Radionuclide Tumor Targeting Using ADAPT Scaffold Proteins: Aspects of Label Positioning and Residualizing Properties of the Label

Abstract

Visualization of cancer-associated alterations of molecular phenotype using radionuclide imaging is a noninvasive approach to stratifying patients for targeted therapies. The engineered albumin-binding domain-derived affinity protein (ADAPT) is a promising tracer for radionuclide molecular imaging because of its small size (6.5 kDa), which satisfies the precondition for efficient tumor penetration and rapid clearance. Previous studies demonstrated that the human epidermal growth factor receptor type 2 (HER2)-targeting ADAPT6 labeled with radiometals at the N terminus is able to image HER2 expression in xenografts a few hours after injection. The aim of this study was to evaluate whether the use of a nonresidualizing label or placement of the labels at the C terminus would further improve the targeting properties of ADAPT6. Two constructs, Cys-ADAPT6 and Cys-ADAPT6, having the (HE)DANS sequence at the N terminus were produced and site-specifically labeled using In-DOTA orI-iodo-((4-hydroxyphenyl)ethyl) maleimide (HPEM). The conjugates were compared in vitro and in vivo. HER2-targeting properties and biodistribution were evaluated in BALB/C mice bearing ovarian carcinoma cell (SKOV-3) xenografts. Specific HER2 binding and high affinity were preserved after labeling. Both Cys-ADAPT6 and Cys-ADAPT6 were internalized slowly by HER2-expressing cancer cells. Depending on the label position, uptake at 4 h after injection varied from 10% to 22% of the injected dose per gram of tumor tissue. Regardless of terminus position, the I-HPEM label provided more than 140-fold lower renal uptake than theIn-DOTA label at 4 after injection. The tumor-to-organ ratios were, in contrast, higher for both of the In-DOTA-labeled ADAPT variants in other organs. Tumor-to-blood ratios forIn-labeled Cys-ADAPT6 and Cys-ADAPT6 did not differ significantly (250-280), but In-DOTA-Cys-ADAPT6 provided significantly higher tumor-to-lung, tumor-to-liver, tumor-to-spleen, and tumor-to-muscle ratios. Radioiodinated variants had similar tumor-to-organ ratios, but I-HPEM-Cys-ADAPT6 had significantly higher tumor uptake and a higher tumor-to-kidney ratio. Residualizing properties of the label strongly influence the targeting properties of ADAPT6. The position of the radiolabel influences targeting as well, although to a lesser extent. Placement of a label at the C terminus yields the best biodistribution features for both radiometal and radiohalogen labels. Low renal retention of the radioiodine label creates a precondition for radionuclide therapy usingI-labeled HPEM-Cys-ADAPT6.

Same-Day Imaging Using Small Proteins: Clinical Experience and Translational Prospects in Oncology

“…Also, several emerging sdAb-and scaffold protein-based probes that target other clinically relevant targets have been developed preclinically, and their clinical translation may impact future patient care. (35,36,38,40 …”

“…Use of the nonresidualizing 125 I-HPEM label provided a tumor-to-kidney ratio of 13 6 3, opening a way for radionuclide therapy (Fig. 5A) (40).…”

On the prevention of kidney uptake of radiolabeled DARPins

“…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].…”