Site-Specific Labeling of F-18 Proteins Using a Supplemented Cell-Free Protein Synthesis System and O-2-[¹⁸F]Fluoroethyl-L-Tyrosine: [¹⁸F]FET-HER2 Affibody Molecule

Purpose: Although a preparation method for F-18-labeled proteins that used a cell-free translation system and 4-[¹⁸F]fluoro-L-proline instead of L-proline has been reported, its introduction depends on amino acid sequences of target proteins. The purpose of the study was to propose site-specific labeling method of F-18 by using cell-free translation systems supplemented with an engineered orthogonal aminoacyl-tRNA synthetase derived from Methanocaldococcus jannaschii (pCNF-RS)/suppressor tRNA (tRNA_CUA^opt) pair, O-2-[¹⁸F]fluoroethyl-L-tyrosine ([¹⁸F]FET), and template DNA inserted with an amber codon. Procedures: [¹⁸F]FET was prepared from the corresponding precursor and determined whether [¹⁸F]FET could be incorporated into an affibody molecule for human epidermal growth factor receptor type 2 (HER2; Z_HER2:342) as the 21st amino acid used with the pCNF-RS-tRNA_CUA^opt pair and template DNA inserted with an amber codon in a cell-free translation system. Using SKOV-3 cells, we performed an in vitro binding assay of [¹⁸F]FET-Z_HER2:342. Furthermore, in vivo positron emission tomography (PET) imaging in SKOV-3 xenograft-bearing mice was performed after the intravenous administration of [¹⁸F]FET-Z_HER2:342. Results: [¹⁸F]FET was successfully incorporated into proteins by using commercially available cell-free protein synthesis reagents with a pCNF-RS-tRNA_CUA^opt pair and template DNA of the desired proteins inserted with an amber codon. The mean radiochemical yield (non-decay-corrected) of [¹⁸F]FET-Z_HER2:342 was 6.5 ± 4.1 %. An in vitro cell binding assay revealed that SKOV-3 cells–bound [¹⁸F]FET-Z_HER2:342 expressed HER2. The in vivo PET imaging in SKOV-3 xenograft-bearing mice revealed that [¹⁸F]FET-Z_HER2:342 accumulated in SKOV-3 xenografts. Conclusion: The method proposed in this study might be useful for preparing proteins with F-18 and molecular imaging in the preclinical development.