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Comparison of a mouse and a novel human scFv-SNAP-auristatin F drug conjugate with potent activity against EGFR-overexpressing human solid tumor cells

Authors Woitok M, Klose D, Di Fiore S, Richter W, Stein C, Gresch G, Grieger E, Barth S, Fischer R, Kolberg K, Niesen J

Received 26 April 2017

Accepted for publication 23 May 2017

Published 6 July 2017 Volume 2017:10 Pages 3313—3327


Checked for plagiarism Yes

Review by Single-blind

Peer reviewer comments 2

Editor who approved publication: Dr Carlos E Vigil

Mira Woitok,1,2 Diana Klose,1 Stefano Di Fiore,1 Wolfgang Richter,3 Christoph Stein,1 Gerrit Gresch,1 Elena Grieger,1 Stefan Barth,1 Rainer Fischer,1,2 Katharina Kolberg,1,* Judith Niesen1,*

1Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen, Germany; 2Institute of Molecular Biotechnology (Biology VII), RWTH Aachen University, Aachen, Germany; 3Tube Pharmaceuticals GmbH, Vienna, Austria

*These authors contributed equally to this work

Abstract: Antibody–drug conjugates (ADCs) can deliver toxins to specific targets such as tumor cells. They have shown promise in preclinical/clinical development but feature stoichiometrically undefined chemical linkages, and those based on full-size antibodies achieve only limited tumor penetration. SNAP-tag technology can overcome these challenges by conjugating benzylguanine-modified toxins to single-chain fragment variables (scFvs) with 1:1 stoichio­metry while preserving antigen binding. Two (human and mouse) scFv-SNAP fusion proteins recognizing the epidermal growth factor receptor (EGFR) were expressed in HEK 293T cells. The purified fusion proteins were conjugated to auristatin F (AURIF). Binding activity was confirmed by flow cytometry/immunohistochemistry, and cytotoxic activity was confirmed by cell viability/apoptosis and cell cycle arrest assays, and a novel microtubule dynamics disassembly assay was performed. Both ADCs bound specifically to their target cells in vitro and ex vivo, indicating that the binding activity of the scFv-SNAP fusions was unaffected by conjugation to AURIF. Cytotoxic assays confirmed that the ADCs induced apoptosis and cell cycle arrest at nanomolar concentrations and microtubule disassembly. The SNAP-tag technology provides a platform for the development of novel ADCs with defined conjugation sites and stoichiometry. We achieved the stable and efficient linkage of AURIF to human or murine scFvs using the SNAP-tag technology, offering a strategy to improve the development of personalized medicines.

Keywords: epidermal growth factor receptor, EGFR, antibody–drug conjugate, ADC, SNAP-tag technology, single-chain fragment variable, scFv, BG-modified auristatin F, AURIF

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