title: EphB4-targeted imaging with antibody h131, h131-F(ab')2 and h131-Fab.
abstract: Accumulating evidence suggests that overexpression of the tyrosine kinase receptor EphB4, a mediator of vascular development, is a novel target for tumor diagnosis, prognosis and therapy. Noninvasive imaging of EphB4 expression could therefore be valuable for evaluating disease course and therapeutic efficacy at the earliest stages of anti-EphB4 treatment. In this study, we systematically investigated the use of anti-EphB4 antibody h131 (150 kDa) and its fragments (h131-F(ab')2, 110 kDa; h131-Fab, 50 kDa) for near-infrared fluorescence (NIRF) imaging of EphB4 expression in vivo. h131-F(ab')2 and h131-Fab were produced through pepsin and papain digestion of h131 respectively, whose purity was confirmed by FPLC and SDS-PAGE. After conjugation with Cy5.5, in vivo characteristics of h131, h131-F(ab')2 and h131-Fab were evaluated in EphB4-positive HT29 tumor model. Although h131-Cy5.5 demonstrated highest tumor uptake among these probes, its optimal tumor uptake level was obtained at 2 days post injection (p.i.). For h131-Fab-Cy5.5, maximum tumor uptake was achieved at 4 h p.i. However, no significant difference was observed between h131-Fab-Cy5.5 and hIgG-Fab-Cy5.5, indicating the tumor accumulation was mainly caused by passive targeting. In contrast, h131-F(ab')2-Cy5.5 demonstrated prominent tumor uptake at 6 h p.i. The target specificity was confirmed by hIgG-F(ab')2-Cy5.5 control and immunofluorescent staining. Collectively, h131-F(ab')2 exhibited prominent and specific tumor uptake at early time points, which suggests it is a promising agent for EphB4-targeted imaging.
authors.affiliation: Department of Radiology, The Third Affiliated Hospital of Sun Yat-sen University , Guangzhou, 510630, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.forename: Shuanglong
authors.initials: S
authors.lastname: Liu
authors.forename: Ren
authors.initials: R
authors.lastname: Liu
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhou
authors.forename: Ryan
authors.initials: R
authors.lastname: Park
authors.forename: Kranthi
authors.initials: K
authors.lastname: Naga
authors.forename: Valery
authors.initials: V
authors.lastname: Krasnoperov
authors.forename: Parkash S
authors.initials: PS
authors.lastname: Gill
authors.forename: Zibo
authors.initials: Z
authors.lastname: Li
authors.forename: Hong
authors.initials: H
authors.lastname: Shan
authors.forename: Peter S
authors.initials: PS
authors.lastname: Conti
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D045744:Cell Line, Tumor; D003952:Diagnostic Imaging / Q000379:methods; D019073:HT29 Cells; D006801:Humans; D009369:Neoplasms / Q000175:diagnosis* / Q000276:immunology*; D036224:Receptor, EphB4 / Q000276:immunology*; D014018:Tissue Distribution / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D036224:Receptor, EphB4