PT - JOURNAL ARTICLE AU - Chatalic, Kristell L.S. AU - Veldhoven-Zweistra, Joke AU - Bolkestein, Michiel AU - Hoeben, Sander AU - Koning, Gerben A. AU - Boerman, Otto C. AU - de Jong, Marion AU - van Weerden, Wytske M. TI - A Novel <sup>111</sup>In-Labeled Anti–Prostate-Specific Membrane Antigen Nanobody for Targeted SPECT/CT Imaging of Prostate Cancer AID - 10.2967/jnumed.115.156729 DP - 2015 Jul 01 TA - Journal of Nuclear Medicine PG - 1094--1099 VI - 56 IP - 7 4099 - http://jnm.snmjournals.org/content/56/7/1094.short 4100 - http://jnm.snmjournals.org/content/56/7/1094.full SO - J Nucl Med2015 Jul 01; 56 AB - 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 111In-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-c-myc-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 111In at room temperature. 111In-JVZ007-c-myc-his and 111In-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 111In-JVZ-007-c-myc-his and 111In-JVZ-007-cys. Renal uptake of 111In-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 (&lt;4 percentage injected dose per gram) for 111In-JVZ-007-cys already at 3 h after injection. Conclusion: We developed an 111In-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.