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

[Image: see text] 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 (64)Cu-DOTA-Z(EGFR:1907) and (18)F-FBEM-Z(EGFR:1907) can discriminate between high and low EGFR-expression tumors and have the potential for patient selection for EGFR-targeted therapy. Compared with (64)Cu, (18)F may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of (18)F-FBEM-Z(EGFR:1907) requires a long synthesis time. The aim of the present study is to develop a new generation of (18)F labeled affibody probes (Al(18)F-NOTA-Z(EGFR:1907) and (18)F-CBT-Z(EGFR:1907)) and to determine whether they are suitable agents for imaging of EGFR expression. The first approach consisted of conjugating Z(EGFR:1907) with NOTA and radiolabeling with Al(18)F to produce Al(18)F-NOTA-Z(EGFR:1907). In a second approach the prosthetic group (18)F-labeled-2-cyanobenzothiazole ((18)F-CBT) was conjugated to Cys-Z(EGFR:1907) to produce (18)F-CBT-Z(EGFR:1907). Binding affinity and specificity of Al(18)F-NOTA-Z(EGFR:1907) and (18)F-CBT-Z(EGFR:1907) to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of Al(18)F-NOTA-Z(EGFR:1907) or (18)F-CBT-Z(EGFR:1907) with or without coinjection of unlabeled affibody proteins. The radiosyntheses of Al(18)F-NOTA-Z(EGFR:1907) and (18)F-CBT-Z(EGFR: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 (18)F-CBT-Z(EGFR:1907) showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al(18)F-NOTA-Z(EGFR: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 Al(18)F-NOTA-Z(EGFR:1907) and (18)F-CBT-Z(EGFR:1907) could clearly identify EGFR positive tumors with good contrast. Two strategies for (18)F-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. Al(18)F-NOTA-Z(EGFR:1907) and (18)F-CBT-Z(EGFR:1907) can be reliably obtained in a relatively short time. Biodistribution and PET studies demonstrated that Al(18)F-NOTA-Z(EGFR:1907) is a promising PET probe for imaging EGFR expression in living mice.