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 determine 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, ^99mTc 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.