Targeting T-Cell Immunoglobulin and Mucin Domain 3 with a <scp>D</scp> -Configured Peptide Radiotracer for Tumor Positron Emission Tomography Imaging

T-cell immunoglobulin and mucin domain 3 (TIM-3), a critical immunosuppressive checkpoint receptor, regulates antitumor immunity within the tumor microenvironment (TME). Noninvasive quantification of TIM-3 expression could be helpful for guiding immunotherapy and monitoring treatment response. We developed ⁶⁸Ga-DOTA-D-P24, a novel D-configured peptide radiotracer designed for enhanced protease resistance and optimized TIM-3 targeting. Radiolabeling yielded high radiochemical purity (RCP) (>98%) and excellent <i>in vitro</i>/<i>in vivo</i> stability. Positron emission tomography (PET)/CT imaging across six tumor models demonstrated that ⁶⁸Ga-DOTA-D-P24 showed high specific uptake in MGC-803 gastric carcinoma. Comparative PET studies showed that the D-configured tracer exhibited 1.6-fold higher tumor uptake than that of ⁶⁸Ga-DOTA-L-P24. Furthermore, ⁶⁸Ga-DOTA-D-P24 successfully visualized the interleukin-15 (IL-15)-triggered elevation of TIM-3 expression in tumors, demonstrating its potential as a noninvasive tool for assessing target engagement and treatment response in TIM-3-associated malignancies.