Department of Medical Physics, University of Wisconsin - Madison, 1111 Highland Avenue, Madison, WI 53705-2275, USA.
Eur J Nucl Med Mol Imaging. 2013 May;40(5):759-67. doi: 10.1007/s00259-012-2334-2. Epub 2013 Jan 24.
The goal of this study was to generate and characterize the Fab fragment of TRC105, a monoclonal antibody that binds with high affinity to human and murine CD105 (i.e., endoglin), and investigate its potential for PET imaging of tumor angiogenesis in a small-animal model after (61/64)Cu labeling.
TRC105-Fab was generated by enzymatic papain digestion. The integrity and CD105 binding affinity of TRC105-Fab was evaluated before NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) conjugation and (61/64)Cu labeling. Serial PET imaging and biodistribution studies were carried out in the syngeneic 4T1 murine breast cancer model to quantify tumor targeting efficiency and normal organ distribution of (61/64)Cu-NOTA-TRC105-Fab. Blocking studies with unlabeled TRC105 were performed to confirm CD105 specificity of the tracer in vivo. Immunofluorescence staining was also conducted to correlate tracer uptake in the tumor and normal tissues with CD105 expression.
TRC105-Fab was produced with high purity through papain digestion of TRC105, as confirmed by SDS-PAGE, HPLC analysis, and mass spectrometry. (61/64)Cu labeling of NOTA-TRC105-Fab was achieved with about 50 % yield (specific activity about 44 GBq/μmol). PET imaging revealed rapid uptake of (64)Cu-NOTA-TRC105-Fab in the 4T1 tumor (3.6 ± 0.4, 4.2 ± 0.5, 4.9 ± 0.3, 4.4 ± 0.7, and 4.6 ± 0.8 %ID/g at 0.5, 2, 5, 16, and 24 h after injection, respectively; n = 4). Since tumor uptake peaked soon after tracer injection, (61)Cu-labeled TRC105-Fab was also able to provide tumor contrast at 3 and 8 h after injection. CD105 specificity of the tracer was confirmed with blocking studies and histological examination.
We report PET imaging of CD105 expression using (61/64)Cu-NOTA-TRC105-Fab, which exhibited prominent and target-specific uptake in the 4T1 tumor. The use of a Fab fragment led to much faster tumor uptake (which peaked at a few hours after tracer injection) compared to radiolabeled intact antibody, which may be translated into same-day immunoPET imaging for clinical investigation.
本研究旨在生成并鉴定 TRC105 的 Fab 片段，该片段是一种与人及鼠源 CD105（即内皮糖蛋白）高亲和力结合的单克隆抗体，并研究其在经过（61/64）Cu 标记后用于小动物模型肿瘤血管生成 PET 成像的潜力。
通过酶木瓜蛋白酶消化生成 TRC105-Fab。在 NOTA（1,4,7-三氮杂环壬烷-1,4,7-三乙酸）偶联和（61/64）Cu 标记之前，评估 TRC105-Fab 的完整性和 CD105 结合亲和力。通过在同源 4T1 乳腺癌模型中进行连续的 PET 成像和生物分布研究，定量评估（61/64）Cu-NOTA-TRC105-Fab 的肿瘤靶向效率和正常器官分布。通过未标记的 TRC105 进行阻断研究，以确认体内示踪剂的 CD105 特异性。还进行了免疫荧光染色，以将肿瘤和正常组织中的示踪剂摄取与 CD105 表达相关联。
通过木瓜蛋白酶消化 TRC105 可高纯度地产生 TRC105-Fab，这一点通过 SDS-PAGE、HPLC 分析和质谱得到确认。NOTA-TRC105-Fab 的（61/64）Cu 标记产率约为 50%（比活度约为 44GBq/μmol）。PET 成像显示，（64）Cu-NOTA-TRC105-Fab 在 4T1 肿瘤中的摄取迅速（分别在注射后 0.5、2、5、16 和 24 小时时为 3.6±0.4、4.2±0.5、4.9±0.3、4.4±0.7 和 4.6±0.8%ID/g；n=4）。由于肿瘤摄取在示踪剂注射后很快达到峰值，因此（61）Cu 标记的 TRC105-Fab 也能够在注射后 3 和 8 小时提供肿瘤对比度。通过阻断研究和组织学检查证实了示踪剂的 CD105 特异性。
我们报告了使用（61/64）Cu-NOTA-TRC105-Fab 进行 CD105 表达的 PET 成像，该示踪剂在 4T1 肿瘤中表现出明显的、靶向特异性的摄取。与放射性标记完整抗体相比，使用 Fab 片段可导致更快的肿瘤摄取（在示踪剂注射后数小时达到峰值），这可能转化为用于临床研究的同日免疫 PET 成像。
