Boogerd Leonora S F, Boonstra Martin C, Prevoo Hendrica A J M, Handgraaf Henricus J M, Kuppen Peter J K, van de Velde Cornelis J H, Fish Alexander, Cordfunke Robert A, Valentijn A Rob P M, Terwisscha van Scheltinga A G, MacDonald Glen C, Cizeau Jeannick, Premsukh Arjune, Vinkenburg van Slooten Maaike L, Burggraaf Jacobus, Sier Cornelis F M, Vahrmeijer Alexander L
Leiden University Medical Centre, Department of Surgery, Leiden, the Netherlands.
Netherlands Cancer Institute, Division of Biochemistry, Amsterdam, the Netherlands.
Surg Oncol. 2019 Mar;28:1-8. doi: 10.1016/j.suronc.2018.10.004. Epub 2018 Oct 23.
Tumor-specific fluorescent imaging agents are moving towards the clinic, supporting surgeons with real-time intraoperative feedback about tumor locations. The epithelial cell adhesion molecule (EpCAM) is considered as one of the most promising tumor-specific proteins due its high overexpression on epithelial-derived cancers. This study describes the development and evaluation of EpCAM-F800, a novel fluorescent anti-EpCAM antibody fragment, for intraoperative tumor imaging. Fab production, conjugation to the fluorophore IRDye 800CW, and binding capacities were determined and validated using HPLC, spectrophotometry and cell-based assays. In vivo, dose escalation-, blocking-, pharmacokinetic- and biodistribution studies (using both fluorescence and radioactivity) were performed, next to imaging of clinically relevant orthotopic xenografts for breast and colorectal cancer. EpCAM-F800 targets EpCAM with high specificity in vitro, which was validated using in vivo blocking experiments with a 10x higher dose of unlabeled Fab. The optimal dose range for fluorescence tumor detection in mice was 1-5 nmol (52-260 μg), which corresponds to a human equivalent dose of 0.2-0.8 mg/kg. Biodistribution showed high accumulation of EpCAM-F800 in tumors and metabolizing organs. Breast and colorectal tumors could clearly be visualized within 8 h post-injection and up to 96 h, while the agent already showed homogenous tumor distribution within 4 h. The blood half-life was 4.5 h. This study describes the development and evaluation of a novel EpCAM-targeting agent and the feasibility to visualize breast and colorectal tumors by fluorescence imaging during resections. EpCAM-F800 will be translated for clinical use, considering its abundance in a broad range of tumor types.
肿瘤特异性荧光成像剂正迈向临床应用，为外科医生提供有关肿瘤位置的实时术中反馈。上皮细胞粘附分子（EpCAM）因其在上皮源性癌症中高度过表达，被认为是最有前景的肿瘤特异性蛋白之一。本研究描述了一种新型荧光抗EpCAM抗体片段EpCAM-F800用于术中肿瘤成像的研发与评估。通过高效液相色谱法、分光光度法和基于细胞的试验，确定并验证了Fab的产生、与荧光团IRDye 800CW的偶联以及结合能力。在体内，除了对乳腺癌和结直肠癌的临床相关原位异种移植瘤进行成像外，还进行了剂量递增、阻断、药代动力学和生物分布研究（同时使用荧光和放射性）。EpCAM-F800在体外以高特异性靶向EpCAM，这通过使用10倍高剂量未标记Fab的体内阻断实验得到验证。小鼠荧光肿瘤检测的最佳剂量范围为1-5 nmol（52-260 μg），相当于人体等效剂量0.2-0.8 mg/kg。生物分布显示EpCAM-F800在肿瘤和代谢器官中高度蓄积。注射后8小时内直至96小时，乳腺癌和结直肠癌肿瘤均可清晰可视化，而该制剂在4小时内已显示出肿瘤内均匀分布。血液半衰期为4.5小时。本研究描述了一种新型EpCAM靶向制剂的研发与评估，以及在切除术中通过荧光成像可视化乳腺癌和结直肠癌肿瘤的可行性。鉴于EpCAM-F800在多种肿瘤类型中均有表达，它将转化为临床应用。
