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Inverse electron demand Diels–Alder click chemistry for pretargeted PET imaging and radioimmunotherapy

Abstract

Radiolabeled antibodies have shown promise as tools for both the nuclear imaging and endoradiotherapy of cancer, but the protracted circulation time of radioimmunoconjugates can lead to high radiation doses to healthy tissues. To circumvent this issue, we have developed an approach to positron emission tomography (PET) imaging and radioimmunotherapy (RIT) predicated on radiolabeling the antibody after it has reached its target within the body. This in vivo pretargeting strategy is based on the rapid and bio-orthogonal inverse electron demand Diels–Alder reaction between tetrazine (Tz) and trans-cyclooctene (TCO). Pretargeted PET imaging and RIT using TCO-modified antibodies in conjunction with Tz-bearing radioligands produce high activity concentrations in target tissues as well as reduced radiation doses to healthy organs compared to directly labeled radioimmunoconjugates. Herein, we describe how to prepare a TCO-modified antibody (humanized A33-TCO) as well as how to synthesize two Tz-bearing radioligands: one labeled with the positron-emitting radiometal copper-64 ([64Cu]Cu-SarAr-Tz) and one labeled with the β-emitting radiolanthanide lutetium-177 ([177Lu]Lu-DOTA-PEG7-Tz). We also provide a detailed description of pretargeted PET and pretargeted RIT experiments in a murine model of human colorectal carcinoma. Proper training in both radiation safety and the handling of laboratory mice is required for the successful execution of this protocol.

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Fig. 1: In vivo pretargeting based on the IEDDA reaction.
Fig. 2: Several ligation mechanisms have been leveraged to facilitate in vivo pretargeting.
Fig. 3
Fig. 4
Fig. 5
Fig. 6: In vivo pretargeted radioimmunotherapy data.
Fig. 7: Representative radio-iTLC chromatograms for radiosynthesis.
Fig. 8: Representative pretargeted PET images.

Data availability

The data described in ‘Anticipated results’ were derived from refs. 21 and 26, which are available in the public domain from the National Library of Medicine Database at https://pubmed.ncbi.nlm.nih.gov. Source data are provided with this paper.

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Acknowledgements

The authors thank the National Institutes of Health (B.M.Z.: R01CA240963, U01CA221046, R01CA204167 and R01244327), the Academy of Finland (OMK) and the Tow Foundation (GDLR) for their generous financial support.

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Correspondence to Brian M. Zeglis.

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Key references using this protocol

Membreno, R. et al. Mol. Pharm. 15, 1729–1734 (2018): https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.8b00093

Zeglis, B. M. et al. Mol. Pharm. 12, 3575–3587 (2015): https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5b00294

Adumeau, P. et al. Theranostics 6, 2267–2277 (2016): https://www.thno.org/v06p2267.htm

Houghton, J. L. et al. Mol. Cancer Ther. 16, 124–133 (2017): https://mct.aacrjournals.org/content/16/1/124

Keinänen, O. et al. Mol. Pharm. 16, 4416–4421 (2019): https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.9b00746

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Source Data Fig. 7

Organ activity concentration data for each mouse to go along with biodistribution data.

Source Data Fig. 8

Organ activity concentration data for each mouse to go along with biodistribution data.

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Sarrett, S.M., Keinänen, O., Dayts, E.J. et al. Inverse electron demand Diels–Alder click chemistry for pretargeted PET imaging and radioimmunotherapy. Nat Protoc 16, 3348–3381 (2021). https://doi.org/10.1038/s41596-021-00540-2

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