T-cell immunoglobulin and mucin domain-3 (TIM3) is an immune checkpoint that plays a negative regulatory role in the immune response. TIM3-targeted drugs inhibit this negative regulation, thereby modulating the level of immune response activation. In the previous investigation, several peptides targeting TIM3 were identified through screening from a phage peptide library. In this research, three peptides were selected to construct the radioactive molecular probes according to the characteristic that targeting TIM3 drugs would lead to the increase of interferon-γ (IFN-γ) secretion. Molecular docking was performed to assess the binding properties of the selected peptides with the TIM3 protein. To further enhance the targeting properties, one of the peptides with a higher-affinity peptide was structurally modified. Then, 68 Ga was used to construct the peptide probe 68 Ga-DOTApeptide by labeling the six peptides with 68 Ga riboprobes, and the binding affinity and specificity were assessed using TIM3 overexpressing cell line A549 TIM3 and the parental A549 cells. In addition, in Micro-PET/ CT imaging, transfected model mice were dynamically imaged for 30 min after injection of 3.7−7.4 MBq 68 Ga-DOTA-peptides via the tail vein. Meanwhile, the same dose of molecular probes was injected in the MC38 model (colorectal cancer in mice) and the CCRCC (clear cell renal cell carcinoma) xenografted model, followed by static scans at 15, 30, and 60 min postinjection. Finally, immunohistochemical (IHC) staining was performed to assess TIM3 expression in the dissected tumor tissues. The molecular docking results showed that the binding energy of P26 to TIM3 protein was −6.5 kcal/mol, which was lower than that of P24 to TIM3 protein, −3.6 kcal/mol, indicating that the affinity of P26 peptide to TIM3 protein was higher than that of P24 and P20 peptide. After structural modification of the P26 peptide, P26NH 2 , r-NH 2 , and P26X 2 were obtained, and the above peptides were successfully constructed into six targeting TIM3 peptide probes by 68 Ga labeling. Cellular uptake experiments demonstrated that 68 Ga-DOTA-P26, 68 Ga-DOTA-P26NH 2 , and 68 Ga-DOTA-r-NH 2 showed significantly higher uptake in A549 TIM3 cells than in A549 cells and could be blocked by the unlabeled peptide. Micro-PET imaging experiments showed that the uptake of each probe in the A549 TIM3 model tumor tissue was significantly higher than that in the A549 model tumor tissue, and a comparison of the tumor-tocardiac uptake ratios of each group showed that the 68 Ga-DOTA-P26 had a better tumor-to-cardiac uptake ratio in the A549 TIM3 model than several other molecular probes, and in the MC38 model, similar results were obtained, with the difference that the 68 Ga-DOTA-P26NH 2 had the highest tumor-to-cardiac uptake ratio in the CCRCC model. Finally, validation by IHC showed that A549 TIM3 , MC38, and CCRCC tumor tissues had varying degrees of TIM3 expression. Upon comparison of ex vivo and in vivo studies, one of them, the 68 Ga-DOTA-P26 probe, demonstrated significan...
