Bimodal nanobody agents for cancer imaging and potential intraoperative guidance: a systematic review

Bimodal imaging tracers that combine nuclear and optical modalities are gaining increasing relevance for in vivo applications in oncology, particularly for surgical guidance, where both real-time visualization and preoperative deep-tissue localization are crucial. Nanobodies are heavy-chain antibody fragments that offer unique advantages in this context, such as high specificity and rapid clearance, which allows for precise tumor localization and real-time surgical navigation. In this systematic review, we evaluate research studies reporting nanobody-based tracers for dual-modality imaging and analyze their design strategies, preclinical imaging performance, and translational progress. The analyses revealed that molecular targets commonly overexpressed in cancer cells, such as HER2, EGFR, and CEA have been the primary focus in the design of these tracers, together with widely used fluorophores like Cy5 and IRDye800CW combined with radionuclides such as gallium-68, technetium-99m, and copper-64. The preclinical performance of the reported tracers was highly promising, both in absolute tumor uptake and ability to achieve high-contrast images rapidly, as highlighted by a CD38-targeting tracer that produced a ~ 96-fold tumor-to-muscle ratio within hours of injection. While achieving stable and site-specific dual labeling remains a technical challenge, the combination of high target specificity and rapid background clearance makes nanobody-based systems particularly well-suited for generating high-contrast images on the same day of administration. This positions nanobodies as a versatile platform to develop tracers that enhance real-time image-guided surgery in oncology and ultimately improve patient outcomes.