Tumour Targeting Laboratory, Olivia Newton-John Cancer Research Institute, Level 5 ONJ Centre, 145 Studley Road, Heidelberg, VIC, 3084, Australia.
School of Cancer Medicine, La Trobe University, Melbourne, VIC, Australia.
Eur J Nucl Med Mol Imaging. 2024 Nov;51(13):3863-3873. doi: 10.1007/s00259-024-06854-z. Epub 2024 Jul 26.
CI-8993 is a fully human IgG1κ monoclonal antibody (mAb) that binds specifically to immune checkpoint molecule VISTA (V-domain Ig suppressor of T-cell activation). Phase I safety has been established in patients with advanced cancer (NCT02671955). To determine the pharmacokinetics and biodistribution of CI-8993 in patients, we aimed to develop Zr-labelled CI-8993 and validate PET imaging and quantitation in preclinical models prior to a planned human bioimaging trial.
CI-8993 and human isotype IgG1 control were conjugated to the metal ion chelator p-isothiocyanatobenzyl-desferrioxamine (Df). Quality of conjugates were assessed by SE-HPLC, SDS-PAGE, and FACS. After radiolabelling with zirconium-89 (Zr), radioconjugates were assessed for radiochemical purity, immunoreactivity, antigen binding affinity, and serum stability in vitro. [Zr]Zr-Df-CI-8993 alone (1 mg/kg, 4.6 MBq) or in combination with 30 mg/kg unlabelled CI-8993, as well as isotype control [Zr]Zr-Df-IgG1 (1 mg/kg, 4.6 MBq) were assessed in human VISTA knock-in female (C57BL/6 N-Vsir, huVISTA KI) or control C57BL/6 mice bearing syngeneic MB49 bladder cancer tumours; and in BALB/c nu/nu mice bearing pancreatic Capan-2 tumours.
Stable constructs with an average chelator-to-antibody ratio of 1.81 were achieved. SDS-PAGE and SE-HPLC showed integrity of CI-8993 was maintained after conjugation; and ELISA indicated no impact of conjugation and radiolabelling on binding to human VISTA. PET imaging and biodistribution in MB49 tumour-bearing huVISTA KI female mice showed specific localisation of [Zr]Zr-Df-CI-8993 to VISTA in spleen and tumour tissues expressing human VISTA. Specific tumour uptake was also demonstrated in Capan-2 xenografted BALB/c nu/nu mice.
We radiolabelled and validated [Zr]Zr-Df-CI-8993 for specific binding to huVISTA in vivo. Our results demonstrate that Zr-labelled CI-8993 is now suitable for targeting and imaging VISTA expression in human trials.
CI-8993 是一种全人源 IgG1κ 单克隆抗体（mAb），特异性结合免疫检查点分子 VISTA（T 细胞激活的 V 域免疫球蛋白抑制剂）。在晚期癌症患者中已确定了 I 期安全性（NCT02671955）。为了确定 CI-8993 在患者中的药代动力学和生物分布，我们旨在开发 Zr 标记的 CI-8993，并在计划进行人体生物成像试验之前，在临床前模型中验证 PET 成像和定量。
CI-8993 和人同种型 IgG1 对照物与金属离子螯合剂对异硫氰酸苄基去铁胺（Df）缀合。通过 SE-HPLC、SDS-PAGE 和 FACS 评估缀合物的质量。用锆-89（Zr）标记后，评估放射性标记物的放射化学纯度、免疫反应性、抗原结合亲和力和体外血清稳定性。单独使用 [Zr]Zr-Df-CI-8993（1mg/kg，4.6MBq）或与 30mg/kg 未标记的 CI-8993 联合使用，以及同种型对照物 [Zr]Zr-Df-IgG1（1mg/kg，4.6MBq）在携带同源 MB49 膀胱癌肿瘤的人 VISTA 敲入雌性（C57BL/6N-Vsir，huVISTA KI）或对照 C57BL/6 小鼠中进行评估；并在携带胰腺 Capan-2 肿瘤的 BALB/c nu/nu 小鼠中进行评估。
实现了平均螯合剂与抗体比为 1.81 的稳定构建体。SDS-PAGE 和 SE-HPLC 表明，缀合后 CI-8993 的完整性得以维持；ELISA 表明，缀合和放射性标记对与人 VISTA 的结合没有影响。在携带 MB49 肿瘤的 huVISTA KI 雌性小鼠中进行的 PET 成像和生物分布显示，[Zr]Zr-Df-CI-8993 特异性定位于表达人 VISTA 的脾脏和肿瘤组织中的 VISTA。在 Capan-2 异种移植 BALB/c nu/nu 小鼠中也证明了特异性肿瘤摄取。
我们对 [Zr]Zr-Df-CI-8993 进行了放射性标记和验证，以证明其在体内与 huVISTA 的特异性结合。我们的结果表明，Zr 标记的 CI-8993 现在适合用于靶向和成像人类试验中的 VISTA 表达。
