The Edward Mallinckrodt Institute of Radiology, Washington University, St Louis, Missouri 91320-1799, USA.
J Nucl Med. 2011 Jun;52(6):942-9. doi: 10.2967/jnumed.110.086157. Epub 2011 May 13.
Conatumumab is a fully human monoclonal antibody that binds to and activates human death receptor 5 (DR5; also known as TRAIL receptor 2). The purpose of this study was to characterize (64)Cu-labeled conatumumab as a PET tracer for imaging DR5 in tumors.
DOTA-conatumumab was synthesized by incubating conatumumab with 2,2',2″-(10-(2-(2,5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTA-NHS). The absolute numbers of DOTA molecules per conatumumab molecules were determined by matrix-assisted laser desorption ionization mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry. (64)Cu-DOTA-conatumumab was prepared by incubating (64)CuCl(2) (33-222 MBq) with DOTA-conatumumab at 37°C for 1 h. Binding of conatumumab and DOTA-conatumumab to Fc-coupled human DR5 (huTR2-Fc) was tested in a kinetic analysis assay, and the biologic activity of copper-DOTA-conatumumab was measured using a caspase-3/7 luminescent assay. In vivo evaluation of DOTA-conatumumab and copper-DOTA-conatumumab was done in severe combined immunodeficiency mice bearing Colo205 xenografts: tissue uptake was determined with biodistribution studies, and small-animal PET and autoradiography were used to determine the uptake of (64)Cu-DOTA conatumumab into tumors and other tissues.
DOTA-conatumumab was prepared with an average of 5 DOTA molecules per conatumumab molecule. The in vitro median effective concentration required to induce a 50% effect of DOTA-conatumumab and conatumumab from the assay were 389 and 320 pM, respectively. The median effective dose (±SD) of DOTA-conatumumab and conatumumab via the caspase assay was 135 ± 31 and 128 ± 30 pM, respectively. In female CB17 severe combined immunodeficiency mice bearing Colo205 xenografts, DOTA-conatumumab and conatumumab inhibited tumor growth to the same extent. Small-animal PET studies showed tumor uptake at 24 h after injection of the tracer, with a mean standardized uptake value of 3.16 (n = 2). Tumor uptake was decreased by the coadministration of 400 μg of unlabeled conatumumab (mean standardized uptake value, 1.55; n = 2), suggesting saturable uptake. Tissue uptake determined by biodistribution studies was in agreement with the small-animal PET findings.
These results suggest that (64)Cu-DOTA-conatumumab is a potential PET tracer for imaging DR5 in tumors and may be useful for measuring on-target occupancy by conatumumab.
表征（64）Cu 标记的 conatumumab 作为一种 PET 示踪剂，用于在肿瘤中成像 DR5。
通过将 conatumumab 与 2,2',2″-(10-(2-(2,5-二氧代吡咯烷-1-基氧基)-2-氧代乙基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸（DOTA-NHS）孵育来合成 DOTA-conatumumab。通过基质辅助激光解吸电离质谱和电喷雾四极杆飞行时间质谱确定 conatumumab 分子上 DOTA 分子的绝对数量。通过在 37°C 下将（64）CuCl2（33-222MBq）与 DOTA-conatumumab 孵育 1 小时来制备（64）Cu-DOTA-conatumumab。在动力学分析测定中测试 conatumumab 和 DOTA-conatumumab 与人 Fc 偶联的 DR5（huTR2-Fc）的结合，并用 caspase-3/7 发光测定法测量铜-DOTA-conatumumab 的生物学活性。在携带 Colo205 异种移植物的严重联合免疫缺陷小鼠中进行 DOTA-conatumumab 和铜-DOTA-conatumumab 的体内评估：通过生物分布研究确定组织摄取，并用小动物 PET 和放射自显影确定（64）Cu-DOTA conatumumab 进入肿瘤和其他组织的摄取。
用平均每个 conatumumab 分子 5 个 DOTA 分子制备 DOTA-conatumumab。诱导 DOTA-conatumumab 和 conatumumab 达到 50%效应的体外半数有效浓度分别为 389 和 320pM。通过 caspase 测定的 DOTA-conatumumab 和 conatumumab 的中位数有效剂量（±SD）分别为 135±31 和 128±30pM。在携带 Colo205 异种移植物的雌性 CB17 严重联合免疫缺陷小鼠中，DOTA-conatumumab 和 conatumumab 抑制肿瘤生长的程度相同。小动物 PET 研究显示，在注射示踪剂后 24 小时即可检测到肿瘤摄取，平均标准化摄取值为 3.16（n=2）。用 400μg 未标记的 conatumumab 共给药降低了肿瘤摄取（平均标准化摄取值，1.55；n=2），表明摄取是饱和的。生物分布研究确定的组织摄取与小动物 PET 研究结果一致。
这些结果表明，（64）Cu-DOTA-conatumumab 是一种潜在的用于在肿瘤中成像 DR5 的 PET 示踪剂，并且可能用于测量 conatumumab 的靶标占有率。
