Ghosh, PrithaDavies, Lani J.Nitsche, Christoph2025-05-312025-05-311433-7851WOS:001357864300001PubMed:39481115ORCID:/0000-0003-1834-0452/work/172891163ORCID:/0000-0002-3704-2699/work/172891288https://hdl.handle.net/1885/733755876Targeted theranostics heavily rely on metal isotopes conjugated to antibodies. Single-domain antibodies, known as nanobodies, are much smaller in size without compromising specificity and affinity. The conventional way of conjugating metals to nanobodies involves non-specific modification of amino acid residues with bifunctional chelating agents. We demonstrate that mutagenesis of a single residue in a nanobody creates a triple cysteine motif that selectively binds bismuth which is, for example, used in targeted alpha therapy. Two mutations create a quadruple cysteine mutant specific for gallium and indium used in positron emission tomography and single-photon emission computed tomography, respectively. Labelling is quantitative within a few minutes. The metal nanobodies maintain structural integrity and stability over weeks, resist competition from endogenous metal binders like glutathione, and retain functionality.We gratefully acknowledge the Australian Research Council for funding support, including a Discovery Project (DP230100079) and a Future Fellowship (FT220100010). We thank Dr. Doug Lawes (NMR Suite, Research School of Chemistry, ANU) for his invaluable assistance with NMR acquisition. We also extend our appreciation to the staff of the Joint Mass Spectrometry Facility (ANU) for their guidance in training, sample preparation, and instrument handling. We thank Dr. Shouvik Aditya (Research School of Biology, ANU) for his support in SPR training and analysis and Dr. Yang Wu (Research School of Earth Sciences, ANU) for his support in ICP-MS sample preparation and instrumentation.9en© 2024 Wiley-VCH GmbHBismuthGalliumIndiumNanobodyTheranostics2024-10-3110.1002/anie.20241945585209824063