Side-by-Side Comparison of Different Thiol Bioconjugation Strategies for the Chemoselective Radiolabeling of Human Serum Albumin with Zirconium-89

Author(s): Julia Kronberger, Barbora Neuzilova, Anja Federa, Manuel Tieber, Amando Palombo, Marie R. Brandt, Petra Heffeter, Christian R. Kowol, Milos Petrik, Thomas L. Mindt
Abstract: Different bioconjugation strategies are available for the cysteine (Cys)-specific functionalization of proteins with different payloads, including imaging probes and (pro-)drugs. Most commonly applied linkers include maleimides (mal); however, because of the sometimes-observed instability of the formed thiosuccinimidyl linkage, its suitability for in vivo applications has been challenged. Consequently, several alternatives have been developed and compared to mal as a benchmark, yet examples of a direct comparison among new methodologies are scarce. We herein report a comparison of the use of mal, phenyloxadiazole methyl sulfone (PODS), and vinylketone (VK) as functional groups for the thiol-specific functionalization of human serum albumin (HSA) via its available free Cys(34). Bifunctional chelating agents (BFCA) based on DFO*, identical in all regards but the functional group for bioconjugation, were prepared and conjugated to HSA and the obtained DFO*-HSA conjugates were radiolabeled with Zirconium-89 (Zr-89) for positron emission tomography (PET). The efficiency of the conjugation of DFO*-X (X = mal, PODS, VK) to HSA differed significantly, with mal > PODS > VK. Stability studies of the Zr-89-labeled HSA-conjugates indicated good stability for [Zr-89]Zr-DFO*malHSA 11 and [Zr-89]Zr-DFO*-POD-HSA 12 in blood serum but only the latter was found stable in cell culture medium. [Zr-89]Zr-DFO*VK-HSA 13 was excluded from biological experiments due to its surprisingly low stability in all media tested. [Zr-89]Zr-DFO*-POD-HSA 12 was further investigated in CT26-tumor-bearing mice by PET/CT imaging and biodistribution studies. Specific uptake of radioactivity in tumors was high (up to 17% ID/g) and the tumors could be clearly visualized by PET at all time points with excellent tumor-to-background signal (tumor-to-blood ratio 3.2 +/- 1.0 after 48 h p.i.). Unexpectedly, the uptake of radioactivity in bones was observed for [Zr-89]Zr-DFO*-POD-HSA 12. Overall, the in vivo performance of [Zr-89]Zr-DFO*-labeled HSA obtained by mal chemistry is the most promising candidate as a companion diagnostic PET imaging probe for the stratification of patients for therapies based on HSA-binding (pro-)drugs.
Organisation(s): Department of Inorganic Chemistry, Joint Applied Medicinal Radiochemistry Facility
External organisation(s): Vienna Doctoral School in Chemistry (DoSChem), Palacký University Olomouc, University of Vienna, Medizinische Universität Wien
Journal: ACS bio & med chem Au : an open access journal of the American Chemical Society
ISSN: 2694-2437
DOI: https://doi.org/10.1021/acsbiomedchemau.6c00002
Publication date: 2026
Peer reviewed: Yes
Austrian Fields of Science 2012: 104003 Inorganic chemistry, 104020 Radiochemistry, 301904 Cancer research
Keywords
Sustainable Development Goals: SDG 3 - Good Health and Well-being
Portal url: https://ucrisportal.univie.ac.at/en/publications/e850db0b-361f-4639-b56e-46eb424b1f9e