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, ZB7–H3_2, ZB7–H3_3, ZB7–H3_4, and ZAC12 (as control), were produced, characterized, and successfully labeled with 68Ga. 68Ga-labeled conjugates bound specifically to B7–H3-expressing cells in vitro and in vivo. Biodistribution showed that [68Ga]Ga-ZB7–H3_2 had the highest tumor accumulation only 2 h after administration, which was 2.8-fold higher than that for the control ZAC12. There was a tendency for higher tumor-to-organ ratios compared to the other variants, resulting in higher imaging contrast using [68Ga]Ga-ZB7–H3_2 for preclinical PET imaging of B7–H3-expressing tumors. Thus, [68Ga]Ga-ZB7–H3_2 could be a promising candidate for further development aimed at clinical PET in the future.