Whole-Body Dynamic PET Imaging of Half-Life Extended Variable Domains of Heavy Chain-Only Antibodies: Beyond Blood Pharmacokinetics

Nanobody molecules are single-domain antibodies derived from stable and fully functional heavy-chain-only (VHH) camelid antibodies. Due to their small size, approximately 15 kDa, their tissue distribution differs from that of conventional antibodies. This study investigates the whole-body pharmacokinetics (WBPK) and specific binding of half-life extended VHHs (HLE-VHHs) using dynamic whole-body immunoPET imaging in nonhuman primates. A HLE-VHH targeting the IL-6 receptor (CD126-VHH) and its nontargeting control (IRR-VHH) were evaluated in vivo after zirconium-89 radiolabeling. We compared microdoses (0.18 ± 0.11 mg/kg, <i>n</i> = 2) and/or pharmacological doses (coinjection, 3.19 ± 0.02 mg/kg, <i>n</i> = 2) of IRR-VHH, alongside microdoses of CD126-VHH (0.25 ± 0.14 mg/kg, <i>n</i> = 2). Initial dynamic whole-body PET scans (240 min) were followed by static PET scans on days 1, 7, and 14. We validated an image-derived input function against the arterial blood sampling method for kinetic modeling. [⁸⁹Zr]IRR-VHHs showed predominantly vascular distribution from day 0 to day 14, with comparable activity patterns and tissue distribution between microdose and pharmacological dose administrations. Early [⁸⁹Zr]CD126-VHH distribution mirrored the nonspecific IRR-VHH pattern, showing strong correlation (<i>R</i>² = 0.87, slope = 1.1) in uptakes of organs, normalized to blood (standard uptake value ratio, SUVR) values at day 0. Interestingly, by days 7 and 14, [⁸⁹Zr]CD126-VHH exhibited higher uptake in bone marrow, spleen, liver, and kidneys, suggesting specific binding. The distribution of [⁸⁹Zr]IRR-VHH in these organs was assumed to reflect the nonspecific uptake of VHHs and was used to estimate the specific binding of [⁸⁹Zr]CD126-VHH. The specific binding of [⁸⁹Zr]CD126-VHH was similarly estimated using either the arterial input function or the image-derived input function (R² = 0.997, <i>p</i> < 0.0001). In plasma, the shorter elimination half-life of [⁸⁹Zr]CD126-VHH (2.0-1.8 days) compared to microdose [⁸⁹Zr]IRR-VHH (5.1-5.0 days) or pharmaco-dose [⁸⁹Zr]IRR-VHH (4.9-4.8 days) suggested target-mediated drug disposition. This study demonstrates the potential of immunoPET for evaluating the target-mediated drug disposition and WBPK of VHH molecules in vivo, providing valuable insights into VHH-based therapeutic strategies.