PURPOSE: CS-1008 (tigatuzumab; phase I/II), an antihuman death receptor 5 (DR5) agonist, induces apoptosis and has cytotoxic activity against human cancer cell lines. This study reports on the preclinical validation of (111)In-labeled anti-DR5 humanized antibody CS-1008 as a diagnostic tool to study the DR5 occupancy in patients with cancer and establish dose ranges for receptor saturation kinetics in vivo. EXPERIMENTAL DESIGN: CS-1008 was radiolabeled and characterized for DR5 binding and labeling efficiency on TRAIL-sensitive DR5-positive colorectal cancer cells (COLO 205 and WiDr). Pharmacokinetic and biodistribution studies were conducted in BALB/c nu/nu mice bearing COLO 205, WiDr, or DR5-negative CT26 colon tumors. Planar gamma camera imaging and computerized tomography (CT) images were obtained to study receptor occupancy in vivo. RESULTS: Scatchard analysis showed high and specific binding affinity (Kd, 1.05 ± 0.12 nmol/L) of (111)In-labeled CS-1008. (111)In-labeled CS-1008 was specifically taken up in mice bearing COLO 205 and WiDr tumors with prolonged tumor retention (26.25 ± 2.85%ID/g vs. 12.20 ± 2.24 at 168 hours post injection; n = 5, SD), and uptake correlated both with DR5 expression on tumor cells and antitumor activity. DR5 saturation was shown in vivo via both biodistribution studies and planar gamma camera imaging/CT imaging of (111)In-labeled CS-1008. Saturation of DR5 corresponded to maximal in vivo antitumor efficacy. CONCLUSIONS: Imaging of DR5 receptor occupancy in vivo correlates with tumor concentration and in vivo efficacy, and is a novel molecular imaging technique that can be used to determine receptor occupancy and effective dose levels of DR5 agonist antibodies in the clinic.