Comparison of Two Site-Specifically 18F-Labeled Affibodies for PET Imaging of EGFR Positive Tumors

The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous positron emission tomography (PET) studies showed that the EGFR-targeting affibody molecules Cu-64-DOTA-ZEGFR:1907 and F-18-FBEM-ZEGFR:1907 can discriminate between high and low EGFR-expression tumors and have the potential for patient selection for EGFR-targeted therapy. Compared with Cu-64, F-18 may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of F-18-FBEM-ZEGFR:1907 requires a long synthesis time. The aim of the present study is to develop a new generation of F-18 labeled affibody probes ((AlF)-F-18-NOTA-ZEGFR:1907 and F-18-CBT-ZEGFR:1907) and to determine whether they are suitable agents for imaging of EGFR expression. The first approach consisted of conjugating ZEGFR:1907 with NOTA and radiolabeling with (AlF)-F-18 to produce (AlF)-F-18-NOTA-ZEGFR:1907. In a second approach the prosthetic group F-18-labeled-2-cyanobenzothiazole (F-18-CBT) was conjugated to Cys-ZEGFR:1907 to produce F-18-CBT-ZEGFR:1907. Binding affinity and specificity of (AlF)-F-18-NOTA-ZEGFR:1907 and F-18-CBT-ZEGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of (AlF)-F-18-NOTA-ZEGFR:1907 or F-18-CBT-ZEGFR:1907 with or without coinjection of unlabeled affibody proteins. The radiosyntheses of (AlF)-F-18-NOTA-ZEGFR:1907 and F-18-CBT-ZEGFR:1907 were completed successfully within 40 and 120 min with a decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 cells. Although F-18-CBT-ZEGFR:1907 showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, (AlF)-F-18-NOTA-ZEGFR:1907 demonstrated high in vitro and in vivo stability, high tumor uptake, and relative low uptake in most of the normal organs except the liver and kidneys at 3 h after injection. The specificity of both probes for A431 tumors was confirmed by their lower uptake on coinjection of unlabeled affibody. PET studies showed that (AlF)-F-18-NOTA-ZEGFR:1907 and F-18-CBT-ZEGFR:1907 could clearly identify EGFR positive tumors with good contrast. Two strategies for F-18-labeling of affibody molecules were successfully developed as two model platforms using NOTA or CBT coupling to affibody molecules that contain an N-terminal cysteine. (AlF)-F-18-NOTA-ZEGFR:1907 and F-18-CBT-ZEGFR:1907 can be reliably obtained in a relatively short time. Biodistribution and PET studies demonstrated that (AlF)-F-18-NOTA-ZEGFR:1907 is a promising PET probe for imaging EGFR expression in living mice.