{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T18:03:53Z","timestamp":1788804233863,"version":"build-2803163510"},"reference-count":39,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,9]],"date-time":"2026-01-09T00:00:00Z","timestamp":1767916800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100012331","name":"Vlaamse Agentschap voor Innovatie en Ondernemen","doi-asserted-by":"publisher","award":["HBC.2021.0814"],"award-info":[{"award-number":["HBC.2021.0814"]}],"id":[{"id":"10.13039\/100012331","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background\/Objectives: NANOBODY\u00ae molecules (VHHs) are attractive vectors for radiopharmaceuticals due to their small size and high target affinity, but rapid clearance and pronounced kidney retention limit their therapeutic applicability. Binding to serum albumin is a widely used strategy to prolong circulation, yet the respective contributions of albumin-binding affinity and molecular format remain insufficiently defined. This study aimed to systematically evaluate how affinity and valency modulate VHH pharmacokinetics. Methods: Four monovalent albumin-binding VHHs spanning nanomolar to micromolar affinities and two bivalent constructs were engineered, generated by fusing an albumin-binding VHH to an irrelevant non-binding VHH. All constructs incorporated a site-specific cysteine for DFO* conjugation, enabling uniform zirconium-89 labeling with high radiochemical purity. Pharmacokinetics were assessed in healthy mice using serial blood sampling and positron emission tomography. Blood and kidney exposure were quantified by non-compartmental analysis. Results: All albumin-binding constructs showed increased systemic exposure and reduced kidney uptake relative to a non-binding control. Nanomolar-affinity binders reached maximal exposure, and further affinity increases (KD &lt; ~100 nM) did not improve pharmacokinetics, suggesting a threshold. The micromolar binder showed intermediate exposure but still reduced renal retention compared with control. Valency effects were affinity-dependent. They were negligible at high affinity but pronounced at low affinity, where bivalency reduced systemic exposure and increased kidney uptake toward control levels. Conclusions: Albumin binding enables tuning of VHH pharmacokinetics in an affinity-dependent manner. Above an apparent affinity threshold, pharmacokinetics become format independent, whereas below this threshold, molecular format substantially influences systemic and renal disposition.<\/jats:p>","DOI":"10.3390\/ph19010120","type":"journal-article","created":{"date-parts":[[2026,1,9]],"date-time":"2026-01-09T16:03:16Z","timestamp":1767974596000},"page":"120","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Affinity- and Format-Dependent Pharmacokinetics of 89Zr-Labeled Albumin-Binding VHH Constructs"],"prefix":"10.3390","volume":"19","author":[{"given":"Simon","family":"Leekens","sequence":"first","affiliation":[{"name":"Laboratory for Radiopharmaceutical Research, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter","family":"Casteels","sequence":"additional","affiliation":[{"name":"Sanofi Ghent, 9000 Ghent, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-7530-3722","authenticated-orcid":false,"given":"Tom","family":"Van Bogaert","sequence":"additional","affiliation":[{"name":"Sanofi Ghent, 9000 Ghent, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pieter","family":"Deschaght","sequence":"additional","affiliation":[{"name":"Sanofi Ghent, 9000 Ghent, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Veronique","family":"De Brabandere","sequence":"additional","affiliation":[{"name":"Sanofi Ghent, 9000 Ghent, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5975-0354","authenticated-orcid":false,"given":"Christopher","family":"Cawthorne","sequence":"additional","affiliation":[{"name":"Department of Imaging and Pathology, Nuclear Medicine and Molecular Imaging, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0335-7190","authenticated-orcid":false,"given":"Guy","family":"Bormans","sequence":"additional","affiliation":[{"name":"Laboratory for Radiopharmaceutical Research, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3692-1608","authenticated-orcid":false,"given":"Frederik","family":"Cleeren","sequence":"additional","affiliation":[{"name":"Laboratory for Radiopharmaceutical Research, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Bodei, L., Lewis, J.S., and Zeglis, B.M. 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