Comparative Evaluation of Two DARPin Variants: Effect of Affinity, Size, and Label on Tumor Targeting Properties

Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins that can be selected for binding to desirable molecular targets. High affinity and small size of DARPins render them promising probes for radionuclide molecular imaging. However, detailed knowledge on many factors influencing their imaging properties is still lacking. We have evaluated two human epidermal growth factor 2 (HER2)-specific DARPins with different size and binding properties. DARPins 9_29-H₆ and G3-H₆ were radiolabeled with iodine-125 and tricarbonyl technetium-99m and evaluated in vitro. A side-by-side comparison of biodistribution and tumor targeting was performed. HER2-specific tumor accumulation of G3-H₆ was demonstrated. A combination of smaller size and higher affinity resulted in a higher tumor uptake of G3-H₆ in comparison to 9_29-H₆. Technetium-99m labeled G3-H₆ demonstrated a better biodistribution profile than 9_29-H₆, with several-fold lower uptake in liver. Radioiodinated G3-H₆ showed the best tumor-to-organ ratios. The combined effect of affinity, molecular weight, scaffold composition, and nonresidualizing properties of iodine label provided radioiodinated G3-H₆ with high clinical potential for imaging of HER2.