Original Articles An EGF receptor targeting Ranpirnase-diabody fusion protein mediates potent antitumour activity in vitro and in vivo Stefan Kiesgen a , Michaela A.E. Arndt a,b , Christoph Körber c , Ulrich Arnold d , Tobias Weber a , Niels Halama a , Armin Keller a , Benedikt Bötticher a , Anne Schlegelmilch a , Nora Liebers a , Martin Cremer a , Christel Herold-Mende e,f , Gerhard Dyckhoff f , Philippe A. Federspil g , Alexandra D. Jensen h,i , Dirk Jäger a , Roland E. Kontermann j , Walter Mier k , Jürgen Krauss a, * a Department of Medical Oncology, National Center for Tumor Diseases, Heidelberg University Hospital, Im Neuenheimer Feld 460, Heidelberg 69120, Germany b Immunotherapy Program, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg 69120, Germany c Institute of Anatomy and Cell Biology, Heidelberg University, Im Neuenheimer Feld 307, Heidelberg 69120, Germany d Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, Halle 06120, Germany e Division of Experimental Neurosurgery, Department of Neurosurgery, University of Heidelberg, Im Neuenheimer Feld 400, Heidelberg 69120, Germany f Molecular Cell Biology Group, ENT Department, University of Heidelberg, Im Neuenheimer Feld 400, Heidelberg 69120, Germany g Department of Otorhinolaryngology, Head and Neck Surgery, University of Heidelberg, Im Neuenheimer Feld 400, Heidelberg 69120, Germany h Department of Radiation Oncology, University of Heidelberg, Im Neuenheimer Feld 400, Heidelberg 69120, Germany i Heidelberg Ion Therapy Center (HIT), Im Neuenheimer Feld 450, Heidelberg 69120, Germany j Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, Stuttgart 70569, Germany k Department of Nuclear Medicine, University Hospital Heidelberg, Im Neuenheimer Feld 400, Heidelberg 69120, Germany ARTICLE INFO Article history: Received 27 August 2014 Received in revised form 19 November 2014 Accepted 20 November 2014 Keywords: immunoRNase Diabody EGFR Ranpirnase Onconase A B ST R AC T Cytotoxic ribonucleases such as the leopard frog derivative Ranpirnase (Onconase ® ) have emerged as a valuable new class of cancer therapeutics. Clinical trials employing single agent Ranpirnase in cancer patients have demonstrated significant clinical activity and surprisingly low immunogenicity. However, dose-limiting toxicity due to unspecific uptake of the RNase into non-cancerous cells is reached at relatively low concentrations of > 1 mg/m 2 . We have in the present study generated a dimeric anti- EGFR Ranpirnase-diabody fusion protein capable to deliver two Ranpirnase moieties per molecule to EGFR-positive tumour cells. We show that this compound mediated far superior efficacy for killing EGFR-positive tumour cells than a monomeric counterpart. Most importantly, cell killing was restricted to EGFR-positive target cells and no dose-limiting toxicity of Ranpirnase-diabody was observed in mice. These data indicate that by targeted delivery of Ranpirnase non-selective toxicity can be abolished and suggests Ranpirnase-diabody as a promising new drug for therapeutic interventions in EGFR-positive cancers. © 2014 Elsevier Ireland Ltd. All rights reserved. Introduction The epidermal growth factor receptor (EGFR) belongs to the ErbB/ HER family of transmembrane receptors involved in modulation of cell proliferation, survival, migration, and differentiation [1]. Mutations clus- tering in functionally relevant hotspots of the EGF receptor commonly lead to enhanced intrinsic tyrosine kinase activity or dysregulation of suppressive domains and contribute to the pathogenesis and progres- sion of epithelial cancers. Moreover, increased EGFR expression has been linked to an adverse prognosis in patients with head and neck, bladder, ovarian and cervical cancers [2]. Consequently, inhibition of EGFR func- tion is considered to be a promising therapeutic approach. Both small molecule tyrosine kinase inhibitors (TKIs) as well as monoclonal an- tibodies (mAbs) with specificity for the extracellular ligand-binding domain have been approved by the FDA as EGFR-targeted cancer thera- pies. However, EGFR-targeted cancers frequently develop resistance mechanisms towards these compounds, rendering them ineffective over time [3–5]. For example, although TKIs have been shown to impres- sively prolong progression-free survival of non-small cell lung cancer patients harbouring gain-of-function EGFR mutants [6–8], almost all of these patients eventually relapsed due to acquired resistance to anti- EGFR treatment [3,9]. Antineoplastic effects of approved EGFR-targeting mAbs are mediated by cell signalling interference, recruitment of * Corresponding author. Tel.: +49 6221 565922; fax: +49 6221 565317. E-mail address: juergen.krauss@nct-heidelberg.de (J. Krauss). http://dx.doi.org/10.1016/j.canlet.2014.11.054 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved. Cancer Letters 357 (2015) 364–373 Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet