Group Fuchs

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Group Fuchs

Protein-based targeted tumor therapies

Research area

The major goal of the research group is to develop novel recombinant fusion proteins to selectively target tumor cells and to mediate the transfer of the cytotoxic drug to intracellular target proteins. We are focusing on two different strategies. In the first strategy, protein toxins are recombinantly fused via a molecular adapter (Heisler et al., 2003) to the tumor cell-specific ligand, which may be an antibody or fragment thereof, a growth factor or a cytokine. The toxins are plant ribosome-inactivating proteins targeting 28S-rRNA or bacterial ADP ribosylating proteins targeting eukaryotic elongation factor 2. The adapter is designed to improve cytosolic uptake, retain the toxin inside the cytosol and detoxify the drug after cell death (Fig. 1). In the second strategy, a particular yeast enzyme not possessing an analog in humans is fused to the ligand (Panjideh et al., BBE, 2008). After the fusion protein has bound to the tumor target cells, an inert small molecule prodrug is added that is converted at the tumor by the enzyme into an active cytotoxic compound (antibody-directed enzyme prodrug therapy = ADEPT) (Panjideh et al., IJC, 2008).

Uptake Of Adaptertoxins
Abb.1) Schematische Darstellung der zellulären Aufnahme von Adaptertoxinen. PTD, Proteintransduktionsdomäne; CCP, cytosolisch spaltbares Peptid; ECP, endosomal spaltbares Peptid; CEnz, cytosolisch lokalisierte Protease; EEnz, endosomal lokalisierte Protease. Der Ligand hindert die PTD daran, eine unspezifische Aufnahme in Zellen zu ermöglichen (1). Nach Endozytose des Fusionsproteins (2) führt eine enzymatische Spaltung im Endosom zur Freisetzung des Liganden (3) und damit zur Freilegung der PTD, die nunmehr den Transfer ins Zytosol vermitteln kann (4), wo die PTD durch eine weitere enzymatische Spaltung, diesmal innerhalb der CCP, freigesetzt wird (5) und das aktive Enzym zurückbleibt, das die Proteinbiosynthese inhibiert (6). Da das Toxin keine PTD mehr besitzt, verbleibt es innerhalb der Zelle und kann selbst nach Zelltod und Zelllyse keine normal differenzierten Zellen mehr erreichen. Dieser Mechanismus bewirkt also eine Detoxifizierung, ohne den zytotoxischen Effekt innerhalb der Zielzelle zu beeinflussen.

In both strategies, the proteins will be optimized including improvement of production, purification and stability, reduction of immunogenicity and minimizing side effects. Treatment regimens are being developed in different mouse models. We demonstrated in vitro (Bachran et al., 2008; Heisler et al., 2005) and in vivo (Bachran et al., BJP, 2009; Bachran et al., JI, 2009) that a combined application of the chimeric adaptertoxins with certain glycosylated triter­penoids (saponins from Gypsophila paniculata (Bachran et al., 2006)) drastically enhances the specific cytotoxicity on tumor target cells (Fig. 2). Dependent on the cell line, the cytotoxicity was enhanced up to 385000-fold (Heisler et al., 2005) in cell culture by low non-permeabilizing saponin concentrations in a clearly synergistic manner, i.e. the single compounds were not toxic at the same concentrations. Subcutaneous application at distant sites (neck and flank) of BALB/c mice bearing a solid tumor resulted in 94% tumor volume reduction with a 50-fold lower adaptertoxin concentration than used for the adaptertoxin alone, which resulted in 71% tumor reduction (Bachran et al., BJP, 2009; Bachran et al., JI, 2009; Fuchs et al., 2007).

Dose Response Combination Therapy
Abb. 2) Dosis-Wirkungs-Beziehungen bei der kombinierten Anwendung eines chimären Adaptertoxins und eines Saponins. Jeder Punkt repräsentiert eine Gruppe von fünf Mäusen, die jeweils viermal mit der entsprechenden Kombination behandelt wurden. Anhand der Wirkung und der Nebenwirkungen wurden sechs Kategorien unterschieden, die farblich gekennzeichnet sind. Die Flächen wurden interpoliert. Die Prozentzahlen beschreiben beim therapeutischen Effekt den Rückgang im Tumorvolumen, bei der Letalität die Anzahl der Mäuse.

In the antibody-directed enzyme prodrug therapy, we are optimizing the stability of the fusion proteins by adding glycosylation sites, by PEGylation and by site-directed mutagenesis at protease-sensitive sites. The different proteins will be tested for pharmacodynamics and pharmacokinetics in a mouse model for colon cancer.

If you are interested in our research please contact hendrik.fuchs(at)charite.de. Doctoral theses can be accomplished in our group in medicine, veterinary medicine and natural sciences.

Projects

  • Optimization of recombinant fusion proteins for antibody-directed enzyme-prodrug-therapy (ADEPT) using an in vivo model of colon carcinoma (Deutsche Krebshilfe)
  • Characterization, purification, isolation and scale up of the isolation procedure for plant saponins valuable as synergistic enhancers for the therapeutic efficacy of chimeric toxins directed against tumors (Alexander von Humboldt Stiftung)
  • Development of tumor-activated targeted toxins and their investigation in combination with glycosylated triterpenoids.
  • Further research projects in our group, e.g. revolving around signal transduction, are accessible via our homepage (regrettably only available in German at this moment).

Selected publications


The complete publication list will be found via the homepage of the group.