofer IME, Department of Pharmaceutical Product Development, Forckenbeckstr. 6, 52074 AACHEN, Germany (e-mail: [email protected]). Received for publication November 19, 2007; accepted January 20, 2008 Stefan Barth and Michael Stöcker equally contributed to principle investigatorship. Journal of Immunotherapy: May 2008 - Volume 31 - Issue 4 - p 370-376 doi: 10.1097/CJI.0b013e31816a2d23 Buy Metrics Abstract The most potent immunotoxins (ITs) developed to date contain bacterial or plant cytotoxic components. As these are potentially immunogenic in man, human proteins are preferred for the long-term treatment of cancer. We have developed the first humanized IT for the treatment of CD64+ malignancies such as acute myeloid leukemia. The bacterially expressed IT is composed of a humanized anti-CD64 single chain fragment [h22(scFv)] genetically fused to the human RNase angiogenin. As angiogenin lacks a dedicated translocation domain responsible for the higher potency of bacterial and plant-derived toxins, we have incorporated a recombinant adapter that contains a synthetic translocation domain flanked by proteolytically cleavable endosomal and cytosolic consensus sites. Although insertion of the adapter increased the cytotoxicity by up to 20-fold, serum stability was markedly reduced. Therefore, we designed a modified adapter variant with the endosomal-cleavable peptide deleted. The IT containing the truncated adapter showed significantly higher cytotoxicity than the adapter-free IT and superior serum stability to facilitate the potential applications in patients. © 2008 Lippincott Williams & Wilkins, Inc.