caa a22 4500 606175490 CHVBK 20210128100743.0 cr unu---uuuuu 210128e20150401xx s 000 0 eng 10.1007/s00018-014-1751-8 doi (NATIONALLICENCE)springer-10.1007/s00018-014-1751-8 A new prodrug form of Affibody molecules (pro-Affibody) is selectively activated by cancer-associated proteases [Elektronische Daten] [Lisa Sandersjöö, Andreas Jonsson, John Löfblom] Affinity proteins have advanced the field of targeted therapeutics due to their generally higher specificity compared to small molecular compounds. However, side effects caused by on-target binding in healthy tissues are still an issue. Here, we design and investigate a prodrug strategy for improving tissue specificity of Affibody molecules in future in vivo studies. The prodrug Affibody (pro-Affibody) against the HER2 receptor was constructed by fusing a HER2-specific Affibody (ZHER2) to an anti-idiotypic Affibody (anti-ZHER2). The linker was engineered to comprise a substrate peptide for the cancer-associated matrix metalloprotease 1 (MMP-1). The hypothesis was that the binding surface of ZHER2 would thereby be blocked from interacting with HER2 until the substrate peptide was specifically hydrolyzed by MMP-1. Binding should thereby only occur where MMP-1 is overexpressed, potentially decreasing on-target toxicities in normal tissues. The pro-Affibody was engineered to find a suitable linker and substrate peptide, and the different constructs were evaluated with a new bacterial display assay. HER2-binding of the pro-Affibody was efficiently masked and proteolytic activation of the best variant yielded over 1,000-fold increase in apparent binding affinity. Biosensor analysis revealed that blocking of the pro-Affibody primarily affected the association phase. In a cell-binding assay, the activated pro-Affibody targeted native HER2 on cancer cells as opposed to the non-activated pro-Affibody. We believe this prodrug approach with proteolytic activation is promising for improving tissue specificity in future in vivo targeting applications and can hopefully be extended to other Affibody molecules and similar affinity proteins as well. Springer Basel, 2014 HER2 nationallicence Bacterial display nationallicence Combinatorial protein engineering nationallicence Staphylococcal display nationallicence Directed evolution nationallicence Cell surface display nationallicence Flow cytometry nationallicence MMP-1 nationallicence Sandersjöö Lisa Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, 106 91, Stockholm, Sweden aut Jonsson Andreas Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, 106 91, Stockholm, Sweden aut Löfblom John Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, 106 91, Stockholm, Sweden aut Cellular and Molecular Life Sciences Springer Basel 72/7(2015-04-01), 1405-1415 1420-682X 72:7<1405 2015 72 18 https://doi.org/10.1007/s00018-014-1751-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00018-014-1751-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Sandersjöö Lisa Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, 106 91, Stockholm, Sweden aut NATIONALLICENCE 700 1- Jonsson Andreas Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, 106 91, Stockholm, Sweden aut NATIONALLICENCE 700 1- Löfblom John Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, 106 91, Stockholm, Sweden aut NATIONALLICENCE 773 0- Cellular and Molecular Life Sciences Springer Basel 72/7(2015-04-01), 1405-1415 1420-682X 72:7<1405 2015 72 18