RESUMEN
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 1-5â¯nmol (52-260⯵g), which corresponds to a human equivalent dose of 0.2-0.8â¯mg/kg. Biodistribution showed high accumulation of EpCAM-F800 in tumors and metabolizing organs. Breast and colorectal tumors could clearly be visualized within 8â¯h post-injection and up to 96â¯h, while the agent already showed homogenous tumor distribution within 4â¯h. The blood half-life was 4.5â¯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.
Asunto(s)
Bencenosulfonatos/farmacocinética , Neoplasias de la Mama/diagnóstico , Neoplasias Colorrectales/diagnóstico , Molécula de Adhesión Celular Epitelial/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Indoles/farmacocinética , Imagen Óptica/métodos , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Femenino , Humanos , Persona de Mediana Edad , Espectroscopía Infrarroja Corta , Distribución Tisular , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Incomplete resections and damage to critical structures increase morbidity and mortality of patients with cancer. Targeted intraoperative fluorescence imaging aids surgeons by providing real-time visualization of tumors and vital structures. This study evaluated the tumor-targeted zwitterionic near-infrared fluorescent peptide cRGD-ZW800-1 as tracer for intraoperative imaging of multiple cancer types. cRGD-ZW800-1 was validated in vitro on glioblastoma (U-87 MG) and colorectal (HT-29) cell lines. Subsequently, the tracer was tested in orthotopic mouse models with HT-29, breast (MCF-7), pancreatic (BxPC-3), and oral (OSC-19) tumors. Dose-ranging studies, including doses of 0.25, 1.0, 10, and 30 nmol, in xenograft tumor models suggest an optimal dose of 10 nmol, corresponding to a human equivalent dose of 63 µg/kg, and an optimal imaging window between 2 and 24 h post-injection. The mean half-life of cRGD-ZW800-1 in blood was 25 min. Biodistribution at 4 h showed the highest fluorescence signals in tumors and kidneys. In vitro and in vivo competition experiments showed significantly lower fluorescence signals when U-87 MG cells (minus 36%, p = 0.02) or HT-29 tumor bearing mice (TBR at 4 h 3.2 ± 0.5 vs 1.8 ± 0.4, p = 0.03) were simultaneously treated with unlabeled cRGD. cRGD-ZW800-1 visualized in vivo all colorectal, breast, pancreatic, and oral tumor xenografts in mice. Screening for off-target interactions, cRGD-ZW800-1 showed only inhibition of COX-2, likely due to binding of cRGD-ZW800-1 to integrin αVß3. Due to its recognition of various integrins, which are expressed on malignant and neoangiogenic cells, it is expected that cRGD-ZW800-1 will provide a sensitive and generic tool to visualize cancer during surgery.