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

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

Supplementary material

Figure S1 Cytotoxic effects of free AURIF and mock-SNAP-AURIF.

Notes: The cytotoxicity of free AURIF and mock-SNAP-AURIF assessed using an XTT-based viability assay after incubation for 72 h. Representative data from one assay are shown. Cells were incubated with decreasing concentrations of free AURIF and mock-SNAP-AURIF. The EC₅₀ values relative to cells treated with PBS (negative control) and Zeocin (positive control) were calculated using GraphPad Prism v5. The data are shown as mean ± SD of each measurement.

Abbreviations: AURIF, auristatin F; EC₅₀, concentration required to achieve a 50% reduction of cell viability; PBS, phosphate-buffered saline.

Acknowledgments

Mira Woitok was supported by the RWTH Aachen University scholarship of Young Researchers at the RWTH Aachen University (RFwN). The data in this publication were produced during the TheraSECOURE project, which was funded by the Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung [BMBF]) under project number 03V0348. Responsibility for the content of this publication lies with the authors. We thank Frank Schmelter for providing A549-SNAP-TubB3 cells. The authors would like to thank Kai Fuhrmann for excellent technical support and Dr Richard M Twyman for critical reading of the manuscript.

The present address of Stefan Barth is University of Cape Town, South African Research Chair in Cancer Biotechnology, Institute of Infectious Disease and Molecular Medicine (IDM), Faculty of Health Sciences, Department of Integrative Biomedical Sciences, Anzio Road, Observatory, 7925 Cape Town, South Africa. The present adress of Judith Niesen is Research Institute Children’s Cancer Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Disclosure

Wolfgang Richter is an employee of Tube Pharmaceuticals GmbH. The authors report no other conflicts of interest in this work.