Diabody Pretargeting with Click Chemistry In Vivo

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)-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-bearing mice with AVP04-07-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 (177)Lu-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 (111)In-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 (177)Lu-tetrazine with respect to the diabody showed the most promising biodistribution. Specifically, high (177)Lu-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.