An Efficient Method for Labeling Single Domain Antibody Fragments with ¹⁸F Using Tetrazine-Trans-Cyclooctene Ligation and a Renal Brush Border Enzyme-Cleavable Linker

Single domain antibody fragments (sdAbs) labeled with ¹⁸F have shown promise for assessing the status of oncological targets such as the human epidermal growth factor receptor 2 (HER2) by positron emission tomography (PET). Earlier, we evaluated two residualizing prosthetic agents for ¹⁸F-labeling of anti-HER2 sdAbs; however, these methods resulted in poor labeling yields and high uptake of ¹⁸F activity in the kidneys. To potentially mitigate these limitations, we have now developed an ¹⁸F labeling method that utilizes the trans-cyclooctene (TCO)-tetrazine (Tz)-based inverse-electron demand Diels–Alder reaction (IEDDAR) in tandem with a renal brush border enzyme-cleavable glycine-lysine (GK) linker in the prosthetic moiety. The HER2-targeted sdAb 2Rs15d was derivatized with TCO-GK-PEG₄-NHS or TCO-PEG₄-NHS, which lacks the cleavable linker. As an additional control, the non HER2-specific sdAb R3B23 was derivatized with TCO-GK-PEG₄-NHS. The resultant sdAb conjugates were labeled with ¹⁸F by IEDDAR using [¹⁸F]­AlF-NOTA-PEG₄-methyltetrazine. As a positive control, the 2Rs15d sdAb was radioiodinated using the well-characterized residualizing prosthetic agent, N-succinimidyl 4-guanidinomethyl-3-[¹²⁵I]­iodobenzoate ([¹²⁵I]­SGMIB). Synthesis of [¹⁸F]­AlF-NOTA-Tz-TCO-GK-2Rs15d was achieved with an overall radiochemical yield (RCY) of 17.8 ± 1.5% (n = 5) in 90 min, a significant improvement over prior methods (3–4% in 2–3 h). In vitro assays indicated that [¹⁸F]­AlF-NOTA-Tz-TCO-GK-2Rs15d bound with high affinity and immunoreactivity to HER2. In normal mice, when normalized to coinjected [¹²⁵I]­SGMIB-2Rs15d, the kidney uptake of [¹⁸F]­AlF-NOTA-Tz-TCO-GK-2Rs15d was 15- and 28-fold lower (P < 0.001) than that seen for the noncleavable control ([¹⁸F]­AlF-NOTA-Tz-TCO-2Rs15d) at 1 and 3 h, respectively. Uptake of [¹⁸F]­AlF-NOTA-Tz-TCO-GK-2Rs15d in HER2-expressing SKOV-3 ovarian carcinoma xenografts implanted in athymic mice was about 80% of that seen for coinjected [¹²⁵I]­SGMIB-2Rs15d. On the other hand, kidney uptake was 5–6-fold lower, and as a result, tumor-to-kidney ratios were 4-fold higher for [¹⁸F]­AlF-NOTA-Tz-TCO-GK-2Rs15d than those for [¹²⁵I]­SGMIB-2Rs15d. SKOV-3 xenografts were clearly delineated even at 1 h after administration of [¹⁸F]­AlF-NOTA-Tz-TCO-GK-2Rs15d by Micro-PET/CT imaging with even higher contrast observed thereafter. In conclusion, this strategy warrants further evaluation for labeling small proteins such as sdAbs because it offers the benefits of good radiochemical yields and enhanced tumor-to-normal tissue ratios, particularly in the kidney.