Efficient 68Ga Labeling of a B7-H3-Targeting Affibody Molecule via Acyclic Tris(hydroxypyridinone) Chelator: Effects on Biodistribution in a Preclinical Model.

B7-H3 (CD276), an immune checkpoint protein, is overexpressed in malignant tumors, while its expression in normal tissues is low, and several B7-H3-targeting therapies are under clinical evaluation. Radionuclide molecular imaging offers a non-invasive method for determining B7-H3 expression levels and may aid in improved patient selection. The feasibility of the use of Affibody molecules for the visualization of B7-H3 was demonstrated earlier. The selection of an approach for routine labeling providing high radiochemical yields and reproducibility is, however, critical for successful clinical translation. The optimal combination of a targeting protein, chelator/linker, and radionuclide should provide high-contrast visualization. In this study, we evaluated an acyclic chelator, tris(3,4-hydroxypyridinone) (THP), for labeling of the Affibody molecule ZB7-H3_2 with 68Ga and compared its impact on radiolabeling efficiency and targeting properties with the impact of the cyclic chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). Maleimide derivatives of THP and NOTA were site-specifically coupled to the C-terminal cysteine of ZB7-H3_2. THP-ZB7-H3_2 was successfully labeled with 68Ga within 5 min of incubation at room temperature, achieving a 100% radiochemical yield. NOTA-ZB7-H3 required heating at 60 °C for 10 min, and the radiochemical yield was lower. Both radioconjugates exhibited specific binding to B7-H3-expressing cells with similar binding strength, and both tracers demonstrated similar tumor uptake (p > 0.05) in a murine model. The biodistribution was similar, although [68Ga]Ga-NOTA-ZB7-H3_2 provided slightly but significantly higher tumor-to-liver and tumor-to-spleen ratios. Nonetheless, the advantages of THP include rapid and mild radiolabeling with high efficiency, eliminating the need for heating and a post-purification step, which suggests a potential for streamlined clinical translation of ZB7-H3_2.