Claudia Kessler1,2, Alessa Pardo2, Mehmet K Tur3, Stefan Gattenlöhner3, Rainer Fischer1,4, Katharina Kolberg1,2, Stefan Barth5. 1. Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074, Aachen, Germany. 2. Institute of Applied Medical Engineering, University Hospital RWTH Aachen, Aachen, Germany. 3. Institute for Pathology, Justus-Liebig University, Giessen, Germany. 4. Institute of Molecular Biotechnology (Biology VII), RWTH Aachen University, Aachen, Germany. 5. South African Research Chair in Cancer Biotechnology, Department of Integrative Biomedical Sciences, Faculty of Health Sciences, Institute of Infectious Disease and Molecular Medicine (IDM), University of Cape Town, Anzio Road, Observatory, 7925, South Africa. stefan.barth@uct.ac.za.
Abstract
PURPOSE: Despite great progress in the diagnosis and treatment of localized prostate cancer (PCa), there remains a need for new diagnostic markers that can accurately distinguish indolent and aggressive variants. One promising approach is the antibody-based targeting of prostate stem cell antigen (PSCA), which is frequently overexpressed in PCa. Here, we show the construction of a molecular imaging probe comprising a humanized scFv fragment recognizing PSCA genetically fused to an engineered version of the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT), the SNAP-tag, enabling specific covalent coupling to various fluorophores for diagnosis of PCa. Furthermore, the recombinant immunotoxin (IT) PSCA(scFv)-ETA' comprising the PSCA(scFv) and a truncated version of Pseudomonas exotoxin A (PE, ETA') was generated. METHODS: We analyzed the specific binding and internalization behavior of the molecular imaging probe PSCA(scFv)-SNAP in vitro by flow cytometry and live cell imaging, compared to the corresponding IT PSCA(scFv)-ETA'. The cytotoxic activity of PSCA(scFv)-ETA' was tested using cell viability assays. Specific binding was confirmed on formalin-fixed paraffin-embedded tissue specimen of early and advanced PCa. RESULTS: Alexa Fluor® 647 labeling of PSCA(scFv)-SNAP confirmed selective binding to PSCA, leading to rapid internalization into the target cells. The recombinant IT PSCA(scFv)-ETA' showed selective binding leading to internalization and efficient elimination of target cells. CONCLUSIONS: Our data demonstrate, for the first time, the specific binding, internalization, and cytotoxicity of a scFv-based fusion protein targeting PSCA. Immunohistochemical staining confirmed the specific ex vivo binding to primary PCa material.
PURPOSE: Despite great progress in the diagnosis and treatment of localized prostate cancer (PCa), there remains a need for new diagnostic markers that can accurately distinguish indolent and aggressive variants. One promising approach is the antibody-based targeting of prostate stem cell antigen (PSCA), which is frequently overexpressed in PCa. Here, we show the construction of a molecular imaging probe comprising a humanized scFv fragment recognizing PSCA genetically fused to an engineered version of the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT), the SNAP-tag, enabling specific covalent coupling to various fluorophores for diagnosis of PCa. Furthermore, the recombinant immunotoxin (IT) PSCA(scFv)-ETA' comprising the PSCA(scFv) and a truncated version of Pseudomonas exotoxin A (PE, ETA') was generated. METHODS: We analyzed the specific binding and internalization behavior of the molecular imaging probe PSCA(scFv)-SNAP in vitro by flow cytometry and live cell imaging, compared to the corresponding IT PSCA(scFv)-ETA'. The cytotoxic activity of PSCA(scFv)-ETA' was tested using cell viability assays. Specific binding was confirmed on formalin-fixed paraffin-embedded tissue specimen of early and advanced PCa. RESULTS: Alexa Fluor® 647 labeling of PSCA(scFv)-SNAP confirmed selective binding to PSCA, leading to rapid internalization into the target cells. The recombinant IT PSCA(scFv)-ETA' showed selective binding leading to internalization and efficient elimination of target cells. CONCLUSIONS: Our data demonstrate, for the first time, the specific binding, internalization, and cytotoxicity of a scFv-based fusion protein targeting PSCA. Immunohistochemical staining confirmed the specific ex vivo binding to primary PCa material.
Entities:
Keywords:
Immunotoxin (IT); Prostate cancer; Prostate stem cell antigen (PSCA); Pseudomonas exotoxin A (ETA′); SNAP-tag; Single-chain fragment variable (scFv)
Authors: S Barth; M Huhn; B Matthey; R Schnell; S Tawadros; T Schinköthe; J Lorenzen; V Diehl; A Engert Journal: Int J Cancer Date: 2000-06-01 Impact factor: 7.396
Authors: Florian Kampmeier; Markus Ribbert; Thomas Nachreiner; Sofia Dembski; Florent Beaufils; Andreas Brecht; Stefan Barth Journal: Bioconjug Chem Date: 2009-05-20 Impact factor: 4.774
Authors: Richard Cathomas; Christian Rothermundt; Dirk Klingbiel; Lukas Bubendorf; Rolf Jaggi; Daniel C Betticher; Peter Brauchli; Denise Cotting; Cornelia Droege; Ralph Winterhalder; Daniele Siciliano; Dominik R Berthold; Miklos Pless; Ralph Schiess; Roger von Moos; Silke Gillessen Journal: Clin Cancer Res Date: 2012-09-12 Impact factor: 12.531
Authors: Chaoyu Zhang; Wenjie Sheng; Marwah Al-Rawe; T M Mohiuddin; Marcus Niebert; Felix Zeppernick; Ivo Meihold-Heerlein; Ahmad Fawzi Hussain Journal: Int J Mol Sci Date: 2022-05-30 Impact factor: 6.208