Prostate-specific membrane antigen (PSMA) is an extensively studied antigen for imaging prostate cancer. We prepared a single-chain variable fragment (scFv) of J591, a monoclonal antibody that recognises an external epitope of PSMA, incorporating a His-tag for labelling with 99mTc tricarbonyl, and evaluated its binding using human PCa cell lines.Methods
J591(scFv) was expressed in HEK-293T cells and purified by metal ion affinity chromatography, followed by size exclusion chromatography. Stability and monomer/dimer ratios of purified scFv under different storage conditions were analysed by SDS-PAGE and analytical size exclusion chromatography. J591(scFv) was labelled withMethods
at 37°C for 60 min. The stability of 99mTc-scFv in human serum was analysed by SDS-PAGE with autoradiography. Cell-binding studies were carried out using PC3LN3 (PSMA negative) and PC3LN3-PSMA (a variant engineered to express PSMA) cell lines.Results
J591(scFv) was most stable to dimerisation on storage at −80°C compared with −20 and 4°C. Radiochemical yields of 85–90% were obtained with the final radiochemical purity of more than 99% after purification by gel filtration. In these small-scale studies, the maximum specific activity achieved was 7 MBq/μg. Liquid chromatography–mass spectrometry showed the formation of 99mTc-J591(scFv), which was radiochemically stable in serum, with no dissociation of 99mTc over 24 h. Cell-binding assays showed specific binding to PSMA-positive cells.Conclusion
J591(scFv) can be radiolabelled withConclusion
conveniently and efficiently. The labelled product was stable in serum. It showed selective binding to PSMA-positive cells compared with PSMA-negative cells. This potential radiotracer warrants evaluation in PCa xenograft models.