CC49 diabody pretargeting using click chemistry in vivo

Abstract

Radioimmunotherapy and nuclear imaging (Immuno-PET/SPECT) of cancer using radiometal-labeled antibody fragments or peptides is hampered by low tumor-to-kidney ratios due to 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 tumor targeting diabody, AVP04-07, and a low-molecular weight radiolabeled tetrazine probe, which previously showed low kidney retention and relative fast renal clearance. Methods: AVP04-07 diabodies were functionalized with TCO tags and in vitro immunoreactivity towards bovine submaxillary mucin and tetrazine reactivity was assessed. Next, pretargeting biodistribution studies were performed in LS174T-tumor bearing mice with AVP04-07-TCO(n) and radiolabeled tetrazine to optimize the TCO modification grade (0 vs. 1.8 vs. 4.7 TCOs/diabody) and ¹⁷⁷Lu-tetrazine dose (0.1 vs. 1.0 vs. 10 equivalents with respect to diabody). Radiolabeled tetrazine was injected 47h post-diabody injection and mice were euthanized 3h later. A pretargeting SPECT/CT study with ¹¹¹In-tetrazine was carried out employing the optimized conditions. Results: Immunoreactivity for native and TCO-functionalized AVP04-07 was similar and the latter reacted efficiently with radiolabeled tetrazine in vitro. The combination of the pretargeting components AVP04-07 functionalized with 4.7 TCOs and 1 equivalent of ¹⁷⁷Lu-tetrazine with respect to diabody showed the most promising biodistribution. Specifically, high ¹⁷⁷Lu-tetrazine tumor uptake (6.9 %ID/g) was observed with low renal retention yielding a tumor-to-kidney ratio of 5.7. SPECT/CT imaging confirmed predominant accumulation of radiolabeled tetrazine in tumor with low non-tumor retention. Conclusion: Pretargeting provides an alternative radioimmunotherapy strategy by overcoming the high renal retention of low molecular weight radiometal tumor-homing agents through the separate administration of the tumor-homing agent and a fast clearing radioactive probe.