目的:RANKL在肿瘤微环境中的表达已被确定为免疫抑制的生物标志物,否定某些癌症免疫疗法的效果。以前,我们开发了一种基于FDA批准的RANKL特异性抗体denosumab的放射性示踪剂,[89Zr]Zr-DFO-denosumab,使成功的免疫PET成像。放射性标记的denosumab,然而,表现出长血液循环和肿瘤摄取延迟,可能会限制其应用。在这里,我们旨在开发一个更小的放射性标记的denosumab片段,[64Cu]Cu-NOTA-denos-Fab,理想情况下,这将显示更快的肿瘤积累和更好的扩散到肿瘤中,用于RANKL的可视化。
方法:使用木瓜蛋白酶从denosumab制备Fab片段,并与NOTA螯合剂缀合用于用64Cu放射性标记。使用SDS-PAGE分析在体外表征生物缀合物,并且使用放射性示踪剂细胞结合测定法评估结合亲和力。小动物PET成像评估了转导的RANKL-ME-180异种移植物中的肿瘤靶向和生物分布。
结果:[64Cu]Cu-NOTA-denos-Fab的放射性标记产率为58±9.2%,比活性为0.79±0.11MBq/µg(n=3)。放射性示踪剂结合测定证明了RANKL在体外的特异性靶向。PET显像显示,早在1hp.i.(2.14±0.21%ID/mL),血液清除快,肿瘤积聚高。在5小时p.i.达到峰值(2.72±0.61%ID/mL)。相比之下,[64Cu]Cu-NOTA-denosumab在24hp.i.(6.88±1.12%ID/mL)时达到最高肿瘤摄取。与阻断组和阴性ME-180异种移植模型相比,[64Cu]Cu-NOTA-denos-Fab特异性靶向转导的ME-180异种移植物中的人RANKL。组织学分析证实RANKL-ME-180异种移植物中的RANKL表达。
结论:这里,我们报道了一种新型的RANKLPET显像剂,[64Cu]Cu-NOTA-denos-Fab,与抗体对应物相比,可以进行快速肿瘤成像,提高成像对比度,有望成为未来临床应用的潜在PETRANKL成像工具。
OBJECTIVE: RANKL expression in the tumor microenvironment has been identified as a biomarker of immune suppression, negating the effect of some cancer immunotherapies. Previously we had developed a radiotracer based on the FDA-approved RANKL-specific antibody denosumab, [89Zr]Zr-DFO-denosumab, enabling successful immuno-PET imaging. Radiolabeled denosumab, however, showed long blood circulation and delayed tumor uptake, potentially limiting its applications. Here we aimed to develop a smaller radiolabeled denosumab fragment, [64Cu]Cu-NOTA-denos-Fab, that would ideally show faster tumor accumulation and better diffusion into the tumor for the visualization of RANKL.
METHODS: Fab fragments were prepared from denosumab using papain and conjugated to a NOTA chelator for radiolabeling with 64Cu. The bioconjugates were characterized in vitro using SDS-PAGE analysis, and the binding affinity was assessed using a radiotracer cell binding assay. Small animal PET imaging evaluated tumor targeting and biodistribution in transduced RANKL-ME-180 xenografts.
RESULTS: The radiolabeling yield of [64Cu]Cu-NOTA-denos-Fab was 58 ± 9.2%, with a specific activity of 0.79 ± 0.11 MBq/µg (n = 3). A radiotracer binding assay proved specific targeting of RANKL in vitro. PET imaging showed fast blood clearance and high tumor accumulation as early as 1 h p.i. (2.14 ± 0.21% ID/mL), which peaked at 5 h p.i. (2.72 ± 0.61% ID/mL). In contrast, [64Cu]Cu-NOTA-denosumab reached its highest tumor uptake at 24 h p.i. (6.88 ± 1.12% ID/mL). [64Cu]Cu-NOTA-denos-Fab specifically targeted human RANKL in transduced ME-180 xenografts compared with the blocking group and negative ME-180 xenograft model. Histological analysis confirmed RANKL expression in RANKL-ME-180 xenografts.
CONCLUSIONS: Here, we report on a novel RANKL PET imaging agent, [64Cu]Cu-NOTA-denos-Fab, that allows for fast tumor imaging with improved imaging contrast when compared with its antibody counterpart, showing promise as a potential PET RANKL imaging tool for future clinical applications.