Optimizing antibody PET imaging: a comparative preclinical analysis of nanobody and minibody-like PET tracers

Positron emission tomography remains the most important medical imaging technique for noninvasive imaging. Advances in radiotracer design, particularly those based on antibody(-formats), have broadened diagnostic applications. While minibodies and nanobodies stand out among antibody-type radiotracers due to their unique characteristics and pharmacokinetics, a direct comparison between these formats remains unexplored. In this study, we generated a so-called minabody, a minibody-like protein consisting of a nanobody fused to a CH3 domain, and compared it with its nanobody counterpart in vitro and in vivo upon labeling with various PET radionuclides.

A previously developed human TIGIT-targeting nanobody was reformatted to a minabody format to allow head-to-head comparison of both formats. The minabody and nanobody formats were compared in vitro for binding, specificity and stability. The minabody and/or nanobody formats were labeled with copper-64, gallium-68 or fluor-18 for non-invasive imaging of human TIGIT-expressing tumors by PET/CT over time.

The minabody format showed higher binding affinities compared to the nanobody format. Despite lower binding affinity, the nanobody format outperformed the minabody format as PET imaging tracer showing higher specificity and allowing imaging of human TIGIT expression from 1 to 48 h post-injection upon labeling with copper-64. In addition, copper-64 showed superior tumor uptake and contrast for nanobody imaging compared to gallium-68 and fluor-18.

This study describes the first head-to-head comparison between the minibody and nanobody format for PET imaging. Our results underscore the importance of considering both targeting vector format and the radionuclide to achieve accurate and high-quality PET imaging.