Engineering nanobodies for next-generation molecular imaging

In recent years, nanobodies have emerged as ideal imaging agents for molecular imaging. Molecular nanobody imaging combines the specificity of nanobodies with the sensitivity of state-of-the-art molecular imaging modalities, such as positron emission tomography (PET). Given that modifications of nanobodies alter their pharmacokinetics (PK), the engineering strategies that combine nanobodies with radionuclides deter-mine the effectiveness, reliability, and safety of the molecular imaging probes. In this review, we introduce conjugation strategies that have been applied to engineer nanobodies, including random conjugation, Tc-99m tricarbonyl chemistry, sortase A-mediated site-specific conjugation, maleimide-cysteine chemistry, and click chemistries. We also summarize the latest advances in nanobody molecular imaging tracers, emphasizing their preclinical and clinical use. In addition, we elaborate on nanobody-based near-infrared fluorescence (NIRF) imaging and image-guided surgery.