Preclinical Study of the 89Zr/177Lu-Labeled Engineered VHH-based Antibody for CLDN18.2-Targeted Radiotheranostics

Claudin 18.2 (CLDN18.2) is a tight junction protein aberrantly expressed in gastric and pancreatic cancer. In this study, we developed the novel nanobody 18.2−50H and VHH-Fc fusion antibody 18.2−50C radiolabeled with 89Zr/177Lu for CLDN18.2-targeted radiotheranostics. Zolbetuximab (IMAB362), 18.2−50H, and 18.2−50C were conjugated with DFO and labeled with 89Zr, respectively. Radiochemical purity and in vitro stability were determined by radio-TLC. CLDN18.2-targeting specificity was evaluated using HEK-293T and HEK-293T/CLDN18.2 cells. PET/CT imaging and biodistribution studies were performed in mouse models and cynomolgus monkeys. Subsequently, 177Lu-DOTA-18.2-50C was prepared and assessed for tumor targeting in gastric cancer PDX models. 89Zr-DFO-IMAB362, 89Zr-DFO-18.2-50H, and 89Zr-DFO-18.2-50C were prepared with satisfactory radiochemical yield, specific activity, and stability. Tumors were clearly visible as early as 1 h post-injection of 89Zr-DFO-18.2-50H, with an uptake value of 9.07 ± 2.36 %ID/g and a tumor-to-muscle ratio of 27.35 ± 3.72. Fc-fused 89Zr-DFO-18.2-50C exhibited improved retention and higher tumor uptake than 89Zr-DFO-18.2-50H and 89Zr-DFO-IMAB362, with % ID/g increasing continuously from 5.07 ± 0.29 at 1 h to 32.95 ± 1.57 at 168 h. Notably, the introduction of the Fc chain also markedly increased the radiation exposure in the liver and spleen. Consistently, PET imaging in cynomolgus showed that 89Zr-DFO-18.2-50C is mainly concentrated in the liver, but no apparent abnormality was observed. Furthermore, 177Lu-DOTA-18.2-50C presented remarkable tumor selectivity with prolonged retention (16.92 ± 1.94 % ID/g at 5 d post-injection) in PDX models. Nanobody 89Zr-DFO-18.2-50H demonstrated a superior tumor-to-background ratio, positioning it as a promising tracer for detecting CLDN18.2-overexpressing lesions. 89Zr/177Lu-labeled VHH-Fc antibody 18.2−50C exhibited sustained higher tumor uptake than commercially available IMAB362 and provides a novel potential candidate for CLDN18.2-targeted radionuclide therapy.