@article {van Duijnhoven1422, author = {Sander M.J. van Duijnhoven and Raffaella Rossin and Sandra M. van den Bosch and Michael P. Wheatcroft and Peter J. Hudson and Marc S. Robillard}, title = {Diabody Pretargeting with Click Chemistry In Vivo}, volume = {56}, number = {9}, pages = {1422--1428}, year = {2015}, doi = {10.2967/jnumed.115.159145}, publisher = {Society of Nuclear Medicine}, abstract = {Radioimmunotherapy and nuclear imaging (immuno-PET/SPECT) of cancer with radiometal-labeled antibody fragments or peptides is hampered by low tumor-to-kidney ratios because of high renal radiometal retention. Therefore, we developed and evaluated a pretargeting strategy using click chemistry in vivo to reduce kidney uptake and avoid unwanted radiation toxicity. We focused on the bioorthogonal reaction between a trans-cyclooctene (TCO){\textendash}functionalized TAG72 targeting diabody, AVP04-07, and a low-molecular-weight radiolabeled tetrazine probe that was previously shown to have low kidney retention and relatively fast renal clearance. Methods: AVP04-07 diabodies were functionalized with TCO tags, and in vitro immunoreactivity toward bovine submaxillary mucin and tetrazine reactivity were assessed. Next, pretargeting biodistribution studies were performed in LS174T tumor{\textendash}bearing mice with AVP04-07{\textendash}TCO(n) (where n indicates the number of TCO groups per diabody) and radiolabeled tetrazine to optimize the TCO modification grade (0, 1.8, or 4.7 TCO groups per diabody) and the 177Lu-tetrazine dose (0.1, 1.0, or 10 Eq with respect to the diabody). Radiolabeled tetrazine was injected at 47 h after diabody injection, and mice were euthanized 3 h later. A pretargeting SPECT/CT study with 111In-tetrazine was performed with the optimized conditions. Results: Immunoreactivity for native AVP04-07 was similar to that for TCO-functionalized AVP04-07, and the latter reacted efficiently with radiolabeled tetrazine in vitro. The combination of the pretargeting component AVP04-07 functionalized with 4.7 TCO groups and 1 Eq of 177Lu-tetrazine with respect to the diabody showed the most promising biodistribution. Specifically, high 177Lu-tetrazine tumor uptake (6.9 percentage injected dose/g) was observed with low renal retention, yielding a tumor-to-kidney ratio of 5.7. SPECT/CT imaging confirmed the predominant accumulation of radiolabeled tetrazine in the tumor and low nontumor retention. Conclusion: Pretargeting provides an alternative radioimmunotherapy and nuclear imaging strategy by overcoming the high renal retention of low-molecular-weight radiometal tumor-homing agents through the separate administration of a tumor-homing agent and a radioactive probe with fast clearance.}, issn = {0161-5505}, URL = {https://jnm.snmjournals.org/content/56/9/1422}, eprint = {https://jnm.snmjournals.org/content/56/9/1422.full.pdf}, journal = {Journal of Nuclear Medicine} }