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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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.
The authors declare no competing interests.
Peer review information Nature Production thanks Jennifer Murphy 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
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
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Keinänen, O. et al. Mol. Pharm. 16, 4416–4421 (2019): https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.9b00746
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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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