Effect of a radiolabel biochemical nature on tumor-targeting properties of EpCAM-binding engineered scaffold protein DARPin Ec1

Influence of Residualizing Properties of the Radiolabel on Radionuclide Molecular Imaging of HER3 Using Affibody Molecules

“…Indirect radioiodination of (HE) 3 -Z HER3:08698 -DOTAGA using N-succinimidyl-para-(trimethylstannyl)benzoate was performed as described earlier for affibody molecules [49] and DARPins [48]. An aqueous solution of 0.1% acetic acid (60-90 µL) was added to radioiodine (40)(41)(42)(43)(44)(45). Then, N-succinimidyl-p-(trimethylstannyl)benzoate (6.5 nmoles, 2.5 µg, 2.5 µL of 1 mg/mL in 5% acetic acid in methanol) and chloramine-T (80 µg, 10 µL, 8 mg/mL in water) were added.…”

“…The goal of this study was to evaluate an alternative approach for improving tumor-to-liver ratios for anti-HER3 affibody molecules. Slow internalization after binding to receptors on cancer cells is a common feature of many engineered scaffold proteins (ESPs), such as affibody molecules [33][34][35], ADAPTs [42], and DARPins [43,44]. Due to the slow internalization, good retention of activity is more dependent on high affinity of the targeting protein and to a lesser extent on the residualizing properties of the radiolabel.…”

“…In contrast, internalization after uptake of scaffold proteins in excretory organs is typically rapid, and the use of a non-residualizing radioiodine label results in a rapid leakage of activity from these organs. This has been used to improve tumor-to-organ ratios for ESPs, e.g., tumor-to-liver [45] and tumor-to-kidney [46] ratios for affibody molecules, tumor-to-kidney ratio for ADAPTs [42], as well as tumor-to-kidney and tumor-to-liver ratios for DARPins [43,44]. It should be noted that this approach was applied for ESPs targeting HER2, which has a high level of receptor overexpression in vitro (e.g., 1.6 × 10 6 receptors/cell in SKOV-3 cells [47]) and in tumor models in vivo.…”

“…To obtain a non-residualizing label, the same affibody molecule was used for indirect radioiodination using [ 125 I]-N-succinimidyl-4-iodobenzoate. This labeling approach was selected because the radiometabolites of [ 125 I]-4-iodobenzoate label are rapidly excreted and do no contribute to background [44,48]. Iodine-125 was used in this study for radioiodination.…”

Investigation of a Pharmacological Approach for Reduction of Renal Uptake of Radiolabeled ADAPT Scaffold Protein

“…Clinical studies using two types of ESPs, affibody molecules and Albumin Binding Domain (ABD)-Derived Affinity Proteins (ADAPTs), confirmed their superior specificity and sensitivity for radionuclide molecular imaging of human epidermal growth factor receptor 2 (HER2) [ 8 , 9 , 10 ]. Other ESPs, such as cystine knot miniproteins and designed ankyrin repeat proteins (DARPins), demonstrated promising results in preclinical studies [ 11 , 12 , 13 , 14 , 15 , 16 ]. ADAPTs, novel small scaffold proteins (5 kDa) consisting of 46 amino acids, have been developed from an engineered albumin-binding domain (ABD) [ 17 ].…”