Applications of Nanobodies in Biological Imaging

Background: Nanobodies (Nbs), derived from Camelidae heavy-chain antibodies, are single-domain fragments (15 kDa) with high antigen-binding specificity, enhanced tissue penetration, and low immunogenicity. These attributes address limitations of conventional antibodies, positioning Nbs as pivotal tools for targeted molecular imaging in diagnostics and therapeutics. Methods: Nbs are screened through phage/mRNA display or single B-cell sequencing, expressed in prokaryotic or yeast systems, and humanized via CDR grafting. Functional probes are engineered by conjugating Nbs with radionuclides (⁶⁸Ga, ^99mTc) or fluorophores (IRDye 800CW) for compatibility with PET, SPECT, NIRF, and ultrasound modalities. Results: Clinical trials validated Nb efficacy: ⁶⁸Ga-HER2-Nb PET/CT achieved tumor-specific uptake in HER2+ cancers (NCT04467515), while ^99mTc-PD-L1-Nb enabled quantitative SPECT-guided immunotherapy in NSCLC. NIRF-Nb conjugates (e.g., 11A4-800CW) enhanced intraoperative tumor delineation in murine models. Dual-targeted ultrasound microbubbles demonstrated multi-biomarker imaging via acoustic pressure modulation. Conclusion: Nbs advance biological imaging through superior resolution and rapid pharmacokinetics. Challenges persist in optimizing probe stability, minimizing immunogenicity, and scaling production. Future priorities include integrating multi-modal platforms, expanding applications to neurodegenerative disorders, and refining personalized diagnostic paradigms, underscoring their transformative potential in precision medicine.