Department of Urology, Erasmus MC, Rotterdam, The Netherlands Departments of Nuclear Medicine and Radiology, Erasmus MC, Rotterdam, The Netherlands
Department of Urology, Erasmus MC, Rotterdam, The Netherlands.
J Nucl Med. 2015 Jul;56(7):1094-9. doi: 10.2967/jnumed.115.156729. Epub 2015 May 14.
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 (111)In-radiolabeled Nanobody for SPECT/CT imaging of PCa.
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.
JVZ-007 was successfully conjugated to DTPA for radiolabeling with (111)In at room temperature. (111)In-JVZ007-c-myc-his and (111)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 (111)In-JVZ-007-c-myc-his and (111)In-JVZ-007-cys. Renal uptake of (111)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 (111)In-JVZ-007-cys already at 3 h after injection.
We developed an (111)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.
研究前列腺特异性膜抗原（PSMA）在前列腺癌（PCa）中的过度表达，以及将其作为分子成像和治疗的潜在靶点。纳米体（单域抗体，VHH）是基于抗体的最小片段，具有理想的分子成像特性，如高靶向特异性和快速背景清除。我们开发了一种新型抗 PSMA 纳米体（JVZ-007），用于 PCa 的靶向成像和治疗。在此，我们报告了（111）In 标记的纳米体用于 PCa 的 SPECT/CT 成像。
通过用 4 个人前列腺癌细胞系免疫骆驼，生成了一个纳米体文库。通过在 PSMA 过表达细胞上进行生物淘选，捕获抗 PSMA 纳米体。选择 JVZ-007 作为成像探针进行评估。JVZ-007 最初带有 c-myc-六组氨酸（his）标签，允许进行纯化和检测。随后，his 标签被 C 末端的单个半胱氨酸取代，允许通过定点连接螯合剂进行放射性标记。JVZ-007-c-myc-his 通过赖氨酸与 2-（4-异硫氰酸苄基）-二乙三胺五乙酸（p-SCN-DTPA）偶联，而 JVZ-007-cys 通过 C 末端半胱氨酸与马来酰亚胺-DTPA 偶联。通过流式细胞术、放射自显影和使用各种 PCa 细胞系和患者来源的异种移植物（PDXs）进行的内化实验，在体外分析 PSMA 靶向性。使用携带 PSMA 阳性 PC-310 和 PSMA 阴性 PC-3 肿瘤的裸鼠，在体内生物分布和 SPECT/CT 成像实验中评估放射性标记纳米体的靶向特性。
JVZ-007 成功地与 DTPA 室温下进行（111）In 标记。（111）In-JVZ007-c-myc-his 和（111）In-JVZ007-cys 在 LNCaP 细胞中内化并与表达 PSMA 的 PDX 结合，重要的是，不与 PSMA 阴性 PDX 和人肾脏结合。（111）In-JVZ-007-c-myc-his 和（111）In-JVZ-007-cys 观察到良好的肿瘤靶向和快速血液清除。（111）In-JVZ-007-c-myc-his 的肾脏摄取最初很高，但通过共注射凝胶和赖氨酸可有效降低。C 末端半胱氨酸取代 his 标签不会影响靶向性，但可进一步降低肾脏摄取。两种示踪剂均可通过 SPECT/CT 清楚地显示 PC-310 肿瘤，（111）In-JVZ-007-cys 在注射后 3 小时，肾脏摄取（<4%注射剂量/克）就很低。
我们开发了一种（111）In 放射性标记的抗 PSMA 纳米体，具有良好的肿瘤靶向性、非靶组织摄取低和肾脏保留低的特点，可在注射后数小时内实现 PCa 的优异 SPECT/CT 成像。
