Optimization of Chelator Conjugation to PD-1/VEGF Bispecific Antibody for Zr-ImmunoPET Imaging.

PD-1/PD-L1 and VEGF pathways jointly mediate T-cell dysfunction and immune suppression, limiting the efficacy of immune checkpoint inhibitors. We developed an Zr-labeled bispecific immuno-PET probe, Zr-JS207, for noninvasive imaging of PD-1 and VEGF in tumors. Zr-JS207 was prepared via -isothiocyanatobenzyl-desferrioxamine (-NCS-Bz-DFO) conjugation with high radiochemical purity (>99%) and excellent in vitro stability (>95% at 240 h). It showed high affinity for PD-1 ( = 6.92 nM) and VEGF-A ( = 82.48 nM), with an elimination half-life of 35.14 h. Micro-PET/CT in humanized mice revealed specific tumor uptake blockable by cold JS207, and stronger tumor retention than monospecific probe. NIRF imaging and IHC validated these findings. Zr-JS207 enables dual-targeted imaging with favorable pharmacokinetics, holding great potential for patient stratification and therapeutic monitoring in bispecific antibody immunotherapy.