Development and Preclinical Evaluation of a Novel 68Ga/177Lu-Labeled Nanobody for Radiotheranostics of HER2-Positive Tumors

Human epidermal growth factor receptor 2 (HER2) is overexpressed in various malignancies, making it an attractive target for radiotheranostics. Nanobodies offer an ideal platform for radiopharmaceutical development. Here, we reported a novel HER2-specific nanobody (NB46) engineered with a C-terminal glycine–serine–cysteine (GSC) tripeptide for site-specific NOTA conjugation and radiolabeling with 68 Ga and 177 Lu. [ 68 Ga]Ga/[ 177 Lu]Lu-NOTA-NB46 exhibited high HER2 affinity and specificity in vitro. In HER2-positive tumor-bearing mice, the radiotheranostic agents showed rapid blood clearance, favorable biodistribution, high tumor accumulation, and low nontarget uptake. [ 177 Lu]Lu-NOTA-NB46 demonstrated prolonged tumor retention. Single-dose therapy with [ 177 Lu]Lu-NOTA-NB46 significantly inhibited tumor growth and extended survival in a dose-dependent manner with good tolerability. The kidneys were the primary dose-limiting organ. [ 68 Ga]Ga/[ 177 Lu]Lu-NOTA-NB46 represents a promising radiotheranostic pair for HER2-positive tumors, supporting its further translation into clinical investigation.