Generic semi-automated radiofluorination strategy for single domain antibodies: [¹⁸F]FB-labelled single domain...

Radiofluorination of single domain antibodies (sdAbs) via N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) has shown to be a promising strategy in the development of sdAb-based PET tracers. While automation of the prosthetic group (PG) [¹⁸F]SFB production, has been successfully reported, no practical method for large scale sdAb labelling has been reported. Therefore, we optimized and automated the PG production, enabling a subsequently efficient manual conjugation reaction to an anti-fibroblast activation protein (FAP)-α sdAb (4AH29) and an anti-folate receptor (FR)-α sdAb (2BD42). Both the alpha isoform of FAP and the FR are established tumour markers. FAP-α is known to be overexpressed mainly by cancer-associated fibroblasts in breast, ovarian, and other cancers, while its expression in normal tissues is low or undetectable. FR-α has an elevated expression in epithelial cancers, such as ovarian, brain and lung cancers. Non-invasive imaging techniques, such as PET-imaging, using tracers targeting specific tumour markers can provide molecular information over both the tumour and its environment, which aides in the diagnosis, therapy selection and assessment of the cancer treatment.

[¹⁸F]SFB was synthesized using a fully automated three-step, one-pot reaction. The total procedure time was 54 min and results in [¹⁸F]SFB with a RCP > 90% and a RCY d.c. of 44 ± 4% (n = 13). The manual conjugation reaction after purification produced [¹⁸F]FB-sdAbs with a RCP > 95%, an end of synthesis activity > 600 MBq and an apparent molar activity > 10 GBq/µmol. Overall RCY d.c., corrected to the trapping of [¹⁸F]F^- on the QMA, were 9% (n = 1) and 5 ± 2% (n = 3) for [¹⁸F]FB-2BD42 and [¹⁸F]FB-4AH29, respectively.

[¹⁸F]SFB synthesis was successfully automated and upscaled on a Trasis AllInOne module. The anti-hFAP-α and anti-hFR-α sdAbs were radiofluorinated, yielding similar RCYs d.c. and RCPs, showing the potential of this method as a generic radiofluorination strategy for sdAbs. The radiofluorinated sdAbs showed a favourable biodistribution pattern and are attractive for further characterization as new PET tracers for FAP-α and FR-α imaging.