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

Abstract

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% 6 3.82%, 76.25% 6 5.90%, 98.93% 6 3.75%, and 82.09% 6 4.14% for DOTA-Lys-hAb47, DOTA-Cys-hAb47, DOTASug-hAb47, and DOTA-Lys-hAb131, respectively). Although DOTASug-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 6 1.73 percentage injected dose [%ID]/g and 11.81 6 2.05 %ID/g, respectively). In contrast, 64 Cu-DOTA-LyshIgG had a low tumor accumulation, thus demonstrating the target specificity of EphB4-antibody-based probes. Moreover, 64 Cu-DOTA-Lys-hAb131 (29.48 6 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 6 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.

Evaluation of EphA2 and EphB4 as Targets for Image-Guided Colorectal Cancer Surgery

“…Targeting of Eph receptors for tumor imaging purposes could be achieved using various agents, such as peptides or antibodies [23,24]. Antibodies offer the advantage of high specificity and affinity and, thanks to molecular engineering, recently even bi-and tri-specific antibodies have been developed, which could enhance tumor specificity [13,25,26].…”

“…Antibodies offer the advantage of high specificity and affinity and, thanks to molecular engineering, recently even bi-and tri-specific antibodies have been developed, which could enhance tumor specificity [13,25,26]. Recently, a series of EphB4 antibodies have been evaluated for the use of Positron Emission Tomography (PET) in mice xenografted with human HT-29 colon cancer and MDA-MB-231 breast cells, underscoring the value of EphB4 as a target for tumor imaging [23]. A recent study also developed NIR fluorescence probes for EphB4 targeted imaging using an EphB4 antibody [27].…”

Axl-Targeted Cancer Imaging with Humanized Antibody h173

“…A lung tumor xenograft model was established by injecting 2×10 6 of A549 or NCI-H249 cells subcutaneously in the right shoulder of nude mice as described previously [21–23]. …”

Optimization of Mesenchymal Stem Cells (MSCs) Delivery Dose and Route in Mice with Acute Liver Injury by Bioluminescence Imaging

“…Postmortem tissue assay was performed as described previously [26][27][28]. Organs were dissected at 3-day posttransplantation of MSCs-R.…”