Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer (PCa) and a promising target for molecular imaging and therapy. Nanobodies (single-domain antibodies, VHH) are the smallest antibody-based fragments possessing ideal molecular imaging properties, such as high target specificity and rapid background clearance. We developed a novel anti-PSMA Nanobody (JVZ-007) for targeted imaging and therapy of PCa. Here, we report on the application of the <sup>111</sup>In-radiolabeled Nanobody for SPECT/CT imaging of PCa. Methods: A Nanobody library was generated by immunization of a llama with 4 human PCa cell lines. Anti-PSMA Nanobodies were captured by biopanning on PSMA-overexpressing cells. JVZ-007 was selected for evaluation as an imaging probe. JVZ-007 was initially produced with a c-myc-hexahistidine (his) tag allowing purification and detection. The c-myc-his tag was subsequently replaced by a single cysteine at the C terminus, allowing site-specific conjugation of chelates for radiolabeling. JVZ-007-cmyc- his was conjugated to 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-DTPA) via the lysines, whereas JVZ-007-cys was conjugated to maleimide-DTPA via the C-terminal cysteine. PSMA targeting was analyzed in vitro by cell-binding experiments using flow cytometry, autoradiography, and internalization assays with various PCa cell lines and patient-derived xenografts (PDXs). The targeting properties of radiolabeled Nanobodies were evaluated in vivo in biodistribution and SPECT/CT imaging experiments, using nude mice bearing PSMA-positive PC-310 and PSMA-negative PC-3 tumors. Results: JVZ-007 was successfully conjugated to DTPA for radiolabeling with <sup>111</sup>In at room temperature. <sup>111</sup>In-JVZ007-c-myc-his and <sup>111</sup>In-JVZ007-cys internalized in LNCaP cells and bound to PSMA-expressing PDXs and, importantly, not to PSMA-negative PDXs and human kidneys. Good tumor targeting and fast blood clearance were observed for <sup>111</sup>In- JVZ-007-c-myc-his and <sup>111</sup>In-JVZ-007-cys. Renal uptake of <sup>111</sup>In- JVZ-007-c-myc-his was initially high but was efficiently reduced by coinjection of gelofusine and lysine. The replacement of the c-myc-his tag by the cysteine contributed to a further reduction of renal uptake without loss of targeting. PC-310 tumors were clearly visualized by SPECT/CT with both tracers, with low renal uptake (,4 percentage injected dose per gram) for <sup>111</sup>In-JVZ- 007-cys already at 3 h after injection. Conclusion: We developed an <sup>111</sup>In-radiolabeled anti-PSMA Nanobody, showing good tumor targeting, low uptake in nontarget tissues, and low renal retention, allowing excellent SPECT/CT imaging of PCa within a few hours after injection.

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The Journal of Nuclear Medicine
Department of Nuclear Medicine

Chatalic, K.L.S, Veldhoven-Zweistra, J, Bolkestein, M, Hoeben, S, Koning, G.A, Boerman, O.C, … van Weerden, W.M. (2015). A novel 111In-labeled anti-prostate-specific membrane antigen nanobody for targeted SPECT/CT imaging of prostate cancer. The Journal of Nuclear Medicine, 56(7), 1094–1099. doi:10.2967/jnumed.115.156729