Targeting of VISTA with Cyclic Peptide-Based PET Tracers for Immune Checkpoint Imaging

Given the distinct roles of VISTA in immune regulation, a better visualization of VISTA expression could clarify the tumor immune status and predict the responses to immunotherapy. However, only a few targeted probes are in development and insufficient to fulfill clinical demands. In this study, based on cyclic peptide antagonist AP1049, three novel radiotracers, [ 68 Ga]Ga-AP1049, [ 68 Ga]Ga-AHX-AP1049, and [ 68 Ga]Ga-Pip-AP1049, were designed and developed for targeting and imaging VISTA in cancers. Molecular dynamics simulation revealed the better binding of NOTA-Pip-AP1049 for VISTA protein than that of NOTA-AP1049 and NOTA-AHX-AP1049. Protein binding assays demonstrated their high specificity and affinity with IC 50 values of 58.10, 23.34, and 17.56 nM, respectively. The B16-F10 tumor uptake of three tracers was significant and decreased over time within 120 min, with tumor uptake values of 1.02 ± 0.21, 1.56 ± 0.21, and 1.89 ± 0.29 %ID/cc and tumor/muscle ratios of 2.36 ± 0.36, 3.10 ± 0.51, and 4.06 ± 0.71 at 30 min post-injection, respectively. Their in vivo specificity were verified by blocking studies. Furthermore, [ 68 Ga]Ga-Pip-AP1049 PET could detect varying VISTA expression in B16-F10, 4T1, MC38, and CT26 xenograft models with tumor uptake values of 2.71 ± 0.43, 2.32 ± 0.44, 1.17 ± 0.30, and 0.85 ± 0.17 %ID/cc, which were in consistent with percentages of VISTA + cells ( r = 0.8712, P < 0.0001). Biodistribution data suggested their renal excretion and favorable pharmacokinetics. In conclusion, [ 68 Ga]Ga-Pip-AP1049 is a promising tracer for PET imaging of VISTA expression in tumor microenvironment, indicating its potential as a companion diagnostic of immunotherapy.