Preclinical Positron Emission Tomography Imaging of B7–H3 Expression Using Affibody Molecules Labeled with Gallium-68

Affibody molecules, nonimmunoglobulin scaffold proteins, have a high potential as probes for molecular imaging of different molecular targets. One of the molecular targets for radionuclide diagnosis and therapy is B7-H3 (known as CD276), which is overexpressed in various cancers, whereas its expression is low in most normal organs and tissues. The visualization of expression levels of B7-H3 has been performed using Affibody molecules labeled with Tc-99m. However, radionuclide molecular imaging using PET offers several advantages such as superior sensitivity, quantitation accuracy, and better spatial resolution compared to SPECT. In this study, we aimed to introduce a radiotracer for PET imaging of B7-H3. To design imaging agents for labeling with the generator-produced positron-emitting radionuclide ⁶⁸Ga, the macrocyclic triaza chelator (2-[4,7-bis-(carboxymethyl)-1,4,7-triazonan-1-yl]-acetic acid) (NOTA) was site-specifically coupled to the C-terminal cysteine of the anti-B7-H3 Affibody molecules. Four different variants of Affibody molecules, Z_B7-H3_₂, Z_B7-H3_₃, Z_B7-H3_₄, and Z_AC12 (as control), were produced, characterized, and successfully labeled with ⁶⁸Ga. ⁶⁸Ga-labeled conjugates bound specifically to B7-H3-expressing cells <i>in vitro</i> and <i>in vivo</i>. Biodistribution showed that [⁶⁸Ga]-Ga-Z_B7-H3_₂ had the highest tumor accumulation only 2 h after administration, which was 2.8-fold higher than that for the control Z_AC12. There was a tendency for higher tumor-to-organ ratios compared to the other variants, resulting in higher imaging contrast using [⁶⁸Ga]-Ga-Z_B7-H3_₂ for preclinical PET imaging of B7-H3-expressing tumors. Thus, [⁶⁸Ga]-Ga-Z_B7-H3_₂ could be a promising candidate for further development aimed at clinical PET in the future.