Radiosynthesis and Preclinical Evaluation of [99mTc]Tc-HYNIC-anti-TIGIT for Immuno-SPECT Imaging

T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is a newly identified immune checkpoint involved in tumor immune evasion; it represents a promising target for cancer immunotherapy. Noninvasive in vivo imaging of TIGIT expression may provide valuable insights into the tumor immune microenvironment and facilitate the evaluation of TIGIT-targeted therapies. In this study, tiragolumab (anti-TIGIT monoclonal antibody) was conjugated with HYNIC and radiolabeled with 99mTc using an EDDA/tricine coligand system under optimized mild conditions. Radiochemical purity and stability were assessed by ITLC and SEC-HPLC, while the chelator-to-antibody ratio (CAR) was determined using MALDI-TOF. In vitro binding was evaluated using saturation binding assays in TIGIT-expressing Jurkat cells, and the immunoreactivity was evaluated by Lindmo analysis. Biodistribution and small-animal SPECT imaging were conducted in a Jurkat tumor-bearing mouse model. The radioconjugate was obtained with a radiochemical yield of 64.2 ± 1.7%. It exhibited high radiochemical purity (>98%) along with good stability under physiological conditions. Saturation binding studies showed concentration-dependent and saturable binding to Jurkat cells, with a dissociation constant of Kd = 0.82 ± 0.11 nM and a maximum binding capacity of Bmax = 18.3 ± 1.6 fmol/106 cells, indicating high-affinity interaction. The immunoreactive fraction was 0.85 ± 0.03. In vivo biodistribution demonstrated progressive tumor accumulation, reaching 11.7 ± 1.4% ID/g at 4 h. SPECT imaging enabled clear visualization of tumors with improving contrast over time. [99mTc]Tc-HYNIC-anti-TIGIT was successfully prepared using a HYNIC-based radiolabeling strategy. It enables visualization of TIGIT expression in a preclinical model, supporting its potential as a radiopharmaceutical platform for immuno-SPECT imaging.