A Head-to-Head Comparison of Two Antibodies for HER2 Pretargeted PET Imaging via Bioorthogonal Click Chemistry

Background/Objectives: Antibody selection is critical for advancing pretargeted PET imaging toward clinical translation. This study directly compared two clinically approved anti-HER2 antibodies—trastuzumab and pertuzumab—within an identical pretargeting system, utilizing the specific chemical ligation between tetrazine and trans-cyclooctene (TCO) via the inverse electron-demand Diels–Alder (IEDDA) cycloaddition. Methods: In HER2-positive SKOV3 tumor-bearing mice, TCO-conjugated antibodies were administered 1–13 days prior to injection of a fluorine-18-labeled tetrazine probe ([18F]PEG12-Tz). Results: Both strategies enabled high-contrast imaging, overcoming the kinetic mismatch between slow-clearing antibodies and the short-lived 18F isotope. Distinct profiles were observed: pertuzumab-TCO yielded higher tumor uptake and a broader imaging window (3–13 days), whereas trastuzumab-TCO resulted in lower uptake and a narrower window (1–5 days). These differences are not affinity-driven, as both conjugates showed similar Kd values (~11–14 nM). Instead, pertuzumab-TCO’s superiority stems from a dual mechanism: (1) slower cellular internalization (65.1% vs. 94.0% at 60 min) preserves surface TCO for click reaction; and (2) markedly slower blood clearance (t1/2β = 285.9 h vs. 106.0 h) ensures sustained bioavailability and tumor extravasation. Conclusions: This head-to-head evaluation demonstrates that epitope-dictated internalization and clearance are decisive parameters for antibody selection, providing a mechanism-based framework for optimizing pretargeted imaging.