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In this study, we engineered an antibody prodrug using an anti-P-selectin antibody as a model system, incorporating N-terminal heavy chain extensions comprising a linear P-selectin (CD62P) epitope for shielding, a thrombin-cleavable linker, and variable spacer in the linker. A prodrug antibody library was prepared comprising various N-terminal extensions of the heavy chain including an Ala-scan over the P-selectin epitope and the thrombin cleavage site sequence (50 variants in the library). The shielding library was thoroughly investigated for retained target binding kinetics and cleavage activation kinetics. Antibody prodrug variants achieving complete shielding of target binding to a completely open state were observed in the library (many orders of magnitude reduced binding based on surface plasmon resonance (SPR) kinetic measurement). Upon thrombin activation, the antibody prodrug variants obtained full target binding at low nM affinity. Thus, at such nM antibody prodrug concentrations, the liberated shielding extensions appeared to diffuse effectively away from the paratope upon cleavage based on SPR and fluorescence-activated cell sorting (FACS) analyses. Importantly, binding studies of variants of the linear epitope peptide alone showed many orders-of-difference in reduced binding compared to the fused extensions of the epitope sequence to the antibody prodrug. Comparison of the kinetic data with a structure model of the prodrug antibody reveals aspects of designing an optimal prodrug antibody that may have general applicability as a novel therapeutic format.<\/jats:p>","DOI":"10.1093\/protein\/gzag029","type":"journal-article","created":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T11:40:41Z","timestamp":1790941241000},"source":"Crossref","is-referenced-by-count":0,"title":["Engineering and kinetic profiling of a thrombin-activable prodrug antibody targeting P-selectin"],"prefix":"10.1093","volume":"39","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-1106-6665","authenticated-orcid":false,"given":"Mie M","family":"Mortensen","sequence":"first","affiliation":[{"name":"Research and Development, Novo Nordisk , Novo Nordisk Park 1, 2760 M\u00e5l\u00f8v ,","place":["Denmark"]},{"name":"Faculty of Engineering and Science, University of Aalborg , Fredrik Bajers Vej, 9220 Aalborg 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