Preclinical evaluation of 89Zr-Df-labeled anti-VISTA antibody CI-8993

Introduction: CI-8993 is a fully human IgG1k monoclonal antibody (mAb) that binds specifically to immune checkpoint molecule VISTA (V-domain Ig suppressor of T-cell activation). Murine anti-VISTA antibodies can overcome anti-PD1 and anti-CTLA4 resistance in orthotopic tumor models. Phase I safety was investigated in NCT02671955 and continues to be explored in Curis’s NTC04475523. To determine the pharmacokinetics and biodistribution of CI-8993 in patients, we aimed to develop ⁸⁹Zr-labeled CI-8993 and validate PET imaging and quantitation in preclinical models prior to a planned human bioimaging trial.

Methods: CI-8993 and isotype IgG1 control were conjugated to the metal ion chelator p-isothiocyanatobenzyl-desferrioxamine (Df). Optimal Df-to-mAb ratio, reaction temperature, reaction time and purification method were established. Quality of conjugates were assessed by SE-HPLC, SDS-PAGE, and FACS. After radiolabeling with zirconium-89 (⁸⁹Zr), radioconjugates were assessed for radiochemical purity, immunoreactivity, antigen binding affinity, and serum stability in vitro. ⁸⁹Zr-Df-CI-8993 alone (1 mg/kg, 4.6 MBq) or in combination with 30 mg/kg unlabeled CI-8993, as well as isotype control ⁸⁹Zr-Df-IgG1 (1 mg/kg, 4.6 MBq) were assessed in huVISTA knock-in female (C57BL/6N-Vsir^{tm1.1(VSIR)Geno}) or control C57BL/6 mice bearing syngeneic MB49 bladder cancer tumors; and in BALB/c nu/nu mice bearing pancreatic tumors. Whole-body animal PET/MRI imaging was performed on day of radioconjugate synthesis, day 1 and day 3 (huVISTA KI model) or day 2 and day 7 (Capan-2 model) p.i. Biodistribution was assessed by image analyses and ex vivo biodistribution studies.

Results: Conjugation of Df to CI-8993 for 60 minutes at room temperature followed by purification via gel filtration resulted in stable constructs with an average chelator-to-antibody ratio of 1.81. SDS-PAGE showed integrity of CI-8993 was maintained after chelation; and ELISA indicated no impact of conjugation on binding to human VISTA. Radiochemical purity (iTLC) and protein integrity (SE-HPLC) at end of synthesis were > 99% and 93%, respectively. PET imaging and biodistribution in MB49 tumor-bearing huVISTA KI female mice showed specific localisation of ⁸⁹Zr-Df-CI-8993 to VISTA expressing organs (liver: 14.98 ± 0.50 %ID/g; spleen: 292.00 ± 14.51 %ID/g; n = 3) compared to ⁸⁹Zr-Df-IgG1 control (liver: 4.615 ± 0.15 %ID/g; spleen: 6.37 ± 0.22 %ID/g; n = 4) or in the presence of competing unlabeled CI-8993 (liver: 8.14 ± 0.50 %ID/g; spleen: 41.14 ± 3.00 %ID/g; n = 5). Tumor-to-blood ratios indicated specific tumor targeting of ⁸⁹Zr-Df-CI-8993 in the presence of unlabeled CI-8993 (20.47 ± 3.09) compared to trace dose ⁸⁹Zr-Df-CI-8993 (0.97 ± 0.12; P = 0.0001) or ⁸⁹Zr-Df-IgG1 control (1.75 ± 0.11; P < 0.0001). Specific tumor uptake was also demonstrated in Capan-2 human pancreatic tumor-bearing BALB/c nu/nu mice.

Conclusions: We have validated ⁸⁹Zr-Df-CI-8993 for specific binding to huVISTA in vivo. Our results demonstrate that ⁸⁹Zr-labeled CI-8993 is now suitable for use in the bioimaging clinical trial study planned in solid tumor patients.

• Download figure
• Open in new tab
• Download powerpoint