Immuno-PET Imaging of a 68Ga-Labeled Single-Domain Antibody for Detecting Tumor TIGIT Expression

Preclinical studies have shown that the expression of T cell immunoglobulin and the immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) in the tumor microenvironment is associated with the efficacy of anti-TIGIT-based immunotherapy. This study aimed to develop a TIGIT single-domain antibody (sdAb)-based positron emission tomography (PET) radiotracer, [⁶⁸Ga]Ga-NOTA-NABT3A1, and evaluate its characteristics. The NABT3A1 was modified with a NOTA derivative and radiolabeled with ⁶⁸Ga. In vitro stability of [⁶⁸Ga]Ga-NOTA-NABT3A1 was assessed in phosphate-buffered saline (PBS) and fetal bovine serum (FBS), along with its specificity for TIGIT stably transfected A375 Human melanoma cells (A375-TIGIT) was performed. In vivo imaging was conducted on A375-TIGIT tumor-bearing nude mice at different time points after the injection of [⁶⁸Ga]Ga-NOTA-NABT3A1. The synthesized [⁶⁸Ga]Ga-NOTA-NABT3A1 achieved a radiochemical yield of 70.56 ± 1.14% and purity levels of 95.80 ± 0.58% in PBS and 96.79 ± 1.69% in FBS at 2 h. Immuno-PET imaging revealed specific accumulation of [⁶⁸Ga]Ga-NOTA-NABT3A1 in A375-TIGIT tumor-bearing nude mice, with a maximum uptake of 3.86 ± 0.29% injected dose/g at 0.5 h. Biodistribution and immunohistochemical analyses confirmed the in vivo imaging results. In conclusion, we successfully synthesized an NABT3A1-derived PET radiotracer with the potential to noninvasively assess TIGIT expression in tumors.