PET of EGFR Expression with an ¹⁸F-Labeled Affibody Molecule

Abstract

Epidermal growth factor receptor (EGFR) is often overexpressed in a variety of human cancers, and its expression is associated with poor prognosis for many cancer types. However, an accurate technique to noninvasively image EGFR expression in vivo is not available in the clinical setting. In this research, an Affibody analog, anti-EGFR Ac-Cys-ZEGFR:1907, was successfully site-specifically 18F-labeled for PET of EGFR expression. Methods The prosthetic group N-[2-(4-18F-fluorobenzamido) ethyl] maleimide (18F-FBEM) was conjugated to Ac-Cys-ZEGFR:1907 under mild conditions (pH 7) to produce the probe 18F-FBEM-Cys-ZEGFR:1907. The binding affinity and specificity tests of 18F-FBEM-Cys-ZEGFR:1907 to EGFR were conducted using A431 cancer cells. Small-animal PET and biodistribution studies were conducted on various mice tumor xenograft models with EGFR overexpression (6 types) after injection of approximately 2.0 MBq of 18F-FBEM-Cys-ZEGFR:1907 with or without coinjection of unlabeled Ac-Cys-ZEGFR:1907 for up to 3 h after injection. A correlation study between 18F-FBEM-Cys-ZEGFR:1907 small- animal PET quantification and ex vivo Western blot analysis of tumor EGFR expression was conducted in those 6 types of tumor models. Results 18F-FBEM-Cys-ZEGFR:1907 binds to EGFR with low nanomolar affinity (37 nM) in A431 cells. 18F-FBEM-Cys-ZEGFR:1907 rapidly accumulated in the tumor and cleared from most of the normal organs except the liver and kidneys at 3 h after injection, allowing excellent tumor–to–normal tissue contrast to be obtained. In the A431 tumor xenograft model, coinjection of the PET probe with 45 μg of Ac-Cys-ZEGFR:1907 was able to improve the tumor uptake (3.9 vs. 8.1 percentage of the injected radioactive dose per gram of tissue, at 3 h after injection) and tumor imaging contrast, whereas coinjection with 500 μg of Ac-Cys-ZEGFR:1907 successfully blocked the tumor uptake significantly (8.1 vs. 1.0 percentage of the injected radioactive dose per gram of tissue, at 3 h after injection, 88% inhibition, P < 0.05). Moderate correlation was found between the tumor tracer uptake at 3 h after injection quantified by PET and EGFR expression levels measured by Western blot assay (P = 0.007, R = 0.59). Conclusion 18F-FBEM-Cys-ZEGFR:1907 is a novel protein scaffold–based PET probe for imaging EGFR overexpression of tumors, and its ability to differentiate tumors with high and low EGFR expression in vivo holds promise for future clinical translation.

Preparation and Evaluation of 99mTc-Epidermal Growth Factor Receptor (EGFR)-Peptide Nucleic Acid for Visualization of EGFR Messenger RNA Expression in Malignant Tumors

“…Several modalities using PET, SPECT, and MR imaging have been introduced to noninvasively evaluate EGFR. Some noninvasive molecular agents, such as 11 Cerlotinib, 11 C-labeled 4-N-(3-bromoanilino)-6, 7-dimethoxyquinazoline, 177 Lu-nimotuzumab, 18 F-labeled Affibody protein, or anti-EGFR monoclonal antibodies using contrast-enhanced MR imaging, have been developed as molecular imaging biomarkers for evaluating tumor EGFR status (17)(18)(19)(20)(21). Although these EGFR-specific imaging probes have been investigated, they all have been directed either at an external ligand binding domain using antibodies and Affibody molecules or at the internal adenosine triphosphate binding domain with small organic reversible and irreversible inhibitors (22).…”

Interrogating Tumor Metabolism and Tumor Microenvironments Using Molecular Positron Emission Tomography Imaging. Theranostic Approaches to Improve Therapeutics

“…However, there was very high background and the use of SPECT does not allow for the quantification of the results (Blankenberg, 2008). Nevertheless, Annexin V and its derivatives have been extensively evaluated in xenograft models when labeled with PET isotopes (Li et al, 2008;Zhang et al, 2009;Bauwens et al, 2011;Cheng et al, 2012;Hu et al, 2012). The results of one interesting study was recently published by Hu et al (2012), describing the ability of […”

“…5). 64 Cu-Affibody also had extremely high renal uptake and retention, although this issue was partially resolved by 18 F labeling (Miao et al, 2010(Miao et al, , 2012. Surprisingly, liver uptake was reduced and tumor uptake was increased when the labeled Affibody was administered as low SA form, with 45-50 mg of unlabeled Affibody ( Fig.…”

“…Another biomarker for targeting EGFR is anti-EGFR Affibody protein (Z EGFR:1907 ), which has been site specifically labeled with 18 F and 64 Cu and evaluated in tumor-bearing mice (Miao et al, 2010(Miao et al, , 2012. The labeled Affibody molecule showed good pharmacokinetics in vivo with fast accumulation in EGFR-positive tumors but also displayed very high uptake in the liver (Fig.…”

Metabolism of Epidermal Growth Factor Receptor Targeting Probe [11C]PD153035: Impact on Biodistribution and Tumor Uptake in Rats

“…EGFR is expressed in the liver and plays an important role in hepatic development and regeneration (25). Therefore, EGFR-targeting tracers would be expected to and have been shown to have noticeable uptake in the liver (26,27). A tendency for more prolonged retention of radioactivity (Fig.…”