Novel Radiofluorinated Nanobody PET Tracer for Preclinical Studies of TIM3 Expression

T-cell immunoglobulin and mucin domain-3 (TIM3) is an inhibitory checkpoint glycoprotein expressed on immune cells, particularly tumor-infiltrating lymphocytes (TILs), and plays a critical role in suppressing antitumor immune responses. While dual blockade of TIM3 and programmed cell death protein 1 (PD1) has shown promising results in enhancing immune responses in advanced cancers, the lack of reliable, noninvasive methods for detecting TIM3 expression in tumors remains a major challenge. To address this, we developed and characterized a novel positron emission tomography (PET) tracer, [ 18 F]AlF-RESCA-HVCR2N2, based on a TIM3-specific nanobody labeled via [ 18 F]AlF radiochemistry. The tracer demonstrated high specificity and affinity for TIM3 ( K D = 4.644 nM) in vitro, enabling the clear visualization of TIM3-expressing lesions in tumor models through PET imaging. Dynamic imaging and kinetic analyses revealed favorable pharmacokinetics, and a preliminary correlation was observed between PET signal intensity and histologically assessed TIM3 expression in tumor tissues. These results highlight the potential of [ 18 F]AlF-RESCA-HVCR2N2 as a sensitive and noninvasive tool for quantifying TIM3 expression in vivo, with significant implications for patient stratification, treatment monitoring, and the development of TIM3-targeted immunotherapies.