Evaluation of 68Ga Labeling Strategies and Thermostability of HER2-Targeted Nanobody NB46

The HER2-targeted nanobody NB46 was evaluated for labeling strategy flexibility and thermostability. Random and site-specific 68Ga labeling strategies were compared. Both conjugates exhibited high HER2 affinity, comparable to unmodified NB46. Cellular uptake, pharmacokinetics, micro-PET/CT imaging, and biodistribution in HER2-positive and HER2-negative tumor-bearing mice revealed no significant differences between the two strategies. NB46 retained HER2 affinity at temperatures up to 90 °C, demonstrating exceptional thermostability. Both conjugates achieved >95% radiochemical yields at all tested temperatures. Micro-PET/CT imaging showed sustained high tumor uptake with renal clearance as the primary elimination pathway. Blocking studies confirmed that NB46 and trastuzumab competitively bind to HER2. Elevated labeling temperatures did not compromise tumor targeting but resulted in a mild increase in renal uptake, notably for the random conjugate at 90 °C. These findings suggest that random labeling may represent a simpler, equally effective alternative for 68Ga-NB46 preparation, and confirm the excellent thermostability of this nanobody scaffold.