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

Abstract

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-H 6 and G3-H 6 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-H 6 was demonstrated. A combination of smaller size and higher affinity resulted in a higher tumor uptake of G3-H 6 in comparison to 9_29-H 6 . Technetium-99m labeled G3-H 6 demonstrated a better biodistribution profile than 9_29-H 6 , with several-fold lower uptake in liver. Radioiodinated G3-H 6 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-H 6 with high clinical potential for imaging of HER2.

On the prevention of kidney uptake of radiolabeled DARPins

“…Furosemid (Takeda Pharma AB, Sweden), mannitol (Fresenius Kabi AB, Sweden), and Gelofusine (B. Braun Melsungen AG, Germany) were purchased as solutions for injections. The anti-human epidermal growth factor receptor 2 (anti-HER2) DARPins G3 and 9_29 were produced in E. coli strain BL21(DE3) as described previously [5,6]. DARPin G3 consists of four ankyrin repeats (14 kDa), and DARPin 9_29 consists of five ankyrin repeats (18 kDa).…”

“…A new class of targeting molecules, engineered scaffold proteins (ESPs), is promising agents for tumor-targeted delivery of radionuclides [1]. Several types of ESPs, such as affibody molecules [2], ADAPTs [3], and DARPins [4][5][6][7][8][9] demonstrated efficient tumor targeting and high contrast of radionuclide imaging in preclinical studies. DARPins are built of 4-6 repeat modules of 33 amino acids and have a molecular weight of 14-18 kDa [10].…”

“…In the case of rapid internalization in tumors, residualizing labels are preferable, as they provide longer retention of activity and higher absorbed dose to the tumor than non-residualizing labels [11]. However, internalization of ESP such as affibody molecules, ADAPTs, and DARPins is relatively slow, and typically less than 20% are internalized during 24 h [2,3,6]. In this case, good tumor retention is mainly dependent on high affinity to a molecular target, and residualizing properties of a label are less critical for good retention of activity in tumors.…”

“…This puts a limit on the amount of activity administered to a patient. In the case of non-residualizing labels for ESP, the renal activity decreases rapidly due to leakage of radiocatabolites [2,3,6,9].…”

“…In order to deliver the required dose to tumors without exceeding the limiting dose to the kidneys (23 Gy), several strategies have been developed. The use of labeling approaches that provide nonresidualizing radiocatabolites, such as radioiodination [5,6] or the use of some peptide-based chelators for rhenium radioisotopes and technetium-99m [12][13][14][15][16][17], was shown to decrease the retention of activity in the kidneys by several folds and to improve tumor-to-kidney ratios. Another effective strategy is the fusion of an ESP to an albumin-binding domain (ABD).…”