PET Imaging of Colorectal and Breast Cancer by Targeting EphB4 Receptor with64Cu-Labeled hAb47 and hAb131 Antibodies

UNLABELLED: Accumulating evidence suggests that ephrin type B receptor 4 (EphB4) plays a key role in the progression of numerous cancer types. In this study, we developed a series of (64)Cu-labeled antibodies for PET imaging of tumor EphB4 expression. METHODS: Anti-EphB4 antibodies (hAb47 and hAb131) were conjugated with the (64)Cu-chelator DOTA through lysine, cysteine, or oligosaccharide on the antibody. DOTA-human IgG (hIgG) was also prepared as a control, which did not bind to EphB4. The EphB4 binding activity of these probes was evaluated through the bead-based binding assay with EphB4-alkaline phosphatase. The resulting PET probes were further evaluated in both HT29 (colorectal cancer) and MDA-MB-231 (breast cancer) xenografts. RESULTS: All 3 conjugation methods retained most of the EphB4 binding activity of the antibodies (83.85% ± 3.82%, 76.25% ± 5.90%, 98.93% ± 3.75%, and 82.09% ± 4.14% for DOTA-Lys-hAb47, DOTA-Cys-hAb47, DOTA-Sug-hAb47, and DOTA-Lys-hAb131, respectively). Although DOTA-Sug-hAb47 demonstrated the highest receptor binding activity based on a EphB4 binding assay, the corresponding PET probe was trapped in the liver quickly in vivo. In HT29 xenografts, both (64)Cu-DOTA-Lys-hAb47 and (64)Cu-DOTA-Cys-hAb47 demonstrated prominent tumor accumulation, which reached a maximum at 48 h after injection (18.13 ± 1.73 percentage injected dose [%ID]/g and 11.81 ± 2.05 %ID/g, respectively). In contrast, (64)Cu-DOTA-Lys-hIgG had a low tumor accumulation, thus demonstrating the target specificity of EphB4-antibody-based probes. Moreover, (64)Cu-DOTA-Lys-hAb131 (29.48 ± 2.60 %ID/g) demonstrated significantly higher HT29 tumor accumulation than (64)Cu-DOTA-Lys-hAb47. (64)Cu-DOTA-Lys-hAb131 was also found to specifically accumulate in the MDA-MB-231 tumor model (12.96 ± 2.31 %ID/g). CONCLUSION: We have demonstrated that EphB4 can serve as a valid target for colorectal and breast cancer imaging. This approach would be valuable for evaluating disease course and therapeutic efficacy at the earliest stages of anti-EphB4 treatment. Moreover, these newly developed probes may have important applications in other cancer types overexpressing EphB4.