CI-8993 是一种全人源 IgG1κ 单克隆抗体 (mAb)，可与免疫检查点分子 VISTA（T 细胞激活的 V 结构域 Ig 抑制因子）特异性结合。晚期癌症患者的 I 期安全性已得到证实 (NCT02671955)。为了确定 CI-8993 在患者中的药代动力学和生物分布，我们的目标是开发 89Zr 标记的 CI-8993，并在计划的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 对照偶联金属离子螯合剂对异硫氰酸苄基去铁胺 (Df)。通过 SE-HPLC、SDS-PAGE 和 FACS 评估缀合物的质量。用锆 89 (89Zr) 进行放射性标记后，评估放射性结合物的放射化学纯度、免疫反应性、抗原结合亲和力和体外血清稳定性。单独使用 [89Zr]Zr-Df-CI-8993（1 mg/kg，4.6 MBq）或与 30 mg/kg 未标记 CI-8993 以及同种型对照 [89Zr]Zr-Df-IgG1（1 mg/ kg, 4.6 MBq) 在人类 VISTA 敲入雌性 (C57BL/6 N-Vsirtm1.1(VSIR)Geno, huVISTA KI) 或携带同基因 MB49 膀胱癌肿瘤的对照 C57BL/6 小鼠中进行评估；以及在携带胰腺 Capan-2 肿瘤的 BALB/c nu/nu 小鼠中。获得了平均螯合剂与抗体比率为 1.81 的稳定构建体。 SDS-PAGE 和 SE-HPLC 显示 CI-8993 在缀合后保持完整性； ELISA 表明缀合和放射性标记对与人 VISTA 的结合没有影响。携带 MB49 肿瘤的 huVISTA KI 雌性小鼠的 PET 成像和生物分布显示，[89Zr]Zr-Df-CI-8993 在表达人 VISTA 的脾脏和肿瘤组织中特异性定位于 VISTA。 Capan-2 异种移植的 BALB/c nu/nu 小鼠也证明了特异性肿瘤摄取。我们对 [89Zr]Zr-Df-CI-8993 进行放射性标记并验证其在体内与 huVISTA 的特异性结合。我们的结果表明，89Zr 标记的 CI-8993 现在适用于人体试验中 VISTA 表达的靶向和成像。© 2024。作者。

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 89Zr-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 (89Zr), radioconjugates were assessed for radiochemical purity, immunoreactivity, antigen binding affinity, and serum stability in vitro. [89Zr]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 [89Zr]Zr-Df-IgG1 (1 mg/kg, 4.6 MBq) were assessed in human VISTA knock-in female (C57BL/6 N-Vsirtm1.1(VSIR)Geno, 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 [89Zr]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 [89Zr]Zr-Df-CI-8993 for specific binding to huVISTA in vivo. Our results demonstrate that 89Zr-labelled CI-8993 is now suitable for targeting and imaging VISTA expression in human trials.© 2024. The Author(s).