68Ga-NOTA-m-SNA006: A Next-Generation CD8-Targeting Nanobody Probe to Enhance Renal and Hepatic Clearance in Noninvasive ImmunoPET Imaging

The ⁶⁸Ga-labeled nanobody SNA006 marks a significant advancement in noninvasive immunoPET imaging of CD8⁺ T cells, facilitating real-time tracking of cellular immune responses in cancer, yet its pharmacokinetic properties remain suboptimal. This study aimed to develop a next-generation CD8-targeting immunoPET nanobody probe by incorporating a PEGylated brush border membrane enzyme-cleavable linker to improve pharmacokinetics and to evaluate its characterization in CD8-positive intrapulmonary tumors. A precursor based on SNA006, containing a PEGylated brush border membrane enzyme-cleavable linker, was designed, synthesized, and radiolabeled with gallium-68 to yield ⁶⁸Ga-NOTA-m-SNA006. The probe was subsequently assessed both in vitro and in vivo. The probe exhibited high radiochemical yield, purity, and favorable stability, and demonstrated binding to the CD8 protein with high affinity. PET/CT imaging and biodistribution studies revealed that ⁶⁸Ga-NOTA-m-SNA006 exhibited favorable pharmacokinetic properties, including rapid clearance from the kidneys, reduced liver uptake, and sustained retention in the tumor, compared with ⁶⁸Ga-NODAGA-SNA006. ⁶⁸Ga-NOTA-m-SNA006 exhibited high uptake in lung lesions during in vivo PET imaging, reflecting CD8 expression in an intrapulmonary tumor model. In summary, we present a novel ⁶⁸Ga-labeled SNA006 radiotracer with an optimized linker moiety, ⁶⁸Ga-NOTA-m-SNA006, which effectively decreases renal and hepatic uptake while maintaining tumor uptake, thereby enhancing the tumor-to-background ratio. This approach represents a critical advancement in addressing the persistent challenge of radioactivity in the kidneys and liver associated with nanobody-based radiopharmaceuticals.