A new prodrug form of Affibody molecules (pro-Affibody) is selectively activated by cancer-associated proteases
2015 (English)In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 72, no 7, 1405-1415 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2015. Vol. 72, no 7, 1405-1415 p.
Bacterial display, Cell surface display, Combinatorial protein engineering, Directed evolution, Flow cytometry, HER2, MMP-1, Staphylococcal display
IdentifiersURN: urn:nbn:se:kth:diva-160032DOI: 10.1007/s00018-014-1751-8ISI: 000351455100014PubMedID: 25287047ScopusID: 2-s2.0-84907611403OAI: oai:DiVA.org:kth-160032DiVA: diva2:788314
FunderSwedish Research Council
QC 201504192015-02-132015-02-132015-05-21Bibliographically approved