Light-induced synthesis of protein conjugates and its application in photoradiosynthesis of 89Zr-radiolabeled monoclonal antibodies

Abstract

Efficient methods to functionalize proteins are essential for the development of many diagnostic and therapeutic compounds, such as fluorescent probes for immunohistochemistry, zirconium-89 radiolabeled mAbs (89Zr-mAbs) for positron emission tomography and antibody-drug conjugates (ADCs). This protocol describes a step-by-step procedure for the light-induced functionalization of proteins with compounds bearing the photochemically active aryl azide group. As an illustration of the potential utility of our approach, this protocol focuses on the synthesis of 89Zr-mAbs using photoactivatable derivatives of the metal ion binding chelate desferrioxamine B (DFO). The light-induced synthesis of 89Zr-mAbs is a unique, one-pot process involving simultaneous radiolabeling and protein conjugation. The photoradiochemical synthesis of purified 89Zr-mAbs, starting from unmodified proteins, [89Zr][Zr(C2O4)4]4– (89Zr-oxalate), and a photoactivatable DFO derivative, can be performed in <90 min. The method can be easily adapted to prepare other radiolabeled proteins, ADCs or fluorescently tagged proteins by using drug molecules or fluorophores functionalized with photoactive moieties.

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Fig. 1
Fig. 2: Chemical structures of three photoactivatable DFO derivatives.
Fig. 3: Photochemical apparatus and experimental setup.
Fig. 4: Emission spectra and beam shape.
Fig. 5: Simultaneous photoradiosynthesis of monovalent (one-armed) [89Zr]ZrDFO-PEG3-azepin-onartuzumab.
Fig. 6: Analytical data on the synthesis and characterization of [89Zr]ZrDFO-PEG3-azepin-onartuzumab.
Fig. 7: Representative PET images using [89Zr]ZrDFO-PEG3-azepin-onartuzumab.

Data availability

Source data for the analytical chromatography and PET images are available from the corresponding author.

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Acknowledgements

J.P.H. thanks the Swiss National Science Foundation (SNSF Professorship PP00P2_163683 and PP00P2_190093), the Swiss Cancer League (Krebsliga Schweiz; KLS-4257-08-2017) and the University of Zurich (UZH) for financial support. This project has received funding from the European Union’s Horizon 2020 research and innovation programme, from the European Research Council under the Grant Agreement No. 676904, ERC-StG-2015, NanoSCAN. We thank all members of the Radiochemistry and Imaging Science group at UZH for helpful discussions.

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Contributions

J.P.H. designed and supervised the project, provided funding and wrote the original manuscript. A.G., D.F.E. and M.P. synthesized and characterized the photoactivatable DFO compounds. All authors performed the radiochemistry and analysis. J.P.H. is the study director and resource manager on the approved animal experimentation license. J.P.H. performed all PET imaging and biodistribution experiments and was assisted by A.G. and M.P. All authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Jason P. Holland.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks David Watt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Guillou, A., Earley, D. F. & Holland, J. P. Chemistry 26, 7185–7189 (2020): https://chemistry-europe.onlinelibrary.wiley.com/doi/abs/10.1002/chem.202001755

Klingler, S., Fay, R. & Holland, J. P. J. Nucl. Med. (2020): http://jnm.snmjournals.org/content/early/2020/01/09/jnumed.119.237180

Patra, M., Klingler, S., Eichenberger, L. S. & Holland, J. P. iScience 13, 416–431 (2019): https://www.sciencedirect.com/science/article/pii/S2589004219300707

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Guillou, A., Earley, D.F., Patra, M. et al. Light-induced synthesis of protein conjugates and its application in photoradiosynthesis of 89Zr-radiolabeled monoclonal antibodies. Nat Protoc (2020). https://doi.org/10.1038/s41596-020-0386-5

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