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Direct fluorine-18 labeling of heat-sensitive biomolecules for positron emission tomography imaging using the Al18F-RESCA method

Nature Protocols volume 13, pages 2330–2347 (2018)Cite this article

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Abstract

Positron emission tomography (PET) is a quickly expanding, non-invasive molecular imaging technology, and there is high demand for new specific imaging probes. Herein, we present a generic protocol for direct radiolabeling of heat-sensitive biomolecules with the positron-emitting radioisotope fluorine-18 (18F) using the aluminum fluoride restrained complexing agent (Al18F-RESCA) method. The Al18F-RESCA method combines the chemical advantages of a chelator-based radiolabeling method with the unique physical properties of the radionuclide of choice, fluorine-18. Proteins of interest can be conjugated to RESCA via amine coupling using (±)-H3RESCA-TFP, followed by purification using size-exclusion chromatography (SEC). Next, RESCA-derivatized biomolecules can be labeled in one step, at room temperature (~20 °C) in an aqueous medium with aluminum fluoride (Al18F). Al18F-labeled proteins can be obtained with moderate (12–17 GBq/µmol) to good (80–85 GBq/µmol) apparent molar activity, depending on the starting activity of 18F–. In addition, satisfactory radiochemical yields (35–55%, non–decay corrected) and high radiochemical purity (>98%, using gel filtration or solid-phase purification) are obtained. The mild radiolabeling procedure takes 0.5 h to complete and can be used for direct labeling of vector molecules such as peptides, protein scaffolds, and engineered antibody fragments.

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Fig. 1: Chemical structures of the RESCA derivatives (±)-H3RESCA-TFP and (±)-H3RESCA-Mal.
Fig. 2: Schematic representation of nanobody modification with (±)-H3RESCA-TFP and subsequent labeling with Al18F at room temperature.
Fig. 3: SEC radiochromatogram of purified (±)-[18F]AlF(RESCA)-V4m119 nanobody, eluting with a retention time of 26.9 min.

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Acknowledgements

The authors thank J. Cleynhens, J. Cornelis, I. Sannen, J. Peetroons, S. Celen, H.-J. Verhaegen, and P. Haspeslagh from the Laboratory for Radiopharmaceutical Research (University of Leuven); J. De Jonge from the In vivo Cellular and Molecular Imaging Center (Vrije Universiteit Brussel); and S. Muyldermans from the Cellular and Molecular Imaging Laboratory (Vrije Universiteit Brussel). This research received support from IWT Flanders (SBO 130065 MIRIAD, G.B., N.D.; SBO S000218N MATATUM, N.D.), the EU (H2020-MSCA-ITN PET3D, C.X., J.B., N.D.), and the FWO (G0D8817N, G.B.; G066615N, C.X., N.D.). F.C. is a Postdoctoral Fellow of FWO (12R3119N).

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Authors and Affiliations

  1. Radiopharmaceutical Research, Department of Pharmacy and Pharmacology, University of Leuven, Leuven, Belgium

    Frederik Cleeren, Joan Lecina, Térence Tshibangu & Guy Bormans

  2. In Vivo Cellular and Molecular Imaging Center, Vrije Universiteit Brussel, Brussels, Belgium

    Jessica Bridoux, Nick Devoogdt & Catarina Xavier

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  1. Frederik Cleeren
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Contributions

F.C., J.L., N.D., C.X., and G.B. designed the research. J.L. synthesized the RESCA ligands. F.C., T.T., and J.B. performed the experimental work. F.C., T.T., and G.B. wrote the manuscript. G.B. is the corresponding author.

Corresponding author

Correspondence to Guy Bormans.

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Competing interests

A patent application related to this work has been filed (WO/2016/065435; F.C., J.L., G.B.). The RESCA chelator will be made commercially available from Chematech in the near future. F.C., J.L., and G.B. will receive a part of the profit. The remaining authors declare no competing interests.

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Cleeren, F. et al. Theranostics 7, 2924–2939 (2017) http://www.thno.org/v07p2924.htm

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Cleeren, F., Lecina, J., Bridoux, J. et al. Direct fluorine-18 labeling of heat-sensitive biomolecules for positron emission tomography imaging using the Al18F-RESCA method. Nat Protoc 13, 2330–2347 (2018). https://doi.org/10.1038/s41596-018-0040-7

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