⁶⁴Cu-Labeled Repebody Molecules for Imaging of Epidermal Growth Factor Receptor–Expressing Tumors

Abstract

The epidermal growth factor receptor (EGFR) is a member of the erbB family of receptors and is overexpressed in many tumor types. A repebody is a newly designed nonantibody protein scaffold for tumor targeting that contains leucine-rich repeat modules. In this study, 3 Cu-labeled anti-EGFR repebodies with different chelators were synthesized, and their biologic characteristics were assessed in cultured cells and tumor-bearing mice. Repebodies were synthesized with the chelators 2-(-isothiocyanatobenzyl)-1,4,7-triazacyclononane-triacetic acid trihydrochloride ([-SCN-Bn]-NOTA), 2,2',2″-(10-(2-(2,5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA--hydroxysuccinimide ester), or 1-(-isothiocyanatobenzyl)diethylenetriamine pentaacetic acid trihydrochloride ([-SCN-Bn]-DTPA) in 1.0 M NaHCO buffer (pH 9.2) for 24 h. Purified NOTA-, DOTA-, and DTPA-conjugated repebody were radiolabeled with Cu in 0.1 M NHOAc buffer (pH 5.5). To compare the EGFR-binding affinities of the repebodies, cellular uptake studies were performed with the human non-small cell lung cancer cell line H1650 (high expression of EGFR) and the human colon adenocarcinoma cell line SW620 (low expression of EGFR). Biodistribution and small-animal PET imaging studies were performed using H1650 tumor-bearing mice. Radiochemical yields of theCu-labeled repebodies were approximately 70%-80%. Cellular uptake of the NOTA-, DOTA-, and DTPA-repebodies was over 4-fold higher in H1650 cells than in SW620 cells at 1 h. The 3 repebodies had accumulated specifically in H1650 tumor-bearing nude mice by 1 h after intravenous injection and were retained for over 24 h, as measured by the percentage injected dose per gram of tissue (%ID/g). Tumor uptake of all repebodies increased from 1 to 6 h (at 1 h, 6.28, 8.46, and 6.91 %ID/g for NOTA-, DOTA-, and DTPA-repebody, respectively; at 6 h, 9.4, 8.28, and 10.1 %ID/g, respectively). H1650 tumors were clearly visible after injection of each repebody, with high tumor-to-background ratios (at 1 h, 3.43, 4.89, and 2.38 for NOTA-, DOTA-, and DTPA-repebody, respectively; at 6 h, 3.05, 4.36, and 2.08; at 24 h, 3.81, 4.58, and 2.86). The 3Cu-repebody complexes demonstrated specific and rapid uptake in EGFR-expressing tumors within 1 h and may have potential as novel EGFR imaging agents for PET.

Development of smart anti-glycan reagents using immunized lampreys

“…Lampreys last shared a common ancestor with mammals~550 million years ago, and this evolutionary distance may provide the potential for the generation of VLRB proteins that recognize highly conserved proteins and carbohydrates that are not immunogenic in rodents 18 . These reagents have proven useful in a number of critical biological assay platforms, such as glycan microarray, flow cytometry, western blotting, and in probing tissue in immunohistochemistry assays 51,52 . VLRB proteins also have a wide variety of applications in biomedical research, including inhibition of cell signaling pathways 53,54 , as scaffolds for drug delivery 55 , and as diagnostic reagents 11,21,23 .…”

The variable lymphocyte receptor as an antibody alternative

“…Similarly, an anti-idiotype VLR could be used to monitor recurrence of chronic lymphocytic leukemia [37]. VLRs could also be valuable imaging reagents as demonstrated for EGFR-specific repebodies employed for in vivo imaging EGFR-expressing tumors [38,39] and mCherry-specific repebodies employed for tracking protein-protein interaction in live cells [40]. VLRs could also be used to study plant pathology and target disease forming pathogens.…”

PET Imaging of Receptor Tyrosine Kinases in Cancer

“…Repebody, a novel non-antibody protein scaffold (106), has also been engineered to monitor EGFR expression (107,108). One advantage of repebody-based PET imaging probes is their small molecular weight (~30 kDa), which may enable early visualization of various EGFR-expressing solid tumors as early as 1 h after injection of the probes.…”